Textbook of Assisted Reproductive Technologies: Laboratory and Clinical Perspectives

d ir on Th iti Ed

Gardner Weissman Howles

Textbook of Assisted Reproductive Technologies

Shoham

Laboratory and Clinical Perspectives

From reviews of previous editions: ‘Sampling liberally from the wealth of knowledge contained between its covers will be rewarded by affirming knowledge already garnered from experience or, better, augmenting knowledge to improve one’s understanding and practice through exposure to a fresh perspective’ Fertility and Sterility ‘The book’s real value is that it is standing on our shelf in the clinic. We discuss a day-to-day problem in the unit and immediately know where to look’ OBGYN Contains sections on: Establishing and Maintaining an IVF Laboratory • Gamete Collection, Preparation and Selection • Micromanipulation • Culture, Selection and Transfer of the Human Embryo • Cryopreservation • Diagnosis of Genetic Disease in Preimplantation Embryos • Implantation • Quality Management Systems • Patient Investigation and the Use of Drugs • Stimulation Protocols • Technical Procedures and Outcomes • Special Medical Conditions • Complications of Treatment • Egg Donation and Surrogate Motherhood • Future Directions and Clinical Applications • The Support Team • Ethics and Legislation

With over 300 color and black-and-white illustrations

David K Gardner DPhil is Chair of Zoology at the University of Melbourne, Australia, and Scientific Director at the Colorado Center for Reproductive Medicine, USA

Ariel Weissman MD is a senior physician at the IVF Unit, Department of Obstetrics and Gynecology, Edith Wolfson Medical Center, Holon, and Sackler Faculty of Medicine, Tel Aviv University, Israel

Colin M Howles PhD, FRSM

is Vice President, Scientific Affairs Fertility, Global Medical Affairs, Merck Serono International SA, Geneva, Switzerland

Textbook of Assisted Reproductive Technologies

A truly comprehensive manual for the whole team at the IVF clinic, this covers both laboratory aspects and their clinical application. Methods, protocols and techniques of choice are presented by eminent international experts. The third edition has been extensively revised, with the addition of important new chapters on developing techniques.

Textbook of Assisted Reproductive Technologies Laboratory and Clinical Perspectives

Third Edition

Edited by

David K Gardner Ariel Weissman Colin M Howles Zeev Shoham

Zeev Shoham MD is Director, Reproductive Medicine and Infertility Unit, Department of Obstetrics and Gynecology, at Kaplan Medical Center, Rehovot, Israel

Third Edition

ISBN 978-0-415-44894-9

Special Edition

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Textbook of Assisted Reproductive Technologies

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The editors (from left to right: David K Gardner, Colin M Howles, Zeev Shoham and Ariel Weissman) at the annual meeting of ESHRE, Barcelona, 2008 The editors would like to make a special acknowledgment to their respective children, who are a constant reminder of the joy and happiness that working in this field of medicine can bring to families, friends, and communities

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Textbook of Assisted Reproductive Technologies Laboratory and Clinical Perspectives Third Edition

Edited by David K Gardner DPhil Chair of Zoology, University of Melbourne, Victoria, Australia and Scientific Director, Colorado Center for Reproductive Medicine, USA Ariel Weissman MD Senior Physician, IVF Unit, Department of Obstetrics and Gynecology, Edith Wolfson Medical Center, Holon and Sackler Faculty of Medicine, Tel Aviv University Tel Aviv, Israel Colin M Howles PhD, FRSM Vice President, Scientific Affairs Fertility, Global Medical Affairs, Merck Serono International SA, Geneva, Switzerland

Zeev Shoham MD Director, Reproductive Medicine and Infertility Unit, Department of Obstetrics and Gynecology, Kaplan Medical Center, Rehovot, Israel

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© 2009 Informa UK Ltd First published in the United Kingdom in 2004 Third edition published in the United Kingdom in 2009 by Informa Healthcare, Telephone House, 69–77 Paul Street, London, EC2A 4LQ. Informa Healthcare is a trading division of Informa UK Ltd. Registered Office: 37/41 Mortimer Street, London W1T 3JH. Registered in England and Wales number 1072954 Tel: +44 (0)20 7017 5000 Fax: +44 (0)20 7017 6699 Website: www.informahealthcare.com All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior permission of the publisher or in accordance with the provisions of the Copyright, Designs and Patents Act 1988 or under the terms of any licence permitting limited copying issued by the Copyright Licensing Agency, 90 Tottenham Court Road, London W1P 0LP. Although every effort has been made to ensure that all owners of copyright material have been acknowledged in this publication, we would be glad to acknowledge in subsequent reprints or editions any omissions brought to our attention. The Authors have asserted their rights under the Copyright, Designs and Patents Act 1988 to be identified as the Authors of this Work. A CIP record for this book is available from the British Library. Library of Congress Cataloging-in-Publication Data Data available on application ISBN 978-0-415-44894-9 Distributed in North and South America by Taylor & Francis 6000 Broken Sound Parkway, NW, (Suite 300) Boca Raton, FL 33487, USA Within Continental USA Tel: 1 (800) 272 7737; Fax: 1 (800) 374 3401 Outside Continental USA Tel: (561) 994 0555; Fax: (561) 361 6018 Email: [email protected] Book orders in the rest of the world Paul Abrahams Tel: +44 (0) 207 017 6917 Email: [email protected] Composition by C&M Digitals (P) Ltd, Chennai, India Printed and bound in India by Replika Press Pvt. Ltd

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Contents List of Contributors Introduction Robert G Edwards

ix xvii

Establishing and Maintaining an IVF Laboratory 1. Setting up an ART laboratory Jacques Cohen, Antonia Gilligan and John Garrisi 2. Quality control: maintaining stability in the laboratory David H McCulloh 3. The ART laboratory in the era of ISO 1000 and GLP Cecelia Sjöblom and Christoph Keck

1 9 25

Gamete Collection, Preparation and Selection 4. Evaluation of sperm Kaylen Silverberg and Tom Turner 5. Sperm preparation techniques Harold Bourne, Janell Archer, David H Edgar and HW Gordon Baker 6. Sperm chromatin assessment Ashok Agarwal, Juris Erenpreiss and Rakesh Sharma 7. Oocyte retrieval and selection Laura F Rienzi and Filippo M Ubaldi 8. Preparation and evaluation of oocytes for ICSI Irit Granot and Nava Dekel 9. Oocyte in vitro maturation Daniela Nogueira, Sergio Romero, Leen Vanhoutte, Daniel G de Matos and Johan Smitz 10. Use of in vitro maturation in a clinical setting Anne-Maria Suikkari

39 53 67 85 103 111 155

Micromanipulation 11. Equipment and general technical aspects of micromanipulation of gametes and embryos Frank L Barnes 12. Intracytoplasmic sperm injection: technical aspects Gianpiero D Palermo, Queenie V Neri, Takumi Takeuchi, Simon J Hong and Zev Rosenwaks 13. Assisted hatching Anna Veiga, Irene Boiso and Itziar Belil 14. Human embryo biopsy procedures Alan R Thornhill and Alan H Handyside

163 171 181 191

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Culture, Selection and Transfer of the Human Embryo 15. Analysis of fertilization Lynette Scott 16. Culture systems for the human embryo David K Gardner and Michelle Lane 17. Evaluation of embryo quality: new strategies to facilitate single embryo transfer Denny Sakkas and David K Gardner

207 219 241

Cryopreservation 18. The human oocyte: controlled rate cooling Andrea Borini and Giovanni Coticchio 19. The human oocyte: vitrification Masashige Kuwayama 20. The human embryo: slow freezing Lucinda L Veeck Gosden, Rosemary Berrios, Richard Bodine, Robert N Clarke and Nikica Zaninovic 21. The human embryo: vitrification Zsolt Peter Nagy, Gábor Vajta, Ching-Chien Chang and Hilton Kort 22. Managing the cryopreserved embryo bank Phillip Matson 23. Cryopreservation and storage of spermatozoa Eileen A McLaughlin and Allan A Pacey 24. Handling and cryopreservation of testicular sperm Joseph P Alukal, Dolores J Lamb and Larry I Lipshultz 25. Ovarian tissue cryopreservation and other fertility preservation strategies Erkan Buyuk, Ozgur Oktem, Murat Sonmezer and Kutluk H Oktay

255 267 275 289 305 311 323 327

Diagnosis of Genetic Disease in Preimplantation Embryos 26. Severe male factor: genetic consequences and recommendations for genetic testing Inge Liebaers, André Van Steirteghem and Willy Lissens 27. Polar body biopsy Markus Montag, Katrin van der Ven and Hans van der Ven 28. Clinical application of polar body biopsy Yury Verlinsky and Anver Kuliev 29. Preimplantation genetic diagnosis for infertility Santiago Munné 30. Genetic analysis of the embryo Yural Yaron, Veronica Gold, Ronni Gamzu and Mira Malcov 31. Proteomic analysis of the embryo Mandy Katz-Jaffe

343 357 371 381 403 417

Implantation 32. Embryonic and maternal dialogue and the analysis of uterine receptivity Francisco Domínguez, Jose Antonio Horcajadas and Carlos Simón

427

Quality Management Systems 33. Quality management in reproductive medicine Christoph Keck, Cecelia Sjöblom, Robert Fischer, Vera Baukloh and Michael Alper

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Patient Investigation and the Use of Drugs 34. Indications for IVF treatment: from diagnosis to prognosis Nick S Macklon, Frank J Broekmans and Bart CJM Fauser 35. Initial investigation of the patient (female and male) Bulent Gulekli, Tim J Child and Seang Lin Tan 36. Drugs used for controlled ovarian stimulation: clomiphene citrate, aromatase inhibitors, metformin, gonadotropins, gonadotropin-releasing hormone analogs, and recombinant gonadotropins Zeev Shoham and Colin M Howles 37. The role of FSH and LH in ovulation induction: current concepts Juan Balasch

447 459

469 489

Stimulation Protocols 38. Endocrine characteristics of ART cycles Jean-Noël Hugues and Isabelle Cédrin-Durnerin 39. The use of GnRH agonists Judith AF Huirne and Roel Schats 40. GnRH antagonists Michael Ludwig 41. Monitoring IVF cycles Matts Wikland and Torbjörn Hilljensjö 42. Oocyte collection Gab Kovacs 43. The luteal phase: luteal support protocols James P Toner 44. Treatment strategies in assisted reproduction for the low responder patient Ariel Weissman and Colin M Howles 45. Repeated implantation failure: the preferred therapeutic approach Mark A Damario and Zev Rosenwaks

511 529 539 553 559 565 577 617

Technical Procedures and Outcomes 46. Ultrasound in ART Marinko M Biljan 47. Sperm-recovery techniques: clinical aspects Herman Tournaye and Patricio Donoso 48. Gamete intrafallopian transfer (GIFT) and zygote intrafallopian transfer (ZIFT) Machelle M Seibel and Ariel Weissman 49. Embryo transfer Leif Bungum and Mona Bungum 50. Anesthesia and in-vitro fertilization Ethan E Harow 51. Medical considerations of single embryo transfer Outi Hovatta

635 657 673 693 701 707

Special Medical Conditions 52. Endometriosis and ART Andy Huang, Mark Hunter and Alan H DeCherney 53. Polycystic ovaries and ART Thomas H Tang and Adam H Balen

711 721

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54. Prognostic testing for ovarian reserve Frank J Broekmans, Bart CJM Fauser and Nick S Macklon 55. Management of hydrosalpinx Annika Strandell

737 547

Complications of Treatment 56. Severe ovarian hyperstimulation syndrome Zalman Levine and Daniel Navot 57. The environment and reproduction Kenneth Barron and Machelle M Seibel 58. Bleeding, severe pelvic infection, and ectopic pregnancy Raoul Orvieto and Zion Ben-Rafael 59. Iatrogenic multiple pregnancy: the risk of ART Isaac Blickstein

759 773 787 795

Egg Donation and Surrogate Motherhood 60. Egg and embryo donation Mark V Sauer and Matthew A Cohen 61. Gestational surrogacy Peter R Brinsden

807 817

Future Directions and Clinical Applications 62. Human embryonic stem cells Rachel Eiges and Benjamin Reubinoff 63. Microfluidics in ART: current progress and future directions Jason E Swain, Thomas B Pool, Shuichi Takyama and Gary D Smith

827 843

The Support Team 64. The evolving role of the ART nurse: a contemporary review Joanne L Libraro 65. Patient support in the ART program Sharon N Covington 66. The relationship between stress and in vitro fertilization outcome Andrea Mechanick Braverman

859 867 877

Ethics and Legislation 67. The impact of legislation and socioeconomic factors in the access to and global practice of ART Fernando Zegers-Hochschild and Karl G Nygren 68. Recent ethical dilemmas in ART Françoise Shenfield Index

885 895

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List of Contributors Ashok Agarwal Center for Reproductive Medicine, Glickman Urological and Kidney Institute and Obstetrics–Gynecology and Women’s Health Institute Cleveland Clinic Cleveland, Ohio, USA Michael Alper Boston IVF Waltham, Massachusetts, USA Joseph P Alukal Scott Department of Urology Baylor College of Medicine Houston, Texas, USA Janell Archer Reproductive Services The Royal Women’s Hospital and Melbourne IVF Melbourne, Victoria, Australia HW Gordon Baker University of Melbourne Department of Obstetrics and Gynaecology The Royal Women’s Hospital and Melbourne IVF Melbourne, Victoria, Australia Juan Balasch Department of Obstetrics and Gynecology Faculty of Medicine Hospital Clinic University of Barcelona Barcelona, Spain Adam H Balen Reproductive Medicine and Surgery Leeds General Infirmary Leeds, UK Frank L Barnes IVF Labs, LLC Salt Lake City, Utah, USA

Kenneth Barron Department of Obstetrics and Gynecology University of Massachusetts School of Medicine Worcester, Massachusetts, USA Vera Baukloh Fertility Center Hamburg Hamburg, Germany Itziar Belil Reproductive Medicine Service Institut Universitari Dexeus Barcelona, Spain Zion Ben-Rafael Department of Obstetrics and Gynecology Rabin Medical Center, Petah Tikva and Sackler Faculty of Medicine Tel Aviv University Tel Aviv, Israel Rosemary Berrios The Center for Reproductive Medicine and Infertility Weill Medical College of Cornell University New York, New York, USA †Marinko M Biljan Isaac Blickstein Department of Obstetrics and Gynecology Kaplan Medical Center Rehovot, Israel Richard Bodine The Center for Reproductive Medicine and Infertility Weill Medical College of Cornell University New York, New York, USA Irene Boiso Centre de Reproducció Assistida Clinica Sagrada Familia Barcelona, Spain

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Andrea Borini Tecnobios Procreazione Centre for Reproductive Health Bologna, Italy

Robert N Clarke The Center for Reproductive Medicine and Infertility Weill Medical College of Cornell University New York, New York, USA

Harold Bourne Reproductive Services and Melbourne IVF The Royal Women’s Hospital Carlton, Victoria, Australia

Jacques Cohen Galileo Research Laboratories Ansonia Station New York, New York, USA

Andrea Mechanick Braverman Psychological and Complementary Care Reproductive Medicine Associates of New Jersey Morristown, New Jersey, USA

Matthew A Cohen Department of Obstetrics and Gynecology College of Physicians & Surgeons Columbia University New York, New York, USA

Peter R Brinsden Bourn Hall Clinic Bourn, Cambridge, UK Frank J Broekmans Department of Reproduction and Gynaecology University Medical Centre Utrecht Utrecht, The Netherlands Leif Bungum Reproductive Medicine Centre Malmo University Hospital Malmo, Sweden Mona Bungum Reproductive Medicine Centre Malmo University Hospital Malmo, Sweden Erkan Buyuk Department of Obstetrics and Gynecology Albert Einstein College of Medicine of Yeshiva University New York, New York, USA

Giovanni Coticchio Tecnobios Procreazione Bologna, Italy Sharon N Covington Psychological Support Services Shady Grove Fertility Reproductive Science Center Rockville, Maryland, USA Mark A Damario Department of Obstetrics, Gynecology and Women’s Health University of Minnesota Minneapolis, Minnesota, USA Alan H DeCherney Department of Obstetrics and Gynecology David Geffen School of Medicine Los Angeles, California, USA Nava Dekel Department of Biological Regulation The Weizmann Institute of Science Rehovot, Israel

Isabelle Cédrin-Durnerin University of Paris XIII Division of Reproductive Medicine Hôpital Jean Verdier Bondy, France

Daniel G de Matos EMD Serono Reproductive Biology Institute Rockland, Massachusetts, USA

Ching-Chien Chang Reproductive Biology Associates Atlanta, Georgia, USA

Francisco Domínguez Fundación Instituto Valenciano de Infertilidad Instituto Universitario IVI Valencia University Valencia, Spain

Tim J Child Oxford Fertility Unit Nuffield Department of Obstetrics and Gynaecology University of Oxford John Radcliffe Hospital Oxford, UK

Patricio Domoso Centre for Reproductive Medicine Clinica Alemana de Santiago Santiago, Chile

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David H Edgar Reproductive Services The Royal Women’s Hospital and Melbourne IVF Melbourne, Victoria, Australia

Irit Granot IVF Unit, Department of Obstetrics and Gynecology Kaplan Medical Center Rehovot, Israel

Robert G Edwards Duck End Farm Dry Drayton Cambridge, UK

Bulent Gulekli Dokuz Eylul Universitesi Tip Fakultesi Kadin Hastaliklari ve Dogum ABD Balcova-Izmir, Turkey

Rachel Eiges IVF Unit, Department of Obstetrics and Gynecology and Goldyne Savad Institute of Gene Therapy Hadassah University Hospital Jerusalem, Israel Juris Erenpreiss Andrology Laboratory Riga Stradins University Riga, Latvia Bart CJM Fauser Department of Reproductive Medicine University Medical Center Utrecht Utrecht, The Netherlands Robert Fischer Fertility Center Hamburg Hamburg, Germany Ronni Gamzu Department of Obstetrics and Gynecology Lis Maternity Hospital Tel Aviv Sourasky Medical Center Tel Aviv, Israel David K Gardner Department of Zoology University of Melbourne, Victoria, Australia John Garrisi Galileo Research Laboratories LLC New York, New York, USA Antonia Gilligan Alpha Environmental, Inc. Jersey City, New Jersey, USA Veronica Gold Sara Racine In Vitro Fertilization Unit Tel Aviv Sourasky Medical Center Tel Aviv, Israel

Alan H Handyside The London Bridge Fertility, Gynaecology and Genetics Centre London, UK Ethan E Harow Outpatient Surgical Center Edith Wolfson Medical Center Holon, Israel Torbjörn Hillensjö Fertility Centre Scandinavia Carlander’s Hospital Göteborg, Sweden Simon J Hong Andrology and Assisted Fertilization Cornell Institute for Reproductive Medicine New York, New York, USA Jose Antonio Horcajadas Fundación Instituto Valenciano de Infertilidad Instituto Universitario IVI Valencia University Valencia, Spain Outi Hovatta Karolinska Institute Karolinska University Hospital Huddinge Stockholm, Sweden Colin M Howles Global Medical Affairs Merck Serono International SA Geneva, Switzerland Andy Huang Department of Obstetrics and Gynecology David Geffen School of Medicine Los Angeles, California, USA Jean-Noël Hugues University of Paris XIII Division of Reproductive Medicine Hôpital Jean Verdier Bondy, France

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Judith AF Huirne Department of Obstetrics and Gynecology Division of Reproduction and Fertility Investigation IVF Center Vrije Universiteit Medical Center Amsterdam, The Netherlands Mark Hunter Department of Obstetrics and Gynecology David Geffen School of Medicine Los Angeles, California, USA

Joanne L Libraro Center for Reproductive Medicine and Infertility Weill Medical College New York, New York, USA Inge Liebaers Center for Medical Genetics University Hospital VUB Brussels, Belgium

Mandy Katz-Jaffe Colorado Center for Reproductive Medicine Lone Tree, Colorado, USA

Larry I Lipshultz Division of Male Reproductive Medicine and Surgery Baylor College of Medicine Scott Department of Urology Houston, Texas, USA

Christoph Keck Department of Obstetrics and Gynecology University of Freiburg Freiburg, Germany

Willy Lissens Center for Medical Genetics University Hospital VUB Brussels, Belgium

Hilton Kort Reproductive Biology Associates Atlanta, Georgia, USA

Michael Ludwig Centre for Reproductive Medicine and Gynaecological Endocrinology Endokrinologikum Hamburg Hamburg, Germany

Gab Kovacs Monash IVF Richmond, Victoria, Australia Anver Kuliev Reproductive Genetics Institute Chicago, Illinois, USA

Nick S Macklon Division of Reproductive Medicine Department of Obstetrics and Gynecology Erasmus Medical Center Rotterdam, The Netherlands

Masashige Kuwayama Kato Ladies’ Clinic Shinjuku Tokyo, Japan

Mira Malcov Sara Racine In Vitro Fertilization Unit Tel Aviv Sourasky Medical Center Tel Aviv, Israel

Dolores J Lamb Scott Department of Urology Baylor College of Medicine Houston, Texas, USA

Phillip Matson Hollywood Fertility Centre Hollywood Private Hospital Monash Avenue Nedlands, Western Australia, Australia

Michelle Lane Department of Obstetrics and Gynecology University of Adelaide Adelaide, South Australia and Repromed Dulwich, South Australia, Australia Zalman Levine Division of Reproductive Endocrinology and Infertility New York Medical College Fertility Institute of New Jersey and New York Westwood, New Jersey, USA

David H McCulloh University Reproductive Associates, PC Hasbrouck Heights, New Jersey, USA Eileen A McLaughlin ARC Centre of Excellence in Biotechnology and Development School of Environmental and Life Sciences University of Newcastle Callaghan, New South Wales, Australia

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Markus Montag Department of Gynaecological Endocrinology and Reproductive Medicine University Clinics Bonn Bonn, Germany

Allan A Pacey Academic Unit of Reproductive and Developmental Medicine University of Sheffield Sheffield, UK

Santiago Munné Institute for Reproductive Medicine and Science of Saint Barnabus Reprogenetics Livingston, New Jersey, USA

Gianpiero D Palermo Andrology and Assisted Fertilization Cornell Institute for Reproductive Medicine New York, New York, USA

Zsolt Peter Nagy Reproductive Biology Associates Atlanta, Georgia, USA

Thomas B Pool Fertility Center of San Antonio San Antonio, Texas, USA

Daniel Navot Division of Reproductive Endocrinology and Infertility New York Medical College Fertility Institute of New Jersey and New York Westwood, New Jersey, USA

Benjamin Reubinoff IVF Unit, Department of Obstetrics and Gynecology and Goldyne Savad Institute of Gene Therapy Hadassah University Hospital Jerusalem, Israel

Queenie V Neri Andrology and Assisted Fertilization Cornell Institute for Reproductive Medicine New York, New York, USA

Laura F Rienzi Centre for Reproductive Medicine Clinica Valle Giulia Rome, Italy

Daniela Nogueira Follicle Biology Laboratory Center for Reproductive Medicine Vrije Universiteit Brussel Brussels, Belgium

Sergio Romero Follicle Biology Laboratory Center for Reproductive Medicine Vrije Universiteit Brussel Brussels, Belgium

Karl G Nygren Fertility and IVF Unit Sophiahemmet Hospital Stockholm, Sweden

Zev Rosenwaks The Center for Reproductive Medicine and Infertility Weill Medical College of Cornell University New York, New York, USA

Kutluk H Oktay Department of Obstetrics & Gynecology New York Medical College, Valhalla and Institute for Fertility Preservation Center for Human Reproduction and Memorial Sloan Kettering Cancer Center New York, New York, USA Ozgur Oktem The Center for Reproductive Medicine and Infertility Weill Medical College of Cornell University New York, New York, USA Raoul Orvieto Department of Obstetrics and Gynecology Rabin Medical Center, Petah Tikva and Sackler Faculty of Medicine Tel Aviv University Tel Aviv, Israel

Denny Sakkas Department of Obstetrics and Gynecology Yale University School of Medicine New Haven, Connecticut, USA Mark V Sauer Department of Obstetrics and Gynecology College of Physicians & Surgeons Columbia University New York, New York, USA Roel Schats Department of Obstetrics and Gynecology Division of Reproduction and Fertility Investigation IVF Center Vrije Universiteit Medical Center Amsterdam, The Netherlands

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Lynette Scott Fertility Center of New England Reading, Massachusetts, USA Machelle M Seibel Department of Obstetrics and Gynecology University of Massachusetts School of Medicine Worcester, Massachusetts, USA Rakesh Sharma Center for Reproductive Medicine, Glickman Urological and Kidney Institute and Obstetrics–Gynecology and Women’s Health Institute Cleveland Clinic Cleveland, Ohio, USA Françoise Shenfield Reproductive Medicine Unit University College Hospital and Medical School London, UK Zeev Shoham Department of Obstetrics and Gynecology Kaplan Medical Center Rehovot, Israel Kaylen Silverberg Texas Fertility Center Austin IVF Austin, Texas, USA Carlos Simón Fundación Instituto Valenciano de Infertilidad Instituto Universitario IVI Valencia University and Centro de Investigación Príncipe Felipe Valencia, Spain Cecilia Sjöblom NURTURE University of Nottingham Queen’s Medical Centre Nottingham, UK Gary D Smith Department of Obstetrics and Gynecology and Reproductive Medicine Program University of Michigan Ann Arbor, Michigan, USA Johan Smitz Radioimmunology and Reproductive Biology Center for Reproductive Medicine University Hospital VUB Brussels, Belgium

Murat Sonmezer Department of Obstetrics and Gynecology School of Medicine Ankara University Ankara, Turkey Annika Strandell Reproductive Medicine Department of Obstetrics and Gynecology Sahlgrenska University Hospital Göteborg, Sweden Anne-Maria Suikkari Väestöliitto Fertility Clinics Helsinki, Finland Jason E Swain Fertility Center of San Antonio San Antonio, Texas, USA Takumi Takeuchi Andrology and Assisted Fertilization Cornell Institute for Reproductive Medicine New York, New York, USA Shuichi Takyama Department of Obstetrics and Gynecology University of Michigan Ann Arbor, Michigan, USA Seang Lin Tan McGill Reproductive Center Royal Victoria Hospital Department of Obstetrics and Gynecology McGill University Montreal, Quebec, Canada Thomas H Tang Reproductive Medicine and Surgery Leeds General Infirmary Leeds, UK Alan R Thornhill The London Bridge Fertility, Gynaecology and Genetics Centre and Department of Obstetrics and Gynecology University College London London, UK James P Toner Atlanta Center for Reproductive Medicine Woodstock, Georgia, USA

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Herman Tournaye Center for Reproductive Medicine University Hospital of the Dutch Speaking Brussels Free University Brussels, Belgium Tom Turner Texas Fertility Center Austin IVF Austin, Texas, USA

Lucinda L Veeck Gosden The Center for Reproductive Medicine and Infertility Weill Medical College of Cornell University New York, New York, USA Anna Veiga Reproductive Medicine Service Institut Universitari Dexeus Banc de Linies Cellulars Centre de Medicina Regenerativa de Barcelona Barcelona, Spain

Filippo M Ubaldi Centre for Reproductive Medicine Clinica Valle Giulia Rome, Italy

Yury Verlinsky Reproductive Genetics Institute Chicago, Illinois, USA

Gábor Vajta Academic Director PIVET Medical Centre Perth, Western Australia Australia

Ariel Weissman IVF Unit Department of Obstetrics and Gynecology Edith Wolfson Medical Center Holon, Israel

Hans van der Ven Department of Gynaecological Endocrinology and Reproductive Medicine University of Bonn Bonn, Germany

Matts Wikland Fertility Centre Scandinavia Carlander’s Hospital Göteborg, Sweden

Katrin van der Ven Department of Gynaecological Endocrinology and Reproductive Medicine University of Bonn Bonn, Germany Leen Vanhoutte Follicle Biology Laboratory Center for Reproductive Medicine Vrije Universiteit Brussel Brussels, Belgium André Van Steirteghem Center for Medical Genetics University Hospital VUB Brussels, Belgium

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Yural Yaron Prenatal Genetic Diagnosis Division Genetic Institute Tel Aviv Sourasky Medical Center Tel Aviv, Israel Nikica Zaninovic The Center for Reproductive Medicine and Infertility Weill Medical College of Cornell University New York, New York, USA Fernando Zegers-Hochschild Unit of Reproductive Medicine Clínica las Condes Santiago, Chile

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Introduction: the beginnings of human in vitro fertilization Robert G Edwards

In vitro fertilization (IVF) and its derivatives in preimplantation diagnosis, stem cells, and the ethics of assisted reproduction continue to attract immense attention scientifically and socially. All these topics were introduced by 1970. Hardly a day passes without some public recognition of events related to this study, and clinics spread ever further worldwide. Now we must be approaching 1.5 million IVF births, it is time to celebrate what has been achieved by so many investigators, clinical, scientific, and ethical. While much of this Introduction covers the massive accumulation of events between 1960 and 2000, it also briefly discusses new perspectives emerging in the 21st century. Fresh advances also increase curiosity about how these fields of study began and how their ethical implications were addressed in earlier days. As for me, I am still stirred by recollections of those early days. Foundations were laid in Edinburgh, London, and Glasgow in the 1950s and early 1960s. Discoveries made then led to later days in Cambridge, working there with many PhD students. It also resulted in my working with Patrick Steptoe in Oldham. Our joint opening of Bourn Hall in 1980, which became the largest IVF clinic of its kind at the time, signified the end of the beginning of assisted human conception and the onset of dedicated applied studies.

Introduction First of all, I must express in limited space my tributes to my teachers, even if inadequately. These include investigators from far-off days when the fundamental facts of reproductive cycles, surgical techniques, endocrinology, and genetics were elicited by many investigators. These fields began to move in the 20th century, and if one pioneer of these times should be saluted, it must be Gregory Pincus. Famous for the contraceptive pill, he was a distinguished embryologist, and part of his work dealt with the maturation of mammalian oocytes in vitro. He was the first to show how oocytes aspirated from their follicles would begin their maturation in vitro, and how a number

matured and expelled a first polar body. I believe his major work was done in rabbits, where he found that the 10–11-hour timings of maturation in vitro accorded exactly with those occurring in vivo after an ovulatory stimulus to the female rabbit. Pincus et al also studied human oocytes.1 Extracting oocytes from excised ovaries, they identified chromosomes in a large number of oocytes and interpreted this as evidence of the completion of maturation in vitro. Many oocytes possessed chromosomes after 12 hours, the proportion remaining constant over the next 30 hours and longer. Twelve hours was taken as the period of maturation. Unfortunately, chromosomes were not classified for their meiotic stage. Maturing oocytes would be expected to display diakinesis or metaphase-I chromosome pairs. Fully mature oocytes would display metaphase-II chromosomes, signifying they were fully ripe and ready for fertilization. Nevertheless, it is well known that oocytes can undergo atresia in the ovary involving the formation of metaphase-II chromosomes in many of them. These oocytes complicated Pincus’ estimates, even in controls, and were the source of his error which led later workers to inseminate human oocytes 12 hours after collection and culture in vitro.2,3 Work on human fertilization in vitro, and indeed comparable studies in animals, remained in abeyance from then and for many years. Progress in animal IVF had also been slow. After many relatively unsuccessful attempts in several species in the 1950s and 1960s, a virtual dogma arose that spermatozoa had to spend several hours in the female reproductive tract before acquiring the potential to bind to the zona pellucida and achieve fertilization. In the late 1960s Austin and Chang independently identified the need for sperm capacitation, identified by a delay in fertilization after spermatozoa had entered the female reproductive tract.4,5 This discovery was taken by many investigators as the reason for the failure to achieve fertilization in vitro, and why spermatozoa had to be exposed to secretions of the female reproductive tract. At the same time, Chang

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reported that rabbit eggs that had fully matured in vitro failed to produce normal blastocysts, none of them implanting normally.6

Modern beginnings of human IVF, preimplantation genetic diagnosis, and embryo stem cells My PhD began at the Institute of Animal Genetics, Edinburgh University, in 1952, encouraged by Professor Conrad Waddington, the inventor of epigenesis, and supervised by Dr Alan Beatty. At the time, capacitation was gaining in significance. My chosen topic was the genetic control of early mammalian embryology, specifically the growth of preimplantation mouse embryos with altered chromosome complements. Achieving these aims included a need to expose mouse spermatozoa to X-rays, ultraviolet light, and various chemicals in vitro. This would destroy their chromatin and prevent them from making any genetic contribution to the embryo, hopefully without impairing their capacity to fertilize eggs in vivo. Resulting embryos would become gynogenetic haploids. Later, my work changed to exposing ovulated mouse oocytes to colchicine in vivo, in order to destroy their second meiotic spindle in vivo. This treatment freed all chromosomes from their attachment to the meiotic spindle, and they then became extruded from the egg into tiny artificial polar bodies. The fertilizing spermatozoon thus entered an empty egg, which resulted in the formation of androgenetic haploid embryos with no genetic contribution from the maternal side. For three years, my work was concentrated in the mouse house, working at midnight to identify mouse females in estrus by vaginal smears, collecting epididymal spermatozoa from males, and practising artificial insemination with samples of treated spermatozoa. This research was successful, as mouse embryos were identified with haploid, triploid, tetraploid, and aneuploid chromosomes. Moreover, the wide scientific talent in the Institute was a perfect place for fresh collaborative studies. For example, Julio Sirlin and I applied the use of radioactive DNA and RNA precursors to the study of spermatogenesis, spermiogenesis, fertilization, and embryogenesis, and gained knowledge unavailable elsewhere. An even greater fortune beckoned. Allen Gates, newly arrived from the United States, brought commercial samples of Organon’s pregnant mares’ serum (PMS) rich in follicle-stimulating hormone (FSH), and human chorionic gonadotropin (hCG) with its strong luteinizing hormone (LH) activity to induce estrus and ovulation in immature female mice. Working with Mervyn Runner,7 he had used low doses of each hormone at an interval of 48 hours to induce oocyte maturation, mating, and ovulation in immature mouse females. He now wished to measure the viability of 3-day embryos from immature mice by transferring them to an adult host to grow to term.8 I was more

interested in stimulating adult mice with these gonadotropins to induce estrus and ovulation at predictable times of day. This would help my research, and I was by now weary of taking mouse vaginal smears at midnight. My future wife, Ruth Fowler, and I teamed up to test this new approach to superovulating adult mice. We chose pregnant mares’ serum to induce multifolliculation and hCG to trigger ovulation, varying doses and times from those utilized by Allen Gates. PMS became obsolete for human studies some time later, but its impact has stayed with me from that moment, even until today. Opinion in those days was that exogenous hormones such as PMS and hCG would stimulate follicle growth and ovulation in immature female mammals, but not in adults because they would interact badly with an adult’s reproductive cycles. In fact, they worked wonderfully well. Doses of 1–3 IU of PMS induced the growth of numerous follicles, and similar doses of hCG 42 hours later invoked estrus and ovulation a further 6 hours later in almost all of them. Often, 70 or more ovulated oocytes crowded the ampulla, most of them being fertilized and developing to blastocysts.9 Oocyte maturation, ovulation, mating, and fertilization were each closely timed in all adults, another highly unusual aspect of stimulation.10 Diakinesis was identified as the germinal vesicle regressed, with metaphase I a little later and metaphase II, expulsion of the first polar body, and ovulation at 11.5–12 hours after hCG. Multiple fertilization led to multiple implantation and fetal growth to full term, just as similar treatments in anovulatory women resulted in quintuplets and other high-order multiple pregnancies a few years later. Years afterwards, germinal vesicle breakdown and diakinesis were to prove equally decisive in identifying meiosis and ovulation in human oocytes in vivo and in vitro. Even as these results were gained, Ruth and I departed in 1957 from Edinburgh to the California Institute of Technology, where I switched into immunology and reproduction, a topic that was to dominate my life for five or six years on my return to the UK. The Institute at Edinburgh had given me an excellent basis in genetics, but equally in reproduction. I had gained considerable knowledge about the endocrine control of estrus cycles, ovulation, spermatozoa, and the male reproductive tract, artificial insemination, and the stages of embryo growth in the oviduct and uterus, superovulation and its consequences, and the use of radiolabeled compounds. Waddington had also been deeply interested in ethics and in relationships between science and religion, and instilled these topics in his students. I had been essentially trained in reproduction, genetics, and scientific ethics, and all of this knowledge was to prove of immense value in my later career. A visit to the California Institute of Technology widened my horizons into the molecular biology of DNA and the gene, a field then in its infancy.

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After a year in California, London beckoned me, to the National Institute for Medical Research working with Drs Alan Parkes and Colin (Bunny) Austin. I was fortunate indeed to have two such excellent colleagues. After two intense years in immunology, my curiosity returned to maturating oocytes and fertilization in vitro. Since they matured so regularly and easily in vivo, it should be easy to stimulate maturation in mouse oocytes in vitro by using gonadotropins. In fact, to my immense surprise, when liberated from their follicles into culture medium, oocytes matured immediately in vast numbers in all groups, with exactly the same timing as those maturing in vivo following an injection of hCG. Adding hormones made no difference. Rabbit, hamster, and rat oocytes also matured within 12 hours, each at their own speciesspecific rates. But to my surprise, oocytes from cows, sheep, and rhesus monkeys, and the occasional baboon, did not mature in vitro within 12 hours. Their germinal vesicles persisted unmoved, arrested in the stage known as diffuse diplotene. Why had they not responded like those of rats, mice, and rabbits? How would human oocytes respond? A unique opportunity emerged to collect pieces of human ovary, and to aspirate human oocytes from their occasional follicles. I grasped it with alacrity.

Moving to human studies Molly Rose was a local gynecologist in the Edgware and District Hospital who delivered two of our daughters. She agreed to send me slithers or wedges of ovaries such as those removed from patients with polycystic disease, as recommended by Stein and Leventhal, or with myomata or other disorders demanding surgery. Stein–Leventhal wedges were the best source of oocytes, with their numerous small graafian follicles lined up in a continuous rim just below the ovarian surface. Though samples were rare, they provided enough oocytes to start with. These oocytes responded just as the oocytes from cows, sheep, and pigs, their germinal vesicles persisting and diakinesis being absent after 12 hours in vitro. This was disappointing, and especially so for me, since Tjio and Levan, and Ford, had identified 46 diploid chromosomes in humans, while studies by teams in Edinburgh (Scotland) and France had made it clear that many human beings were heteroploid. This was my subject, because chromosomal variations mostly arose during meiosis and this would be easily assessed in maturing oocytes at diakinesis. Various groups also discovered monosomy or disomy in many men and women. Some women were XO or XXX; some men were XYY and XYYY. Trisomy 21 proved to be the most common cause of Down’s syndrome, and other trisomies were detected. All this new information reminded me of my chromosome studies in the Edinburgh mice. For human studies, I would have to obtain diakinesis and metaphase I in human oocytes, and then continue this analysis to metaphase II when the oocytes would

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be fully mature, ready for fertilization. Despite being disappointed at current failure with human oocytes, it was time to write my findings for Nature in 1962.11 There was so much to write regarding the animal work, and describing the new ideas then taking shape in my mind. I had heard Institute lectures on infertility, and realized that fertilizing human oocytes in vitro and replacing embryos into the mother could help to alleviate this condition. It could also be possible to type embryos for genetic diseases when a familial disposition was identified. Pieces of tissue, or one or two blastomeres, would have to be excised from blastocysts or cleaving embryos, but this did not seem to be too difficult. There were few genetic markers available for this purpose in the early 1960s, but it might be possible to sex embryos by their XX or XY chromosome complement by assessing mitoses in cells excised from morulae or blastocysts. Choosing female embryos for transfer would avert the birth of boys with various sex-linked disorders such as hemophilia. Clearly, I was becoming totally committed to human IVF and embryo transfer. While looking in the library for any newly published papers relevant to my proposed Nature manuscript, I discovered those earlier papers of Pincus and his colleagues described above. They had apparently succeeded 30 years earlier in maturing human oocytes cultured for 12 hours, where I had failed. My Nature paper11 became very different from that originally intended, even though it retained enough for publication. Those results of Pincus et al had to be repeated. After trying hard, I failed completely to repeat them, despite infusing intact ovaries in vitro with gonadotropin solutions, using different culture media to induce maturation, and using joint cultures of maturing mouse oocytes and newly released human oocytes. Adding hormones to culture media also failed. It began to seem that menstrual cycles had affected oocyte physiology in a different manner than in nonmenstruating mammalian species. Finally, another line of inquiry emerged after two years of fruitless research on the precious few human oocytes available. Perhaps the timing of maturation in mice and rabbits differed from that of those oocytes obtained from cows, baboons, and humans. Even as my days in London were ending, Molly Rose sent a slither of human ovary. The few oocytes were placed in culture just as before. Their germinal vesicles remained static for 12 hours as I already knew, and then after 20 hours in vitro. Three oocytes remained, and I waited to examine them until they had been in vitro for 24 hours. The first contained a germinal vesicle, so did the second. There was one left and one only. Its image under the microscope was electrifying. I gazed down at chromosomes in diakinesis, and at a regressing germinal vesicle. The chromosomes were superb examples of human diakinesis with their classical chiasmata. At last, I was on the way to human IVF, to completion of the maturation program and the onset of studies on fertilization in vitro. This was the step I had waited for, a marker that Pincus had missed. He never checked for diakinesis,

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and apparently confused atretic oocytes, which contained chromosomes, with maturing oocytes. Endless human studies were opening. It was easy now, even on the basis of one oocyte in diakinesis, to calculate the timing of the final stages of maturation because the post-diakinesis stages of maturation were not too different from normal mitotic cycles in somatic cells. This calculation provided me with an estimate of about 36 hours for full maturation, which would be the moment for insemination. All these gaps in knowledge had to be filled. But now, my research program was stretching far into the future. At this wonderful moment, John Paul, an outstanding cell biologist, invited me to join him and Robin Cole at Glasgow University to study differentiation in early mammalian embryos. This was exciting, to work in biochemistry with a leading cell biologist. He had heard that I was experimenting with very early embryos, trying to grow cell lines from them. He also wanted to grow stem cells from mammalian embryos and study them in vitro. This began one of my most memorable 12 months of research. John’s laboratory had facilities unknown outside, with CO2 incubators, numerous cell lines in constant cultivation, cryopreservation facilities, and the use of media droplets held under liquid paraffin. We decided to start with rabbits. Cell lines did not grow easily from cleaving rabbit embryos. In contrast, stem cells migrated out in massive numbers from cultures of rabbit blastocysts, forming muscle, nerves, phagocytes, blood islands, and other tissues in vitro.12 Stem cells were differentiating in vitro into virtually all the tissues of the body. In contrast, dissecting the inner cell mass from blastocysts and culturing it intact or as disaggregated cells produced lines of cells which divided and divided, without ever differentiating. One line of these embryonic stem cells expressed specific enzymes, diploid chromosomes, and a fibroblastic structure as it grew over 200 and more generations. Another was epithelioid and had different enzymes but was similar in other respects. The ability to make whole-embryo cultures producing differentiating cells was now combined with everlasting lines of undifferentiated stem cells which replicated over many years without changing. Ideas of using stem cells for grafting to overcome organ damage in recipients began to emerge. My thoughts returned constantly to growing stem cells from human embryos to repair defects in tissues of children and adults. Almost at my last moment in Glasgow, with this new set of ideas in my mind, a piece of excised ovary yielded several oocytes. Being placed in vitro, two of them had reached metaphase II and expelled a polar body at 37 hours. This showed that another target on the road to human IVF had been achieved as the whole pattern of oocyte maturation continued to emerge but with increasing clarity. Cambridge University, my next and final habitation, is an astonishing place. Looking back on those days, it seems that the Physiological Laboratory was not the

ideal place to settle in that august university. Nevertheless, a mixture of immunology and reproduction remained my dominant themes as I rejoined Alan Parkes and Bunny Austin there. I had to do immunology to obtain a grant to support my family, but thoughts of human oocytes and embryos were never far away. One possible model of the human situation was the cow and other agricultural species, and large numbers of cow, pig, and sheep oocytes were available from ovaries given to me by the local slaughterhouse. Each species had its own timing, all of them longer than 12 hours.13 Pig oocytes were closest to humans, requiring 37 hours. In each species, maturation timings in vitro were exactly the same as those arising in vivo in response to an hCG injection. This made me suspect that a woman ovulated 36–37 hours after an injection of hCG. Human oocytes also trickled in, improving my provisional timings of maturation, and one or two of them were inseminated, but without signs of fertilization. More oocytes were urgently needed to conclude the timings of oocyte meiosis. Surgeons in Johns Hopkins Hospital, Baltimore, performed the Stein–Leventhal operation, which would allow me to collect ovarian tissue, aspirate oocytes from their follicles, and retain the remaining ovarian tissues for pathology if necessary. I had already met Victor McKusick, who worked in Johns Hopkins, at many conferences. I asked for his support for my request to work with the hospital gynecologists for six weeks. He found a source of funds, made laboratory space available, and, a wonderful invitation, introduced me to Howard and Georgeanna Jones. This significant moment was equal to my meeting with Molly Rose. The Joneses proved to be superb and unstinting in their support. Sufficient wedges and other ovarian fragments were available to complete my maturation program in human oocytes. Within three weeks, every stage of meiosis was classified and timed.14 We also undertook preliminary studies on inseminating human oocytes that had matured in vitro, trying to achieve sperm capacitation by using different media or adding fragments of ampulla to the cultures, and even attempting fertilization in rhesus monkey oviducts. Two nuclei were found in some inseminated eggs, resembling pronuclei, but sperm tails were not identified so no claims could be made.15 During those six weeks, however, oocyte maturation was fully timed at 37 hours, permitting me now to predict with certainty that women would ovulate at 37 hours after an hCG injection. A simple means of access to the human ovary was now essential in order to identify human ovarian follicles in vivo and to aspirate them 36 hours after hCG, just before follicular rupture. Who could provide this? And how about sperm capacitation? Only in hamsters had fertilization in vitro been achieved, using in vivo matured oocytes and epididymal spermatozoa.16 I met Victor Lewis, my third clinical colleague, and we noticed what seemed to be anaphase II in some inseminated eggs. Again, no sperm tails were seen within the eggs.

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An attempt to achieve human capacitation, in Chapel Hill, North Carolina, United States, working with Robert McGaughey and his colleagues, also failed.17 A small intrauterine chamber lined with porous membrane was filled with washed human spermatozoa, sealed, and inserted overnight into the uterus of human volunteers at mid-cycle. Molecules entering it could react with the spermatozoa. No matured human eggs were fertilized. Later evidence indicated that the chamber contained inflammatory proteins, perhaps explaining the failure.

Decisive steps to clinical human in vitro fertilization Back in the United Kingdom, my intention to conceive human children in vitro had grown even stronger. So many medical advantages could flow from it. A small number of human embryos had been flushed from human oviducts or uteri after sexual intercourse, providing slender information on these earliest stages of human embryology. It was time to attain human fertilization in vitro, in order to move close to working with infertile patients. Ethical issues and moral decisions would emerge, one after the other, in full public view. Matters such as cloning and sexing embryos, the risk of abnormalities in the children, the clinical use of embryo stem cells, the ethics of oocyte donation and surrogate pregnancy, and the right to initiate human embryonic life in vitro would never be very far away. These issues were all acceptable, since I was confident that studies of human conception were essential for future medicine, and correct ethically, medically, and scientifically. The increasing knowledge of genetics and embryology could assist many patients if I could achieve human fertilization and grow embryos for replacement into their mothers. Few human oocytes were available in the United Kingdom. Despite this scarcity, one or two of those matured and fertilized in vitro possessed two nuclei after insemination. But there were no obvious sperm tails. I devised a cow model for human fertilization, using in vitro matured oocytes and insemination in vitro with selected samples of highly active washed bull spermatozoa extracted from neat semen. It was a pleasure to see some fertilized bovine eggs, with sperm tails and characteristic pronuclei, especially using spermatozoa from one particular bull. Here was a model for human IVF, and a prelude to a series of events which implied that matters in my research were suddenly changing. A colleague had stressed that formalin fixatives were needed to detect sperm tails in eggs. Barry Bavister joined our team to study for his PhD and designed a medium of high pH, which gave excellent fertilization rates in hamsters. We decided to collaborate by using it for trials on human fertilization in vitro. Finally, while browsing in the library of the Physiological Laboratory, I read a paper in The Lancet which instantly caught my attention. Written by Dr P. C. Steptoe of the Oldham and District General Hospital,18

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it described laparoscopy, with its narrow telescope and instruments and the minute abdominal incisions. He could visualize the ampulla and place small amounts of medium there, in an operation lasting 30 minutes or less and maybe even without using anesthesia. This is exactly what I wanted, because access to the ampulla was equivalent to gaining access to ovarian follicles. Despite advice to the contrary from several medical colleagues, I telephoned him about collaboration and stressed the uncertainty in achieving fertilization in vitro. He responded most positively, just as Molly, Howard and Georgeanna, and Victor had done. We decided to get together. Last but by no means least, Molly Rose sent a small piece of ovary to Cambridge. Its dozen or more oocytes were matured in vitro for 37 hours, when Barry and I added washed spermatozoa suspended in his medium. We examined them a few hours later. To our delight, spermatozoa were pushing through the zona pellucida, into several of the eggs. Maternal and paternal pronuclei were forming beautifully. We saw polar bodies and sperm tails within the eggs. That evening in 1969, we watched in delight virtually all the stages of human fertilization in vitro (Fig 1). One fertilized egg had fragments, as Chang had forecast from his work on oocyte maturation and fertilization in vitro of rabbit eggs. This evidence strengthened the need to abandon oocyte maturation in vitro and replace it by stimulating maturation by means of exogenous hormones. Our 1969 paper in Nature surprised a world unaccustomed to the idea of human fertilization in vitro.19 Incredibly fruitful days followed in our Cambridge laboratory. Richard Gardner, another PhD candidate, and I excised small pieces of trophectoderm from rabbit blastocysts and sexed them by staining the sex chromatin body. Those classified as female were transferred into adult females and were all correctly sexed at term. This work transferred my theoretical ideas of a few years earlier into the practice of preimplantation diagnosis of inherited disease, in this case for sexlinked diseases.20 Alan Henderson, a cytogeneticist, and I analyzed chiasmata during diakinesis in mouse and human eggs, and explained the high frequencies of Down’s syndrome in offspring of older mothers as a consequence of meiotic errors arising in oocytes formed last in the fetal ovary, which were then ovulated last at later maternal ages.21 Dave Sharpe, a lawyer from Washington, joined forces to write an article in Nature22 on the ethics of in vitro fertilization, the first ever paper in the field. I followed this up with a detailed analysis of ethics and law in IVF covering scientific possibilities, oocyte donation, surrogacy by embryo transfer, and other matters.22 So the first ethical papers were written by scientists and lawyers and not by philosophers, ethicists, or politicians.

The Oldham years Patrick and I began our collaboration six months later in the Oldham and District General Hospital, almost

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Fig 1 A composite picture of the stages of fertilization of the human egg. Upper left: an egg with a first polar body and spermatozoa attached to the outer zona pellucida. Upper central: spermatozoa are migrating through the zona pellucida. Upper right: a spermatazoon with a tail beating outside the zona pellucida is attaching to the oocyte vitelline membrane. Lower left: a spermatozoon in the ooplasm, with enlarging head and distinct mid-piece and tail. Lower central: further development of the sperm head in the ooplasm. Lower right: a pronucleate egg with two pronuclei and polar bodies. Notice that the pronuclei are apparently aligned with the polar bodies, although more dimensions must be scored to ensure that polarity has been established in all axes.

200 miles north of Cambridge. He had worked closely with two pioneers, Palmer in Paris23 and Fragenheim in Germany.24 He improved the pneumoperitoneum to gain working space in the abdominal cavity, and used carbon fibers to pass cold light into the abdomen from an external source.25 By now, Patrick was waiting in the wings, ready to begin clinical IVF in distant Oldham. We had a long talk about ethics and found our stances to be very similar. Work started in the Oldham and District General Hospital and moved later to Kershaw’s Hospital, set up by my assistants, especially Jean Purdy. We knew the routine. It was based on my Edinburgh experiences with mice. Piero Donini from Serono Laboratories in Rome had purified urinary human menopausal gonadotropins (hMG) as a source of FSH, and the product was used clinically to stimulate follicle growth in anovulatory women by Bruno Lunenfeld.26 It removed the need for PMS, so avoiding the use of nonhuman hormones. We used low-dosage levels in patients, i.e. 2–3 vials (a total of 150–225 IU) given on days 3 and 5, and 5000–7000 IU of hCG on day 10. Initially, the timing of oocyte maturation in vitro was confirmed, by performing laparoscopic collections of oocytes from ovarian follicles at 28 hours after hCG to check that they were in metaphase I.27 We then moved to 36 hours to aspirate

mature metaphase II oocytes for fertilization. Those beautiful oocytes were surrounded by masses of viscous cumulus cells and were maturing exactly as predicted. We witnessed follicular rupture at 37 hours through the laparoscope. Follicles could be classified from their appearance as ovulatory or nonovulatory, this diagnosis being confirmed later by assaying several steroids in the aspirated follicular fluids (Fig 2). It was a pleasure and a new duty to meet the patients searching for help to alleviate their infertility. We did our best, driving from Cambridge to Oldham and arriving at noon to prepare the small laboratory there. Patrick had stimulated the patients with hMG and hCG, and he and his team led by Muriel Harris arrived to prepare for surgery. Patrick’s laparoscopy was superb. Ovarian stimulation, even though mild, produced five or six mature follicles per patient, and ripe oocytes came in a steady stream into my culture medium for insemination and overnight incubation. The next morning, the formation of two pronuclei and sperm tails indicated fertilization had occurred, even in simple media, now with a near-neutral pH. Complex culture media, Ham’s F10 and others, each with added serum or serum albumin, sustained early and later cleavages,28 and, even more fascinating, the gradual appearance of morulae and then light,

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110.9

Within-group variation

99.3

87.6

29.6

18.0

6.3

1 Ovulatory

Group

2 Nonovulatory

18.798 16.832

Within-group variation

14.866 12.900 10.935 8.969 7.003 5.038 3.072 1.106

Follicle no. Group

12

10 11 25 3 4 1

8 17 5 82 22 12 3 24 7 20 12 14 16 9 2

3

4

Fig 2 Eight steroids were assayed in fluids extracted from human follicles aspirated 36–37 hours after human chorionic gonadotropin (hCG). The follicles had been classified as ovulating or nonovulating by laparoscopic examination in vivo. Data were analyzed by cluster analysis, which groups follicles with similar features. The upper illustration shows data collected during the natural menstrual cycle. Note that two sharply separated groups of follicles were identified, each with very low levels of within-group variance. Attempting to combine the two groups resulted in a massive increase of within-group variation, indicating that two sharply different groups had been identified. These different groups accorded exactly with the two groups identified by means of steroid assays. The lower figure shows the same analysis during stimulated cycles on fluids collected at 36–37 hours after hCG. With this form of stimulation, follicle growth displays considerable variation within groups. Attempts to combine all the groups result in a moderately large increase in variation. This evidence suggests that follicles vary considerably in their state of development in simulated cycles using human menopausal gonadotropin (hMG) and hCG.

translucent blastocysts (Fig 3).29 Here was my reward – growing embryos was now routine, and examinations of many of them convinced me that the time had come to replace them into the mothers’ uteri. I had become highly familiar with the teratologic principles of embryonic development, and knew many teratologists.

The only worry I had was the chance of chromosomal monosomy or trisomy, on the basis of our mouse studies, but these conditions could be detected later in gestation by amniocentesis. Our human studies had surpassed work on all animals, a point rubbed in even more when we grew blastocysts to day 9 after they

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had hatched from their zona pellucida (Fig 4).30 This beautifully expanded blastocyst had a large embryonic disc which was shouting that it was a potential source of embryonic stem cells. When human blastocysts became available, we tried to sex them using the sex chromatin body as in rabbits. Unfortunately, they failed to express either sex chromatin or the male Y body so we were unable to sex them as female or male embryos. Human preimplantation genetic diagnosis would have to wait a little longer. During these years there were very few plaudits for us, as many people spoke against IVF. Criticism was mostly aimed at me, as usual when scientists bring new challenges to society. Criticism came not only from the Pope and archbishops, but also from scientists who should have known better, including James Watson (who testified to a US Senate Committee that many abnormal babies would be born), and Max Perutz, who supported him. These scientist critics knew virtually nothing about my field, so who advised them to make such ridiculous charges? Cloning football teams or intelligentsia was always raised by ethicists, which clearly dominated their thoughts rather than the intense hopes of our infertile patients. Yet one theologian, Gordon Dunstan, who became a close friend, knew all about IVF from us, and wrote an excellent book on its ethics. He was far ahead of almost every scientist in my field of study. Our patients also gave us their staunch support, and so did the Oldham Ethical Committee, Bunny Austin back home in Cambridge, and Elliott Philip, a colleague of Patrick’s. Growing embryos became routine, so we decided to transfer one each to several patients. Here again we were in untested waters. Transferring embryos via the cervical canal, the obvious route to the uterus, was virtually a new and untested method. We would have to do our best. From now on, we worked with patients who had seriously distorted tubes or none whatsoever. This step was essential, since no one would have believed we had established a test-tube baby in a woman with near normal tubes. This had to be a condition of our initial work. Curiously, it led many people to make the big mistake of believing that we started IVF to bypass occluded oviducts. Yet we already knew that embryos could be obtained for men with oligozoospermia or antibodies to their gametes, and for women in various stages of endometriosis. One endocrinological problem did worry me. Stimulation with hMG and hCG shortened the succeeding luteal phase, to a very short time for embryos to implant before the onset of menstruation. Levels of urinary pregnanediol also declined soon after oocyte collection. This condition was not a result of the aspiration of granulosa and cumulus cells, and luteal support would be needed, preferably progesterone. Csapo et al stressed how this hormone was produced by the ovaries for the first 8–10 weeks before the placenta

took over this function.31 Injections of progesterone in oil given over that long period of time seemed unacceptable since it would be extremely uncomfortable for patients. While mulling over this problem, my attention turned to those earlier endocrinologists who believed that exogenous hormones would distort the reproductive cycle, although I doubt they even knew anything about a deficient luteal phase. This is how we unknowingly made our biggest mistake in early IVF days. Our choice of Primulot depot, a progestogen, meant it should be given every 5 days to sustain pregnancies, since it was supposed to save threatened abortions. So, we began embryo transfers to patients in stimulated cycles, giving this luteal phase support. Even though our work was slowed by having to wait to see if pregnancies arose in one group of patients before stimulating the next, enough patients had accumulated after 2–3 years. None of our patients was pregnant, and disaster loomed. Our critics were even more vociferous as the years passed, and mutual support between Patrick and me had to pull us through. Twenty or more different factors could have caused our failure, e.g. cervical embryo transfers, abnormal embryos, toxic culture dishes or catheters, inadequate luteal support, incompatibility between patients’ cycles and that imposed by hMG and hCG, inherent weakness in human implantation, and many others. We had to glean every scrap of information from our failures. I knew Ken Bagshawe in London, who was working with improved assay methods for gonadotropic hormones. He offered to measure blood samples taken from our patients over the implantation period using his new hCG-β assay. He telephoned: three or more of our patients previously undiagnosed had actually produced short-lived rises of hCG-β over this period. Everything changed with this information. We had established pregnancies after all, but they had aborted very early. We called them biochemical pregnancies, a term that still sticks today. It had taken us almost three years to identify the cause of our failure, and the finger of suspicion pointed straight at Primulot. I knew it was luteolytic, but it was apparently also an abortifacient, and our ethical decision to use it had caused much heartache, immense loss of work and time, and despair for some of our patients. The social pressures had been immense, with critics claiming our embryos were dud and our whole program was a waste of time; but we had come through it and now knew exactly what to do next. We accordingly reduced levels of Primulot depot, and utilized hCG and progesterone as luteal aids. Suspicions were also emerging that human embryos were very poor at implanting. We had replaced single embryos into most of our patients, rarely two. Increasingly we began to wonder if more should be replaced, as when we replaced two in a program involving transfers of oocytes and spermatozoa into

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Fig 3 Successive stages of human preimplantation development in vitro in a composite illustration made in Oldham in 1971. Upper left: 4-cell stage showing the crossed blastomeres typical of most mammals. Upper middle: 8-cell stage showing the even outline of blastomeres and a small piece of cumulus adherent to the zona pellucida. Upper right: a 16–32-cell stage, showing the onset of compaction of the outer blastomeres. Often, blastocelic fluid can be seen accumulating between individual cells to give a “stripey” appearance to the embryo. Lower left and middle: two living blastocysts showing a distinct inner cell mass, single-celled trophectoderm, blastocelic cavity, and thinning zona pellucida. Lower right: a fixed preparation of a human blastocyst at 5 days, showing more than 100 even–sized nuclei and many mitoses.

Fig 4 A hatched human blastocyst after 9 days in culture. Notice the distinct embryonic disc and the possible bilaminar structure of the membrane. The blastocyst has expanded considerably, as shown by comparing its diameter with that of the shed zona pellucida. The zona contains dying and necrotic cells and its diameter provides an estimate of the original oocyte end embryo diameters.

the ampulla so that fertilization could occur in vivo. This procedure was later called GIFT (gamete intrafallopian transfer) by Ricardo Asch. We now suspected that single embryo transfers could produce a 15–20% chance of establishing pregnancy, just as our first clinical pregnancy arose after the transfer of a single blastocyst in a patient stimulated with hMG and hCG.32 Then came fantastic news – a human embryo fertilized and grown in vitro had produced a pregnancy. Everything seemed fine, even with ultrasound images. My culture protocols were satisfactory after all. Patrick rang: he feared the pregnancy was ectopic and he had to remove it sometime after 10 gestational weeks. Every new approach we tested seemed to be ending in disaster, yet we would not stop, since the work itself seemed highly ethical, and conceiving a child for our patients was perhaps the most wonderful thing anyone could do for them. In any case, ectopic pregnancies are now known to be a regular feature with assisted conception. I sensed that we were entering the final phase of our Oldham work, seven years after it began. We had to speed up, partly because Patrick was close to retiring

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Mrs. MP ODGH 12/1/73

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Fig 5 The first attempts at gamete intrafallopian transfer (GIFT) were called oocyte recovery with tubal insemination (ORTI). In this treatment cycle, using human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG), including additional injections of hCG for luteal support, a single preovulatory oocyte and 1.6 million sperm were transferred into the ampulla. ODGH, Oldham and District General Hospital; LMP, last menstrual period; RTM indicates stages of the menstrual cycle.

from the National Health Service. Four stimulation protocols were tested in an attempt to avoid problems with the luteal phase: hMG and hCG; clomiphene, hMG, and hCG to gain a better luteal phase; bromocryptine, hMG, and hCG because some patients had high prolactin concentrations; and hCG alone at mid-cycle. We also tested what came to be known as gamete intrafallopian transfer (GIFT), calling it ORTI (oocyte recovery with tubal insemination, by transferring one or two eggs and spermatozoa to the ampulla) (Fig 5). Natural-cycle IVF was introduced, based on collections of urine samples at regular intervals eight times daily, to measure exactly the onset of the LH surge, using a modified HiGonavis assay (Fig 6). Cryopreservation was also introduced, by freezing oocytes and embryos that looked to be in good condition when thawed. A recipient was given a donor egg fertilized by her husband’s spermatozoa, but pregnancy did not occur. Lesley and John Brown came as the second entrants for natural-cycle IVF. Lesley had no oviducts. Her egg was aspirated in a few moments and inseminated simply and efficiently. The embryo grew beautifully and was transferred an hour or so after it became 8-cell. Their positive pregnancy test a few days after transfer was another milestone – surely nothing could now prevent their embryo developing to full term in a normal reproductive cycle, but those nine months lasted a very long time. Three more pregnancies were established using natural-cycle IVF as we abandoned the other approaches. A triploid embryo died in utero – more

bad luck. A third pregnancy was lost through premature labor on a mountain walking holiday, two weeks after the mother’s amniocentesis.32,33 It was a lovely, well-developed boy. Louise Brown’s birth, and then Alistair’s, proved to a waiting world that science and medicine had entered human conception. Our critics declared that the births were a fake, and advised against attending our presentation on the whole of the Oldham work at the Royal College of Obstetricians and Gynaecologists.

IVF worldwide The Oldham period was over. Good facilities were now needed, with space for a large IVF clinic. Bourn Hall was an old Jacobean house in lovely grounds near Cambridge (Fig 7). Facilities on offer for IVF in Cambridge were far too small, so we purchased it mostly with venture capital. It was essential to conceive 100 or 1000 IVF babies to ensure that the method was safe and effective clinically. The immense delays in establishing Bourn Hall delayed our work by two years after Louise’s birth. Finally, on minimal finance, Bourn Hall opened in September 1980 on a shoestring, supported by our own cash and loans. The delay gave the rest of the world a chance to join in IVF. Alex Lopata delivered an IVF baby in Australia, and one or two others were born elsewhere. Natural-cycle IVF was chosen initially at Bourn Hall since it had proved successful in Oldham, and we became experts in it. Pregnancies flowed, at 15% per cycle. An Australian team of Alan Trounson and Carl Wood announced the establishment of several IVF pregnancies after stimulation by clomiphene and hCG and replacing two or three embryos,34 so they had moved ahead of us during the delayed opening of Bourn Hall. Our own effort now expanded prodigiously. Thousands of patients queued for IVF. Simon Fishel, Jacques Cohen, and Carol Fehilly joined the embryology team among younger trainees, and new clinicians joined Patrick and John Webster. Patients and pregnancies increased rapidly, and the world was left standing far behind. Howard and Georgeanna Jones began in Norfolk using gonadotropins for ovarian stimulation. Jean Cohen began in Paris, Wilfred Feichtinger and Peter Kemeter in Vienna, Klaus Diedrich and Hans van der Venn in Bonn, Lars Hamberger and Matts Wikland in Sweden, and Andre van Steirteghem and Paul Devroey in Brussels. IVF was now truly international. The opening of Bourn Hall had not deterred our critics. They put up a fierce rearguard action against IVF, alongside LIFE, SPUC (Society for the Unborn Child), individual gynecologists, and others. Objections raised against IVF included low rates of pregnancy (no one mentioned the similar low rates of pregnancy with natural conception), the possibilities of oocyte and embryo donation, surrogate mothers, unmarried parents, one-sex parents, embryo

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Introduction LH lapy surge

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Fig 6 Recording the progress of the human natural menstrual cycle for in vitro fertilization (IVF). Three patients are illustrated. All three displayed rising 24-hour urinary estrogen concentrations during the follicular phase, and rising urinary pregnanediol concentrations in the luteal phase. Luteinizing hormone (LH) levels were measured several times daily and the data clearly reveal the exact time of onset of the LH surge.

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Bourn Hall (courtesy of Dr P. Brinsden).

cryopreservation, cloning, and endless other objections. LIFE issued a legal action against me for the abortion of an embryo grown for 14 days and longer in vitro. Their action was rejected by the UK Attorney General since the laws of pregnancy began after implantation. We fully respected the intense ethical nature of our proceedings. We also recognized the

need for research, and the necessity to protect or cryopreserve the best embryos for later replacement into their mothers. Those not replaced had to be used for research under strict controls, combined with open publication and discussion of our work. Each year, 1000 rising to almost 2000 patients passed through Bourn Hall. Different stimulation regimens or

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new procedures could be tested in very little time. Clomiphene/hMG was reintroduced. Bourn babies increased: 20, 50, 100, to 1000 after 5–6 years. This was far more than half of the world’s entire IVF babies, including the first born in the United States, Germany, Italy, and many other countries. Detailed studies were performed on embryo culture, implantation, and abortion. We even tried aspirating epididymal spermatozoa for IVF, without achieving successful fertilization. Among the immense numbers of patients, people with astonishingly varied conditions of infertility emerged. Some were poor responders in whom immense amounts of endocrine priming were essential, women with a natural menstrual cycle that was not as it should have been, previous misdiagnoses which had laid the cause of infertility on the wife when the husband had never even been investigated, and men bringing semen samples that we discovered had been obtained from a friend. The collaboration between nurses, clinicians, and scientists was remarkable. Yet trouble – ethical trouble – was never far away. I purchased a freezing machine to resume our Oldham work, but, unknown to me, Patrick talked to officers of the British Medical Association (BMA) and for some reason agreed to delay embryo cryopreservation. Apparently, the BMA felt it would be an unwelcome social development. I did not approve of these reservations: David Whittingham had shown how low-temperature cryostorage was successful with mouse embryos, without causing genetic damage. “Freezing and cloning” became a term of intense approbation at this time. I unwillingly curtailed our cryopreservation program. One weekend, major trouble erupted as a result of this difference between Patrick and me. My duties in Bourn Hall prevented me from attending a conference in London. Trying to be helpful, I telephoned my lecture to London. Reception at the other end was apparently so poor as to lead to misinterpretations of what I had said. Next morning, the press furore about my supposed practice of cryopreserving embryos after IVF was awful, so bad, indeed, that legal action had to be taken. Luckily, my lecture had been recorded, and listening to the tapes with a barrister revealed nothing contentious. I had said nothing improper in my lecture nor during answers to questions. That day, I issued seven libel actions against the cream of British society: the BMA and its secretary, the British Broadcasting Corporation (BBC), The Times, and other leading newspapers. There were seven in one day and another one later! If only one was lost, I could be ruined and disgraced. However, they were all won, even though it took several years with the BMA and its secretary. These legal actions had inhibited our research, the cryopreservation program being shut down for more than a year. Every single embryological note of mine from those days in Oldham and from Bourn Hall was examined in detail for my opponents by someone who was clearly an embryologist. Nothing was found to incriminate me. That wretched period passed. Numbers of babies kept on growing, embryo cryopreservation was resumed,

Fig 8 A happy picture of Patrick and me, standing in our robes after being granted our Hon DSc by Hull University.

and Gerhard Zeilmaker in The Netherlands beat us and the world to the first “ice” baby.35 Colin Howles and Mike Macnamee joined us in endocrinology, and Mike Ashwood Smith and Peter Hollands in embryology, as the old team faded away. Fascinating days had returned. Working with barristers, we designed consent forms which were far in advance of those used elsewhere. Oocyte donation and surrogacy by embryo transfer were introduced. The world’s first paper on embryo stem cells appeared in Science in 1984, sent from Bourn Hall, and the world’s first on human preimplantation diagnosis in 1987 appeared in Human Reproduction. However, embryo research faltered as all normal embryos were cryopreserved for their parents, so almost none were available for study. Alan Handyside, one of our Cambridge PhDs, joined Hammersmith Hospital in London to make major steps in introducing preimplantation genetic diagnosis.36 As we reached 1000 pregnancies, our data showed the babies to be as normal as those conceived in vivo. Test-tube babies (an awful term) were no longer unique and were accepted worldwide, exactly as Patrick and I had hoped. Our work was being recognized (Fig 8). Clinics sprang up everywhere. Ultrasound was introduced to detect follicles for aspiration by the Scandinavians,37 making laparoscopy for oocyte recovery largely redundant. Artificial cycles were introduced in Australia and intracytoplasmic sperm injection (ICSI) in Belgium,38 and gonadotropin releasing hormone (GnRH) agonists were used to inhibit the LH surge. Ian Craft in London showed how postmenopausal women aged 52 or more could establish pregnancies using oocyte donation and endocrine support. Women over 60 years of age conceived and delivered children. This breakthrough was especially welcome to me, since older women surely have the right to have children at ages almost the same as those possible for men. Ethics continues side by side with advancing science and medicine. The UK governmental Warnock report

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Introduction

recommended permitting embryo research, and proposed a Licensing Authority for IVF. A year or so later, the UK House of Lords, in all its finery, responded with a 3:1 vote in favor, decisive support for all we had done in Mill Hill, Cambridge, and Oldham. What a wonderful day! The British House of Commons passed a liberal IVF law after intense debate, and so did the Spanish government, although elsewhere things were not so liberal. Ten years after the birth of Louise Brown, the British Parliament had therefore accepted IVF, research on human embryos until day 14, and establishing research embryos. Cloning and embryo stem cells still bothered the politicians in 1988, to re-emerge in 1998, gray shadows of my earlier times in Glasgow. IVF had also become fundamental to establish embryonic stem cells for organ repair, or cloning. During all this activity, tragedy struck all of us in Bourn Hall. Jean Purdy died in 1986 and Patrick Steptoe in 1988. They at least saw IVF come of age. By the 1990s, burgeoning medical science was digging deeper into endless aspects of human conception in vitro. The intracytoplasmic injection of a single spermatozoon into an oocyte to achieve fertilization, ICSI, was one of the greatest advances since IVF was introduced. It transformed the treatment of male infertility, enabling severely oligozoospermic men to father their own children. It did not stop there, since epididymal spermatozoa and even those aspirated from the testis could be used for ICSI. Spermatids have also been used. ICSI became so simple that many clinics reduced IVF to fewer and fewer cases. New GnRH antagonists introduced novel ways to control the cycle, enabling many oocytes to be stimulated by hMG and, subsequently, using recombinant human FSH. Treatment in the natural cycle could be improved, since these antagonists control LH levels and prevent premature LH surges. My own interests were returning to embryology, as the molecular biology revolution influenced our thinking. I am convinced that the oocyte and egg must be highly programmed, timewise, in embryonic polarities and integrating genetic systems such that the tight systems place every new gene product in its right place in the 1-cell egg and cleaving embryo. This must be right – there can surely be no other explanations for the fabulous modification in embryonic growth in the first week or two of embryonic life. I have been delighted to work with Chris Hansis on identifying a gene (for hCG-β) in one blastomere of 4- and 8-cell human embryos, providing evidence of blastomere differentiation at this early stage of embryogenesis.39 This topic returns me to my scientific origins studying mouse embryos in the Institute of Animal Genetics in Edinburgh, where Waddington reported the amazing story of the gene Aristopedia in Drosophila, which he had induced to grow legs in place of eyes. These unusual flies then bred true, showing he had uncovered a gene that had been silenced for millions of

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years and how this could be an essential component of normal differentiation. He called it epigenesis, and we fear today that some aspects of IVF may lead to deleterious epigenetic changes in children such as Angelmann or Beckwith–Wiedemann syndrome. Risks of epigenetic changes in cattle embryos and those of other species may be heightened by adding serum to media used to culture embryos, to cause for example large-calf syndrome. It would be wise to be well aware of these findings when practicing human IVF, for example by assessing the role of sera in human culture media.

IVF outlook In one sense, opening up human conception in vitro was perhaps among the first examples of applied science in modern “hi tech”. Human IVF has since spread throughout the world, with apparently more than 3.5 million babies born worldwide by 2008 – yet Louise Brown is only just 30 years of age. The need for IVF and its derivatives is greater than ever, since up to 10% of couples may suffer from some form of infertility. Major advances in genetic technologies now identify hundreds of genes in a single cell, and diagnosing genetic disease in embryos promises to help avoid desperate genetic diseases in newborn children. Indeed, the ethics of this field have now become even more serious, since typing embryo genotypes provides detailed predictions of future life and health. IVF has now combined closely with genetics, to eliminate disease or disability genes or lengthen the life span. But most of all, practising IVF teaches a wider understanding of the desire and love for a child and a partner, the wonderful and ancient joys of parenthood, the pain of failure, the deep motivation needed in donating and receiving an urgently needed oocyte or a surrogate uterus. Parenthood is more responsible than ever before. Its complex choices are gathered before couples everywhere by the information revolution, placing family responsibilities on patients themselves, where it really matters. And IVF now reveals more and more about miracles preserved in embryogenesis from flies and frogs to humankind, over 600 million years of evolution. The human genome project is now complete, and will inevitably assist IVF since we will soon understand the genetic aspects of early embryo growth and how to detect abnormal genes in embryos. This textbook contains chapters which describe in detail the many advances and developments which have expanded the possibilities of treating diverse causes of human infertility as well as numerous genetic disorders. Already it is clear that a staggering array of genes operate in preimplantation stages in mammalian including human embryos, and new methods are being introduced to deal with such highly multigenic embryonic systems. We are indeed enmeshed in a field embracing some of the most fundamental evolutionary

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stages of our existence as we pass from oocyte to blastocyst and to implantation.

References 1. Pincus G, Saunders B. The comparative behavior of mammalian eggs in vivo and in vitro. VI. The maturation of human ovarian ova. Anat Rec 1939; 75: 537–45. 2. Menkin MF, Rock J. Am J Obstet Gynecol 1949; 55: 440. 3. Hayashi M. Seventh International Conference of the International Planned Parenthood Federation. Excerpta Medica, 1963, p.505. 4. Austin CR. Adv Biosci 1969; 4: 5. 5. Chang M. Adv Biosci 1969; 4: 13. 6. Chang MC. The maturation of rabbit oocytes in culture and their maturation, activation, fertilization and subsequent development in the fallopian tubes. J Exp Zool 1955; 128: 379–405. 7. Runner M, Gates AH. Sterile, obese mothers. J Hered 1954; 45: 51–5. 8. Gates AH. Viability and developmental capacity of eggs from immature mice treated with gonadotrophins. Nature 1954; 177: 754–5. 9. Fowler RE, Edwards RG. Induction of superovulation and pregnancy in mature mice by gonadotrophins. J Endocrinol 1957; 15: 374–84. 10. Edwards RG, Gates AH. Timing of the stages of the maturation divisions, ovulation, fertilization and the first cleavage of eggs of adult mice treated with gonadotrophins. J Endocrinol 1959; 19: 292–304. 11. Edwards RG. Meiosis in ovarian oocytes of adult mammals. Nature (London)1962; 196: 446–50. 12. Cole R, Edwards RG, Paul J. Cytodifferentiation and embryogenesis in cell colonies and tissue cultures derived from ova and blastocysts of the rabbit. Dev Biol 1966; 13: 385–407. 13. Edwards RG. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 1965; 208: 349–51. 14. Edwards RG. Maturation in vitro of human ovarian oocytes. Lancet 1965; 2: 926–9. 15. Edwards RG, Donahue R, Baramki T, Jones H Jr. Preliminary attempts to fertilize human oocytes matured in vivo. Am J Obstet Gynecol 1966; 96: 192–200. 16. Yanagimachi R, Chang MC. J Exp Zool 1964; 156: 361–76. 17. Edwards RG, Talbert L, Israestam D, et al. Diffusion chamber for exposing spermatozoa to human uterine secretions. Am J Obstet Gynecol 1968; 102: 388–96. 18. Steptoe PC. Laparoscopy and ovulation. Lancet 1968; 2: 913. 19. Edwards RG, Bavister BD, Steptoe PC. Early stages of fertilisation in vitro of human oocytes matured in vitro. Nature (London) 1969; 221: 632–5.

20. Gardner RL, Edwards RG. Control of the sex ratio at full term in the rabbit by transferred sexed blastocysts. Nature (London) 1968; 218: 346–8. 21. Henderson SA, Edwards RG. Chiasma frequency and maternal age in mammals. Nature (London) 1968; 218: 22–8. 22. Edwards RG, Sharpe DJ. Social values and research in human embryology. Nature (London) 1971; 231: 81–91. 23. Palmer R. Acad Chir 1946; 72: 363. 24. Fragenheim H. Geburts Frauenheilkd 1964; 24: 740. 25. Steptoe PC. Laparoscopy in Gynaecology. Edinburgh: Livingstone, 1967. 26. Lunenfeld B. In: Inguilla W, Greenblatt RG, Thomas RB, eds. The Ovary. Springfield, IL: CC Thomas, 1969. 27. Steptoe PC, Edwards RG. Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotrophins. Lancet 1970; 1: 683–9. 28. Edwards RG, Steptoe PC, Purdy JM. Fertilization and cleavage in vitro of preovulatory human oocytes. Nature (London) 1970; 227: 1307–9. 29. Steptoe PC, Edwards RG, Purdy JM. Human blastocysts grown in culture. Nature (London) 1971; 229: 132–3. 30. Edwards RG, Surani MAH. The primate blastocyst and its environment. Uppsala J Med Sci 1978; 22: 39–50. 31. Csapo AI, Pulkkinen MO, Kaihola HL. The relationship between the timing of luteectomy and the incidence of complete abortions. Am J Obstet Gycecol 1974; 118: 985–9. 32. Steptoe PC, Edwards RG. Reimplantation of a human embryo with subsequent tubal pregnancy. Lancet 1976; 1: 880–2. 33. Edwards RG, Steptoe PC, Purdy JM. Clinical aspects of pregnancies established with cleaving embryos grown in vivo. Br J Obstet Gynaecol 1980; 87: 757–68. 34. Trounson AO, Leeton JF, Wood C, et al. Pregnancies in humans by fertilization in vitro and embryo transfer in the controlled ovulatory cycle. Science 1981; 212: 681–2. 35. Zeilmaker GH, Alberda T, Gent I, et al. Two pregnancies following transfer of intact frozen–thawed embryos. Fertil Steril 1984; 42: 293–6. 36. Handyside A, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA application. Nature (London) 1990; 344: 768–70. 37. Wikland M, Enk L, Hamberger L. Transvesical and transvaginal approaches for the aspiration of follicles by use of ultrasound. Ann NY Acad Sci 1985; 442: 182–94. 38. Palermo G, Joris H, Devroey P, et al. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992; 340: 17–18. 39. Hansis C, Edwards RG. Cell differentiation in the preimplantation human embryo. Reprod BioMed Online 2003; 6: 215–20.

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1 Setting up an ART laboratory Jacques Cohen, Antonia Gilligan, John Garrisi

Nowadays, there are so many ways of implementing assisted reproduction that one particularly successful outfit may actually have little in common with another, and yet be equally successful. This important fact should be kept in mind when starting a new clinic for assisted reproduction. Systems may vary from a temporary make-shift drive-in type laboratory to a fully equipped purpose-built institute. Laboratory set-ups in temporary space for occasional use which may combine remote egg retrieval and transport systems of gametes and embryos are not discussed here. While these systems may be productive under some circumstances, there have not been any recent studies suggesting that such uncertain models are really compatible with optimal results. Also not covered here are designs that function as a central laboratory for remote locations where egg retrieval and embryo replacement are carried out. Such ‘transport IVF’ systems can be adequately successful, depending on the distance traveled and the physical conditions of gamete transport.1,2 Both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) can be applied to transported oocytes, and in certain situations ‘transport IVF’ is a welcome alternative for those patients whose reproductive options have been limited by restrictive governmental regulations.3,4 This chapter discusses the more typical purpose-built allinclusive laboratories that are adjacent or in close proximity to oocyte retrieval and embryo transfer facilities, with emphasis on the special problems of construction.

Personnel and experience While the surroundings, housing, and equipment require special consideration in the design of an integrated gamete and embryo treatment facility, it is the staff who will conduct the procedures and who are essential to its success. Successful clinical practice is almost entirely dependent on high levels of experience and qualifications among medical and laboratory personnel. Hospitals usually do best when their human resource departments select key personnel based on personality and experience, rather than official qualifications and status. The same principle

applies to assisted reproduction laboratories, as experience is the key to success and especially because there are very few standard teaching and examination systems in place for the assisted reproductive technologies (ART) environment. Few new programs directed by novices have had flying starts, and although we cannot overestimate the potential of human creativity, inexperienced teams may have poor results and eventually fail altogether. This chapter aims to protect experienced practitioners from unexpected failure during the set-up of a new laboratory, when they are essentially placing themselves in a new environment. Laboratory staff, directors, and embryologists must consider their experience within the context of what will be required of them. Even though humility is sometimes difficult for medical professionals, an adequate clinical outcome requires a cautious and rational reassessment of individual abilities and acceptance that much of the environmental effects on ART are unknown. Programs should develop a collegial system to track performance levels for crucial clinical and laboratory personnel such as transfer efficiency, ICSI efficiency, etc. Certain regulatory bodies such as the College of American Pathologists and the British Human Fertilisation and Embryology Authority provide guidelines and licensing for embryologists, sometimes even for subspecialties such as the performance of ICSI, and for those who are capable of setting up and running IVF laboratories. So far, such licensing has done little more than provoke debate, because the abilities of such licensed personnel are largely unproven and the licenses are not interchangeable between countries. Tradition also plays its role, as in many Asian countries embryology directors are usually medical professionals. So qualifications are often seen to be less important than tradition and habit. What then qualifies someone to be a laboratory director and/or an embryologist? The answer is not a simple one. In general, peer evaluation sidesteps the problem by accepting any individual who qualified as a general pathology laboratory director or a reproductive specialist with an MD or PhD degree. However, pathologists do not necessarily have experience in gamete

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cell culture, and some reproductive specialists, such as urologists and immunologists, may never have worked with gametes and embryos at all. Equally, it is perfectly possible for a medical practitioner to direct a laboratory, without ever having practiced gamete and embryo handling. So how can it have been decided that they were actually qualified to set up and run an IVF laboratory? ‘Eppur si muove’ (‘And yet it moves’), as Galileo said when condemned to life imprisonment for heresy.

Empirical and statistical requirements for staff There is considerable disagreement about the required experience of embryologists. Hands-on experience in all facets of clinical embryology is an absolute requirement when starting a new program; even highly experienced veterinarian or basic science embryologists must be individually supervised by experienced clinical personnel. The period during which detailed supervision must continue depends absolutely on the types of skills required, the daily case-load, and total time. It can be appreciated that 100 cases over 1 year is a very different matter than the same amount during 6 weeks; the period of supervision should vary accordingly. The optimal ratio of laboratory staff to the contemplated number of procedures is debatable, and naturally, economics is the enemy. The ratio should be low, because embryologists can then spend enough time on quality control, training, and procedural details to ensure the high standards required for success. In practice, however, the staff/procedure ratio is often very high to save money, as happens sometimes in commercially oriented clinics, or sometimes it is a consequence of national health systems that must provide a wide range of services on a minimal budget. Needless to say, patients do not always benefit from these economic constraints; this is most obvious when comparing outcomes between the various health service systems in Western countries. The job description for the embryologist ideally includes all possible tasks, excepting initial patient intakes and medical and surgical procedures. Embryologists are often involved in important tasks related to general patient management, such as follicular monitoring, genetic counseling, marketing, administration, and nurse management. However, it should be realized that these tasks seriously detract from their true work. First and foremost, the embryologist’s duty is to perform gamete and embryo handling and culture procedures. Secondly, but equally important, the embryologist should maintain full awareness of quality control standards, both by performing routine checks and tests and by maintaining detailed logs of incidents, changes, unexpected changes, and countermeasures. Across all these duties, the following seven job positions can be clearly defined: director, supervisor,

senior embryologist, embryologist, trainee, assistant, and technician, their actual numbers varying according to the number of annual procedures. There may also be positions for others to do preimplantation genetic diagnosis, research, or secretarial work. Obviously, not all of these functions will apply to smaller centers. Although at first sight a seemingly unimportant detail, one of the most useful functions that ever existed was that of a professional witness that was implemented during the first few years of Bourn Hall Clinic. It effectively preserved a high level of security for embryo handling, even when large numbers of patients were being treated simultaneously. It also ensured that embryologists performed only those procedures and techniques for which they were properly qualified. In general, embryologists should concentrate on only gamete and embryo handling, and any laboratory with a relatively high number of annual procedures should have additional embryology technicians and assistants who can order and maintain equipment, and properly record laboratory data. In short, skimping on staff can be seriously self-defeating in the IVF laboratory.

Facility, budget, and design Historically some clinics were built in remote areas, based on beliefs that environmental factors such as stress would affect the patient and thereby the outcome. Today’s laboratories are commonly placed in city centers and large metropolitan areas in order to service large populations. The recent development of better assays for determining the baseline environmental quality, combined with the awareness that some buildings or building sites could be intrinsically harmful to cell tissue culture, makes today’s choice of a laboratory site even more important for a new program.5,6 A laboratory design should be based on the anticipated case-load and any subspecialty. Obviously, local building and practice permits must be assessed prior to engaging in a fully fledged design. There are five basic types of design: 1. Laboratories using only transport IVF. 2. Laboratories adjacent to clinical outpatient facilities that are only used part of the time. 3. Full-time clinics with intra-facility egg transport using portable breeding chambers. 4. Fully integrated laboratories with clinical areas. 5. Portable, temporary laboratories. Before developing the basic design for a new laboratory, environmental factors must be considered. While the air quality in modern laboratories can be controlled to a degree, it can never be fully protected from the exterior environment and adjoining building spaces. Designers should first determine if the building or the surrounding site will undergo renovations,

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demolition or major changes of any kind, in the foreseeable future. City planning should also be reviewed. Historical environmental data and trends, future construction, and the ability of maintenance staff to sustain the IVF laboratory need to be determined. Activity related to any type of construction can have a significant negative impact on any proposed laboratory. Prevalent wind direction, industrial hazards, and general pollution reports such as ozone measurements should also be determined. Even when these factors are all deemed acceptable, basic air sampling and determination of volatile organic compounds (VOCs) should be made inside and outside the proposed building area. The outcome of these tests will determine which design requirements are needed to remove VOCs from the laboratory area. In most cases an overpressured laboratory (at least 0.10–0.20 inches of water) that uses a high number (7–15) of fresh air changes (FACH) per hour is the best solution, because it also provides for proper medical hygiene. The laboratory walls and ceiling should have the absolute minimum number of penetrations. This generally requires a solid ceiling, sealed lighting, and airtight utility connections. Contrary to many vendors’ representations, commercial suspended ceilings using double-sided tape and clips are not acceptable. Doors will require seals and sweeps, and should be lockable. Ducts and equipment must be laid out in such a way that routine and emergency maintenance and repair work can be performed outside the laboratory with minimal disruption. Air handling must not use an open plenum design. In the ideal case, 100% outside air with chemical and physical filtration will be used with sealed supply and return ducts. Alternatively, the air supply equipment may supplement outside air with recirculated air, with processing to control the known levels of VOCs. Some laboratories will require full-time air recirculation while others may actually find the outside air to be perfectly clean (imagine that!). Outside air is often erroneously judged to be polluted, without proper chemical assessment, while inside air is usually considered ‘cleaner’ based on the presumption that it ‘smells’ better.5 In many laboratory locations, conditions are actually the reverse, and designers should not by any means ‘follow their instincts’ in these matters. Humidity must also be completely controlled according to climate and seasonal variation. The system must be capable of supplying the space with air with a temperature as high as 30–35°C, at less than 40% relative humidity. Air inlets and outlets should be carefully spaced to avoid drafts that can change local ‘spot’ temperatures, or expose certain items of equipment to relatively poor air or changes in air quality. Laminar flow hoods and micromanipulation workstations should not be located too close to air supply fixtures, to avoid disruption of the sterile field and to minimize cooling on the microscope stage. A detailed layout and assessment of all laboratory furniture and equipment is

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therefore essential prior to construction, and has many other benefits. Selection of a knowledgeable architect and a mechanical engineer for the project is essential. Confirm what their past experience has been in building biologically clean rooms. The use of ‘environmentally friendly’ or ‘green’ products has been suggested by some designers. The reliance on ‘natural’ products does not ensure a clean laboratory. In one case, wood casework with a green label was found to be a major source of formaldehyde. Floor coverings using recycled vinyl and rubber were selected for their low environmental impact to the planet, without considering the huge offgassing impact of the material. Supervision of the construction is also critical. Skilled tradesmen using past training and experience may not follow all of the architect’s instructions. The general contractor and builders must be briefed on why these novel construction techniques are being used. The use of untested methods and products can compromise not only the project but also their payment. The organization and flow of persons and things in a world-class restaurant results in a special ambience where more than just the food is considered. In the same way, appropriate modular placement of groups of incubators, gamete handling areas (laminar flow unit or isolette), and micromanipulator stations will minimize distances that dishes and tubes need be moved. Ideally, an embryologist should be able to finish one complete procedure without moving more than 3 meters in any direction; not only is this efficient but also it minimizes possible collisions in a busy laboratory. The number of modules can easily be determined by the expected number of cases and procedure types, the average number of eggs collected, and thereby the number of embryologists expected to work simultaneously. Each person should be provided with sufficient workspace to perform all procedures without delay. Additional areas can contain simple gamete handling stations or areas for concentrating incubators. Cryopreservation and storage facilities are often located in a separate space; these should always be adjacent to the main laboratory. Another separate laboratory can contain an area for culture medium preparation, sterilization, and water treatment. Administration should be performed in separate offices. Last but not least, it is preferable to prepare semen in a separate laboratory altogether, adjacent to a collection room. The semen laboratory should have ample space for microscopes, freezing, and sterile zoning. Some thought should go into planning the semen collection area. This room should be at the end of a hallway with its own exit; it should be soundproofed and not too large, with a sink and clear instructions of how to collect semen in preparation for ART. The room should be adjacent to the semen preparation laboratory with a double-door cupboard-type

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pass-through for samples between them. This passthrough should have a signaling device so the patient can inform the embryologist that the sample is ready; it also permits male patients to leave the area without a sample container in their hand.

Equipment and storage A detailed list of equipment should be prepared and checked against the planned location of each item; it can later be used as the basis of maintenance logs. It is important to consider the inclusion of extra crucial equipment and spare tools in the laboratory design, to allow for the event of sudden malfunction. It is particularly important to have redundant elements of the cryopreservation system – including cryopreservation and storage equipment. Similarly, two or more spare incubators should not be seen as excessive; at least one spare suction device and micromanipulator for micromanipulation should also be included. There are many other items whose malfunction would jeopardize patient care, although some spares need not be kept on hand as manufacturers may always have them available; however, such details need to be repeatedly checked as suppliers’ stock continues to change. It may also be useful to team up with other programs or an embryology research laboratory so that a crucial piece of equipment can be exchanged in case of unexpected failure. Some serious thought is needed when contemplating the number of incubators and incubator spaces. The ratio of cases per incubator varies considerably from program to program, and assuredly affects clinical outcome, depending on the number, type, and length of incubator door openings. In principle, the number should be kept to a minimum; we prefer a limit of four cases per incubator. Several other incubators are used for general purpose during micromanipulation and other generic uses, to limit further the number of incubator openings. Strict guidelines must be implemented and adhered to when maintaining distinct spaces for separating dishes or tubes from patients. Separate compartments may be helpful and can be supplied by certain manufacturers. Servicing and sterilizing of equipment such as incubators may have to occur when the laboratory is not performing procedures. Placement of incubators and other pieces of equipment on large castors may be helpful in programs where downtime is rare. Pieces of equipment can then be serviced outside the laboratory. When there are several options available to the laboratory designer, supply and evacuation routes should be planned in advance. One of the most susceptible aspects of ART is cryopreservation. In case of an emergency such as fire or power failure, it may be necessary to relocate the liquid nitrogen-filled dewars without using an elevator, or to relocate the frozen samples using a temporary container. This may seem an extreme consideration, especially in the larger

laboratories that stockpile thousands of samples, but plans should be made. It may be possible to keep a separate storage closet or space near the building exit, where long-term samples that usually provide the bulk of the storage can be kept, but this would require repeated check-ups of a facility that is not part of the laboratory. Liquid nitrogen-level alarms, with remote notification capability, should be contemplated for all dewars. The route of delivery of liquid nitrogen and other medical gas cylinders must be relatively easy, without stairways between the laboratory and the delivery truck, and should be sensibly planned in advance. Note that the flooring of this route is usually destroyed within months because of liquid nitrogen spills and wear caused by delivery containers, so the possibility of an alternative delivery corridor should be considered for these units. Liquid nitrogen containers and medical gas cylinders are preferentially placed immediately adjacent to the laboratory in a closet or small room with outside access. Pipes and tubes enter the laboratory from this room, and cylinders can be delivered and changed to this room without compromising the laboratory area in any way. This allows liquid nitrogen to be pumped into the cryopreservation laboratory using a manifold system and minimal lining. Lines should be properly installed and insulated to insure that they do not leak or allow condensation, and conserve energy. Medical gases can be directed into the laboratory using prewashed vinyl/Teflon-lined tubing. Alternatively, solid manifolds made from stainless steel with suitable compression fittings can be used. Avoid soldered or brazed copper lines used in domestic plumbing applications wherever possible; copper lining can be used but should be cleaned and purged for a prolonged period prior to laboratory use. Copper line connections should not be soldered as this could cause continuous contamination. This recommendation may conflict with existing building codes, but noncontaminating alternatives must be found. In any case, a number of spare lines hidden behind walls and ceilings should be installed in case of later renovation or facility expansion. Large programs should consider the use of exterior bulk tanks for carbon dioxide and liquid nitrogen. This removes the issues of tanks for incubators or cryopreservation. These tanks are located where delivery trucks can hook onto and deliver directly to the tank. Pressurized gas lines or cryogenic lines then run the carbon dioxide or liquid nitrogen to the IVF laboratory for use. Placement of bulky and difficult pieces of equipment should be considered when designing doorways and electrical panels. Architects should be fully informed of all equipment specifications to avoid that truly classic door-width mistake. Emergency generators should always be installed, even where power supplies are usually reliable; the requirements can easily be determined by an electrical engineer.

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Thankfully, these units can be well removed from the laboratory, but must be placed, mind you, in well-ventilated areas that are not prone to flooding. Additional battery ‘uninterruptible power systems’ (UPS) may be considered as well, but are of very limited ability. Buildings should also be checked for placement of the main power inlets and distribution centers, especially because sharing power lines with other departments or companies may not be advisable. Circuit breakers should be easily accessible to embryologists or building maintenance staff. General knowledge of mechanical and electrical engineering of the building and the laboratory specifically will always be advantageous. Ample storage spaces should always be planned for IVF laboratories. In the absence of dedicated storage space, laboratory space ends up being used instead, filling all cabinets and playing havoc with the original design. This storage area should contain all materials in sufficient quantity to maintain a steady supply. A further reason to include storage areas in laboratory design, sufficient itself to justify the space, is that new supplies, including sterilized disposable items, release multiple compounds for prolonged periods. This ‘out-gassing’ has been determined to be a major cause of adverse air content in a number of laboratories in which supplies were stored. Separate storage space therefore provides the best chance of good air quality, especially when it is supplied by separate air handling equipment. It should be large enough to handle bulky items as well as mobile shelving for boxes. One should be careful to avoid the natural inclination to save extra trips by bringing too many items into the laboratory, or the gains made by careful design may be lost.

Microscopes and visualization of cells Though dissecting microscopes are crucial for the general handling of gametes and embryos, many people still consider inverted microscopes to be a luxury, even though they are in regular use with micromanipulation systems. Proper visualization of embryos is key to successful embryo selection for transfer or freezing; if the equipment is first class, the visualization can be done quickly and accurately.7 Even so, appropriately detailed assessment is still dependent on the use of an oil overlay system to prevent damage by prolonged exposure. Each workstation and microscope should be equipped with a still camera and/or video camera and monitor. Still photos can be placed in the patient file, and video footage permits speedy review of embryonic features with colleagues after the gametes are safely returned to the incubator, as well as helping to train new embryologists, an ever-present task. Interference optics, such as Hoffman and Nomarski, are preferable because they permit the best measure of detail and depth. Novel visualization of internal elements such as spindles using the Pol-Scope

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requires more complicated equipment, but is still largely compatible with the limitations of routine embryo assessment.8 Ideally, the captured photos should be digitally stored for recall at their appropriate location in the clinic’s medical database.

Construction, renovation, and building materials Construction and renovation can introduce a variety of compounds into the environment of the ART laboratory, either temporarily or permanently. Either can have significantly adverse effects on the outcome of operations.5,6,9,10 The impact of the exterior environment on IVF success has been demonstrated. Pollutants can have a significant adverse effect on reproductive success in an IVF laboratory. These effects can range from delayed or abnormal embryonic development, absence of fertilization, and depressed implantation rates to complete reproductive failure. Many of the damaging materials are organic chemicals that are released or out-gassed by paint, adhesives from flooring, cabinets, and general building materials, as well as from laboratory equipment and procedures. It is important to realize that the actual construction phase of the laboratory can cause permanent problems. Furthermore, any subsequent adjacent renovation can also cause similar, or even greater problems. Neighboring tenants can be informed of the sensitivity of cultured in vitro gametes, and at least these nearby changes can be supervised by laboratory personnel to minimize potential damage to a greater or lesser extent. However, new construction immediately outside the building is considerably more problematic. City works such as street construction are very hard to predict and nearly impossible to control. A good relationship with the neighbors is not always an easy task, and working relationships should always be maintained with building owners and city planners, in the hope that, at the very least, the IVF laboratory will be kept informed of upcoming changes. In spite of one’s best wishes, or the fervent assurances of building owners, changes of this sort inevitably take place, so here we present some guidelines, all of which apply to new laboratory construction as well as changes in adjacent areas. First of all, the area to be demolished and then constructed needs to be physically isolated from the IVF laboratory (if this is not the new IVF laboratory itself). The degree of isolation should be equivalent to an asbestos or lead abatement project. The isolation should be done by the following techniques. Physical barriers should be erected, consisting of poly sheeting supported by studding where needed. Access to the construction area should be restricted by the use of an access passageway with two doors in series. All construction waste should be removed via an exterior opening or

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properly bagged before using an interior exit. The construction area should be under a negative air pressure, exhausting to the exterior; naturally, this exhaust should be far removed from the laboratory’s air intake, and properly located with regard to the prevailing winds and exterior air flow. Extra interior fans should be used during any painting or use of adhesives, to maximize removal of noxious fumes. Material Safety Data Sheets (MSDS) for all of the paints, solvents, and adhesives should be obtained, logged, and used to evaluate any potential material and manage industrial hygiene concerns. Follow-up investigations with manufacturers and their representatives may be helpful because specifications of these items are changed without notice. The negative pressurization of the laboratory space requires continuous visual indication, such as a ball and tube pressure indicator, or simply paper strips. Periodic sampling for particulates, aldehydes, and organics could be done outside the demolition and construction site, provided this can be budgeted. Alternatively, tracer gas studies can be used to verify containment. The general contractor of the demolition and construction should be briefed in detail on the need to protect the IVF facility and techniques to accomplish this. When possible, the actual members of the construction crew themselves should be selected and briefed in detail. Large filter units using filter pellets of carbon and permanganate can be placed strategically (GenX International, Madison, CT, USA). Uptake of organics can be assayed, but the tempo of routine filter changes should be increased during periods of construction activity.

Selection of building materials Many materials release significant amounts of volatile organics, and a typical list includes paints, adhesives, glues, sealants, and caulking, which release alkanes, aromatics, alcohols, aldehydes, ketones, and other classes of organic materials. This section outlines steps to be taken in an effort to reduce these outgassing chemicals. Any and all interior painting throughout the facility should only be done on prepared surfaces with water-based paint formulated for low VOC potential.* During any painting, auxiliary ventilation should be provided using large industrial construction fans, with exhaust vented to the exterior. Paints that can significantly influence air quality should be emission tested (some suppliers already

*Water-based paints are generally acceptable. However, in some locations epoxy paints are required for regulatory or policy reasons. These materials may offer improved washability and durability. If they must be used, they should be allowed to cure fully after application. Curing can take 60–90 days to complete. Emission testing on samples is required since amine catalysts can be very persistent, if not used exactly according to the manufacturer’s instructions.

have these tests available). MSDS are generally available for construction materials. Suppliers under these specifications are encouraged to conduct product testing for the emission potential. The variety of materials and applications greatly complicates this testing, but several procedures have been developed to identify and quantify the materials released by building materials and furnishings. The interior paints must be water-based, low-volatile paints with acrylic, vinyl acrylic, alkyd, or acrylic latex polymers. Paints meeting this specification can also contain certain inorganic materials. Paints with low volatiles may still contain low concentrations of certain organics. No interior paint should contain materials such as formaldehyde, acetaldehyde, isocyanates, reactive amines, phenols, and other water-soluble volatile organics. Adhesive glues, sealants, and caulking materials present some of the same problems as paints, but water-based materials are generally not available for these applications, although their composition varies widely. Silicone materials are preferred whenever possible, particularly for sealants and caulking work. No adhesive, glue, sealants, or caulking used in the interior should contain materials such as formaldehyde, benzaldehyde, and phenol (for a complete review of potentially toxic materials, contact Alpha Environmental, New Jersey, USA).

‘Burning in’ of the finished facility New IVF laboratories and new facilities around existing laboratories have often been plagued by complaints of occupants who experience discomfort from the chemicals released by new construction and furnishings. The ambient levels of many of these materials can be reduced by ‘burning in’ the facility. A typical burn-in consists of increasing the temperature of the new area by 10–20°C and increasing the ventilation rate; even higher temperatures are acceptable. The combination of elevated temperature and higher air exchange aids in the removal of the volatile organics. Upon completion of the construction, the air handling system should be properly configured for the burn-in of the newly constructed area. As previously stated, the system must be capable of supplying the space with air with a temperature of 30–35°C, at less than 40% relative humidity. The burn-in period can range from 10 to 28 days, and the IVF laboratory should be kept closed during this time. If these temperatures cannot be reached by the base system, use auxiliary electrical heating to reach the minimum temperature. During burn-in, all lighting and some auxiliary equipment should be turned on and leftrunning the whole time. Naturally, ventilation is critical if redistribution of irritants is to be avoided; the whole purpose is to purge the air repeatedly. Auxiliary equipment should of course be monitored during the burn-in. The same burn-in principle applies to newly purchased incubators. Removal of volatile organics is

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especially important in the critical microenvironment of the incubator. Whenever possible, it is advantageous to purchase incubators months in advance of their intended initial use, and to operate them at an elevated temperature in a clean, protected location. After the burn-in is complete, a commissioning of the IVF suite should be conducted to verify that the laboratory meets the design specifications. The ventilation and isolation of the laboratory should be verified by a series of tests using basic airflow measurements and tracer gas studies. The particulate levels should be determined to verify that the highefficiency particulate air (HEPA) system is functional. Particulate sampling can be done by using Federal Standard 209E. Microbial sampling for aerobic bacteria and fungi is often done in new facilities using an Andersen sampler followed by microbiological culturing and identification. The levels of VOC contamination should be determined. Possible methods are included in the US Environmental Protection Agency (EPA) protocols using gas chromatography/ mass spectroscopy (GC/MS) and high-performance liquid chromatography sensitive at the microgram per cubic meter level.11–13

Maintenance planning The best systems and designs will eventually fail unless they are carefully maintained. The heating, ventilation and air conditioning (HVAC) will require filter changes, coil cleaning, replacement of drive belts, and chemical purification media. The most prevalent failure mode is the initial particulate filter. These are inexpensive filters designed to keep out large dust particles, plant debris, insects, etc. If such filters are not replaced they will fail, allowing the HVAC unit to become fouled. The HEPE filters and chemical media also require inspection and periodic replacement. Maintenance staff should report their findings to the IVF laboratory.

Insurance issues Assisted reproductive technologies have become common practice worldwide, and are regulated by any combination of legislation, regulations, or committeebased ethical standards. The rapid evolution and progress of ART reveal new legal issues that require consideration. Even the patients themselves are changing, as it becomes more acceptable for single mothers and homosexual and lesbian couples to present themselves for treatment. Donation of genetic material, age limitation, selective fetal reduction, preimplantation genetic diagnosis, surrogacy, and cloning each present a legal quagmire; their very definitions vary from country to country, along with types of application, as well as regional social factors, religion, and law. Furthermore, financial and emotional stresses often oppress patients seeking treatment in countries where

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social medicine does not cover infertility treatment, and more especially after a failed cycle. This translates into an increasing number of ART lawsuits, in spite of generally improved success rates. Laboratory personnel and the institution owning the laboratory should therefore obtain an insurance policy of sufficiently high level and quality commencing prior to the first day of operations. Litigation-prone issues need special consideration, and include: • • • •

cancellation of cycle prior to egg retrieval failure to become pregnant patient identification errors interrupted cryostorage events.

These issues occur even when experienced practitioners consider themselves at low risk. Prior to engaging in ART activity, protocols can be established to identify these problem areas and establish countermeasures.

Conclusion All in all, it may be surprising for some how many professionals continue to pursue the establishment of new ART clinics at a time when competition is high, obvious financial benefits are small, and existing ART services may appear to be approaching saturation in most areas. Appearances can be wrong. ART is still a pioneering field, with many alternative aspects such as reprogramming, cell rejuvenation and aging, cell cycle mishaps resembling oncologic events, mitochondria reproduction, and stem cell development and applications, all fields of science and medicine by themselves, still very far from resolution. Regardless, this chapter can provide some guidance to those medical professionals aspiring to independence in the world of ART, although it cannot safeguard from some adverse effects in all cases. It should serve to provide useful suggestions and concepts that have been learned from practical experience for the wide variety of problems and solutions that have been used over many years.

References 1. Jansen CA, van Beek JJ, Verhoeff A, Alberda AT, Zeilmaker GH. In vitro fertilisation and embryo transfer with transport of oocytes. Lancet 1986; 22: 676. 2. Verhoeff A, Huisman GJ, Leerentveld RA, Zeilmaker GH. Transport in vitro fertilization. Fertil Steril 1993; 60: 187–8. 3. Coetsier T, Verhoeff A, De Sutter P, Roest J, Dhont M. Transport in vitro fertilization/intracellular sperm injection: a prospective randomized study. Hum Reprod 1997; 12: 1654–6. 4. De Sutter P, Dozortsev D, Verhoeff A, et al. Transport intracytoplasmic sperm injection (ICSI): a cost-effective alternative. J Assist Reprod Genet 1996; 13: 234–7.

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5. Cohen J, Gilligan A, Esposito W, Schimmel T, Dale B. Ambient air and its potential effects on conception in vitro. Hum Reprod 1997; 12: 1742–9. 6. Cohen J, Gilligan A, Willadsen S. Culture and quality control of embryos. Hum Reprod 1998; 13(Suppl 3): 137–44. 7. Alikani M, Cohen J, Tomkin G, et al. Human embryo fragmentation in vitro and its implications for pregnancy and implantation. Fertil Steril 1999; 7: 836–42. 8. Liu L, Trimarchi JR, Oldenbourg R, Keefe DL. Increased birefringence in the meiotic spindle provides a new marker for the onset of activation in living oocytes. Biol Reprod 2000; 63: 251–8. 9. Hall J, Gilligan A, Schimmel T, Cecchi M, Cohen J. The origin, effects and control of air pollution in

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11.

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laboratories used for human embryo culture. Hum Reprod 1998; 13(Suppl 4): 146–55. Boone WR, Johnson JE, Locke AJ, Crane MM 4th, Price TM. Control of air quality in an assisted reproductive technology laboratory. Fertil Steril 1999; 71: 150–4. Seifert B. Regulating indoor air. Presented at the 5th International Conference on Indoor Air Quality and Climate, Toronto, Canada, 1990; 5: 35–49. Federal Standard 209E. Washington, DC: General Services Administration, US Federal Government, 1992. Compendium of Methods for the Determination of Toxic Organic Compounds in Ambient Air, US EPA 600/4-84-041, April 1984/1988. [Available from the US EPA through the Superintendent of Government Documents, Washington, DC.]

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2 Quality control: maintaining stability in the laboratory David H McCulloh

Introduction The field of assisted reproductive technologies (ART), primarily involving in vitro fertilization, embryo culture, and embryo transfer (collectively known as IVF), is now maturing in its development. Since the birth of Louise Brown in 1978, the field has grown immensely, and its success has improved steadily and impressively (as measured by the incidence of live births per cycle initiated) from less than 1% to a remarkable incidence of roughly 33% in just over 25 years. Equally remarkable is the observation that, throughout the field, many attempts are followed by failure. Hence, despite the improvements of the past quarter century, there remains a need to optimize procedures. Let us concentrate on the steady improvement in success that has been documented over this period. To what can we attribute the improved success, from nearly non-existent to success exceeding 60% in some programs? There are at least three general areas in which changes have impacted on outcome.

New products New products have revolutionized patient treatment. There have been steady improvements in many areas, including the generalized use of gonadotropin-releasing hormone analogs to control the pituitary’s release of gonadotropins (follicle-stimulating hormone and luteinizing hormone). There have been improvements in culture media, both through mass production leading to more uniform and repeatable production of media and through developments in new media leading to stepwise media changes that are currently the standard of care. Production of media for gamete and embryo culture has become a competitive and complicated business. Now it is unusual for laboratories to fabricate their own medium since individual production is prone to error. The introduction of soft catheters has also become extremely competitive, with many manufacturers vying for our programs’ business. The introduction of recombinant gonadotropins has led to much more uniform and stable formulations

that yield more reproducible responses in patients. The use of drugs designed to treat type II diabetes (metformin, rosiglitazone) has begun as an adjunct to treatment of patients with polycystic ovaries. Many of these improvements have benefited from the use of more uniform products, manufactured to more stringent standards requiring quality control measures to assure lack of toxicity.

New procedures New procedures have resulted in such a dramatic improvement in success that our field is experiencing a shift in the type of patients treated. The introduction of the laboratory technique of intracytoplasmic sperm injection (ICSI) has revolutionized the field so that our most optimistic cases are now couples for whom the only diagnosis is a male factor that precludes fertilization. Prior to the inception of ICSI around 1993, these patients were unlikely to achieve fertilization without the use of donor sperm. Microtools previously fabricated in the laboratory are now commercially available. In addition, the improved sensitivity of Kruger’s strict criteria for scoring sperm morphology has resulted in improved detection of males with poor prognosis. The use of ICSI has also advanced the field so that azoospermic men can now be the source of sperm that result in live births, since sperm obtained by surgical techniques are quite effective for use in ART. A second procedural advancement that is revolutionizing the field is extended culture of embryos to the fifth or sixth day after oocyte retrieval. This advancement is founded on research determining that embryos’ metabolic needs vary during the first few days of development. Extended culture has permitted more critical assessment and selection of embryos. The ability to select the most rapidly developing embryos has focused attention on replacing fewer embryos and thereby eliminating the dangerous occurrence of triplet and higherorder multiple pregnancies. A recent trend has been to transfer only one single embryo to avoid the occurrence of twins. The more sensitive assessment of embryos

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possible after extended culture has, no doubt, affected many aspects of IVF treatment, including improvements in embryo culture and even improvements in ovulation induction. Women, too, have benefited from improved protocols for controlled ovarian hyperstimulation and through the development of more critical tests of ovarian reserve prior to treatment.

New legislation New legislation has both directly and indirectly assisted with the improved outcome of ART. In 1988, legislation was enacted in the United States1 that defined standards for all clinical laboratories. In particular it stated that laboratories performing quantitative semen analysis (andrology laboratories) must comply. Standards included qualifications for personnel and specific requirements for a quality assurance program involving frequent quality control measurements. Although the Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88) did not specifically dictate standards for embryology laboratories, they strongly influenced many programs to adopt CLIA’88like standards for their embryology laboratories also. The College of American Pathologists in association with the American Fertility Society (now the American Society for Reproductive Medicine, ASRM) formed its Reproductive Laboratory Accreditation Program and adopted specific standards for accreditation (the first agency achieving deem status with a specific program for andrology/embryology laboratories). Updated recommendations were published by the ASRM2 and included personnel qualifications as well as standards for quality assurance, including quality control. Standards were also defined for personnel involved in direct clinical patient care during ART. The passage of The Fertility Clinic Success Rate and Certification Act of 19923 mandated that all ART programs report their ART success and undergo certification, additionally challenging programs to improve, undergo certification, or face extinction. More recently, in 1998, the ‘Obstetric and gynecologic devices; Reclassification and classification of medical devices used for in vitro fertilization and related assisted reproduction procedures’4 legislated that the Food and Drug Administration (FDA) must regulate the devices used in ART. Their regulation was largely responsible for causing manufacturers to alter their production practices and to require more stringent quality control to assure that the products are not toxic. In addition, the adoption of legislation entitled ‘Human Cellular and Tissue-Based Products or HCT/Ps,’5 effective March 25, 2005, has changed the complexion of donor gamete use in ART. This legislation was designed to improve the safety of donor gametes (oocytes, sperm) by regulating the screening and testing of donors for infectious diseases prior to

obtaining gametes. In addition, several laboratory functions, including labeling of gamete containers and housekeeping, are also regulated. During this same period, facilities have worked feverishly to improve their success through application of stringent standards of practice. It is difficult to determine just what has led to the consistent improvement in IVF success over the years. Has it been federal regulation of the assisted reproduction industry, or has it been patient selection? However, a large portion of the improvements, including new products and practices, are founded on more careful quality control practices in the highly regulated pharmaceutical and medical device industry. Careful quality control can lead to much more stable products with less variability and less likelihood of toxicity. Maintenance of stable conditions from these industries has benefited the practice of ART, and its implementation in our own programs is the focus of this chapter. Quality control is also crucial for the clinical ART program where stability is also advantageous. The stability afforded by careful quality control may effectively confirm that any patient-related success or failure is really specific to the patient or the patient’s treatment rather than an inconsistency in the program.

Introductory comments This chapter is an extension of two previously published reviews on this subject.6,7 An update to the article on quality control and quality assurance that previously appeared6 summarizes new legislation that has occurred since the article was written, including the ASRM’s revised guidelines and the FDA legislation. In addition, there is a section devoted to staffing norms that was available only in the previous edition.8 This chapter begins with a definition of quality control, and continues by describing several features of quality control: record keeping, quality control of personnel, quality control of procedures, quality control of equipment, quality control of computers, and quality control of materials and supplies. Descriptions of process testing, including sperm survival assays and mouse embryo testing, and mention of quality control of the entire process by consideration of patient treatment data, are discussed as overall quality control checks that should be performed by the program. Although written from a US perspective, the contents of this chapter are adaptable and applicable to all IVF laboratories.

Definition of quality control Although ART success has improved remarkably since its inception, the field of human assisted reproduction overall remains relatively poor at providing the patient with demonstrably high levels of success. As practitioners in the field, we must constantly

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attempt to improve our success. Until we can offer nearly 100% success throughout the field, we must strive to maintain a stable level of success so that we may provide our patients with honest, realistic estimates of their chances of achieving their goal of bearing a healthy child. Quality control is the process whereby all aspects of the program are monitored and confirmed to be functioning within limits previously determined to be tolerable. The program must remain within these limits to assure that the program operates in a stable, repeatable fashion. The goal of quality control is to confirm and document that the program maintains stable conditions. This stability provides a constant backdrop against which all patient treatment is performed. Without this stability, it is impossible to know whether an unusual outcome for a particular patient is associated with a patient-specific issue or a programmatic failure. Quality assurance is the overall process (that includes quality control as a subset) by which the program undergoes improvements and corrective actions to maintain or improve its processes. The goal of quality assurance is to improve the outcome. Quality control is a necessary portion of the quality assurance program. It is the quality control assessment of personnel, procedures, equipment, and materials that provides much of the data used in performing quality assurance/improvement activities.

Features of quality control Quality control records should be maintained to demonstrate that quality control was performed and so that data may be analyzed at a later time to detect the source of problems and determine ways to rectify problems. Data should be accumulated in several different categories, as described below.

Record keeping During performance of quality control, the monitored parameters should be recorded and maintained so that they will be available for review in the future. When a problem arises or improvements are desired, the assembled data will be useful in determining what corrective actions should be taken (quality assurance/improvement task). The natural variability in quality control data is a source of variation that can be analyzed to assess trends for improvement.6,9 Records of quality control tasks must be maintained for several years in many localities. In instances of use of donor gametes, you may be required to maintain the records for up to 21 years. Check with your local regulations. Quality control data may be maintained in one of at least two forms, either paper records or electronic records. Paper records have been used for years as the major method of data recording. When analysis of data is required, the data must be transferred from the

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paper records into a form for analysis. This often involves conversion of data from paper records to electronic records. Electronic records (maintenance in a computer spreadsheet or database) are quickly replacing paper records as the preferred method of data recording, since the data recorded electronically are ready for analysis nearly immediately, much more quickly than data in paper records. This will require off-site back up of all data.

Quality control of laboratory personnel Employees in ART laboratories must adhere to predetermined standards. They must have appropriate training and must be able to demonstrate competence with the procedures that they will perform. Standards have been provided within CLIA ’881 describing the educational requirements for personnel in different positions within laboratories performing high-complexity testing. Personnel in andrology laboratories (any laboratory performing semen analysis) must adhere to these standards, because laboratories performing semen analysis are specifically designated as laboratories performing high-complexity testing. The qualifications for personnel in laboratories performing high-complexity testing (specifically andrology laboratories) are summarized in Table 2.1. Each laboratory must have a laboratory director, a technical supervisor, a general supervisor, a clinical consultant, and testing personnel. One individual may share multiple positions. While legally defined standards exist for andrology laboratories, similar standards for embryology laboratories do not exist in the United States. Embryology laboratories are neither specifically included in the CLIA ’88 documents defining high-complexity testing, nor excluded as waived testing. Moreover, the US congress has not defined embryology procedures as high-complexity testing, opting rather to consider embryology procedures as medical procedures. However, the ASRM has provided guidelines for staffing.2 These guidelines are summarized in Table 2.2 and are applicable globally. Personnel hired to perform an andrology and/or embryology procedure, once they satisfy the educational requirements for the position, are also required to demonstrate competency to perform the procedure. CLIA do not indicate a particular mechanism for this other than the requirement to participate in routine, periodic proficiency testing, thereby demonstrating competence to perform the procedure. The ASRM guidelines suggest that personnel should perform a minimum of 30 complete procedures in order to qualify as a technologist, or 60 complete procedures to qualify as a laboratory director or a supervisor. This competency should be documented in the quality control records. At the present time, there are very few academic programs in the United States offering training for

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Table 2.1 Personnel qualifications required for high-complexity testing in andrology laboratories as specified by the Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88)1 Laboratory Director (or Clinical Consultant) must possess Laboratory Director License in the state (if available) and satisfy one complete row Degreea

Certification

Experienceb

Supervisionc

MD, or DO, and licensed to practice medicine in the state MD, DO, or DPM, and licensed to practice medicine, osteopathy, or podiatric medicine in the state Doctoral Degreed

Anatomic pathology, or clinical pathology, or both (ABP or AOBP) None

≥1 year

≥2 years

2 yearse

2 yearse

2 years

2 years

No doctoral degree

Certified by ABMM, ABCC, ABB, or ABMLI Certification prior to 2/28/1992

Technical Supervisor: must possess Technical Supervisor License in state (if available) and satisfy one complete rowf Degreea

Certification

Experienceg

MD, DO, or DPM and licensed to practice medicine, osteopathy, or podiatric medicine in the state Doctoral Degreed Master’s Degreed Bachelor’s Degreed

Certified in both anatomic and clinical pathology by ABP, AOBP, or equivalent None None None

1 year 2 years 4 years

General Supervisor: must possess General Supervisor License in state (if available) and satisfy one complete row Education

Qualification as:

Experienceb

(see above) (see above) (see below) Independent of education Graduate of MLT/CLT programa High School Graduate (or GED)

Laboratory Director Technical Supervisor Testing Personnel Qualified as General Supervisor prior to 2/28/92 Qualified as General Supervisor prior to 9/1/92 Qualified as General Supervisor prior to 9/1/92

1 year 1 year 2 years None None None

Testing Personnel: must possess Testing Personnel License in state (if available) and satisfy one complete row Educationa

Experienceb

Further qualifications

MD, DO, DPM, licensed to practice in the state Doctoral Degree,d Master’s Degree,d Bachelor’s Degree,d or in Medical Technology Associate Degreeh Education equivalent to an Associate Degree Education equivalent to an Associate Degree

None None

None None

None None Completion of CLT programi

None 24 semester hours of MLT courses 24 semester hours of science – specifically 6 hours chemistry, 6 hours biology, 12 hours chemistry, biology, or MLT 24 semester hours of science – specifically 6 hours chemistry, 6 hours biology, 12 hours chemistry, biology, or MLT Prior qualification as a technologist before 2/28/1992 None

Education equivalent to an Associate Degree

3 months of laboratory training in each specialty

Education equivalent to an Associate Degree

None

High School Graduate or equivalent

Completion of CLT programi,j

Abbreviations: MD, doctor of medicine; DO, doctor of osteopathy; DPM, doctor of podiatric medicine; ABP, American Board of Pathology; AOBP, American Osteopathic Board of Pathology; ABMM, American Board of Medical Microbiology; ABB, American Board of Bioanalysis; ABCC, American Board of Clinical Chemistry; ABMLI, American Board of Medical Laboratory Immunology; GED, high school equivalency demonstrated by examination; MLT, medical laboratory training, CLT, clinical laboratory training. a Degree must be granted from an accredited institution. b Training or experience must be gained in clinical laboratory. c Experience as a director or supervisor of high complexity testing. d The degree must be in chemical, physical, biological, or clinical laboratory science. e Effective 4/24/2003. f Other than state licensure, no specific qualifications for Technical Supervisors are listed in the field of andrology. If andrology is considered a discipline in hematology, these qualifications apply. g Experience must be in the specific field to be supervised (hematology/andrology). h Associate Degree in a laboratory science or medical laboratory technology. i Completion of a clinical laboratory training program accredited or approved by the Accreditation Bureau of Health Education Schools (ABHES), the Committee on Allied Health Education and Accreditation (CAHEA), or other program approved by HHS. j Successful completion of an official US military medical laboratory procedures training course and hold the enlisted occupational specialty of Medical Laboratory Specialist.

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Table 2.2

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Personnel qualifications recommended for embryology programs by the American Society for Reproductive Medicine2

Laboratory Director: must satisfy one complete row Degreea

Certification

Experienceb

ART proceduresc

PhD,d,e MD,e or DOe No doctoral degree

In embryology In embryology, qualification prior to January 1992

2 years 2 years

60 ART procedures 60 ART procedures

Supervisorf: must satisfy one complete row Degreea

Experienceb

ART proceduresc

Master’s or Bachelor’s Degreed

6 months

60 ART procedures

Technologist: must satisfy the complete row Degreea

ART proceduresc

Bachelor’s Degreed

30 ART procedures, under supervision

a

Degree should be from an accredited institution. Documented pertinent experience in a program performing IVF-related procedures, including quality control, detailed knowledge of cell culture, ART, and andrology procedures. c Number of ART procedures completed (each procedure including examination of follicular aspirates, insemination, documentation of fertilization, and preparation for embryo transfer) in a program performing at least 100 IVF procedures per year with a minimum annual 10% IVF live birth rate per retrieval. d Degree should be from an accredited institution, in a chemical, physical, or biological science as the major subject. e Education should include expertise and/or special training in biochemistry, cell biology, and physiology of reproduction with experience in experimental design, statistics, and problem solving/trouble shooting. f If the laboratory director is also the medical director, there should be a qualified designated supervisor. b

individuals in the fields of andrology and/or embryology. Therefore, it is difficult to receive formal training in these fields. Most training occurs ‘on the job’ at facilities where the procedures are performed. Good practices should be in place to assure that trainees are overseen throughout their training process and that the training is documented. It is wise for each staff member to keep track of the number of procedures personally performed. Further demonstration of competency should be achieved by each staff member’s participation in proficiency testing exercises. A rotation should be instated that assures that each laboratory staff member performs the proficiency testing, thereby demonstrating competency using an unknown analyte at least periodically.

Laboratory staffing norms In addition to covering all the functions required by law/guidelines, sufficient personnel should be available to perform all the andrology/embryology work without subjecting either the personnel or the patients to undue risk. Staffing norms are not widely available. However, it is clearly a quality control issue to be certain that enough staff are available to provide the desired volume of laboratory services safely and efficiently. Reference and hospital laboratories provide many broadly distributed laboratory services (especially for stat testing) 7 days per week. To do this they

have large numbers of staff who are broadly crosstrained. However, ART laboratories are generally staffed with fewer individuals, often with a repertoire limited specifically to the field of gamete testing and manipulation, and yet the staff are expected to provide services 7 days per week for extended periods of weeks. The effect of fatigue in this environment has not been evaluated, despite the possibly severe consequences of an error such as specimen misidentification. For many years, staffing norms were circulated by word of mouth throughout the field, without any published standards. The circulated norm suggested that one embryologist is necessary for each 100 IVF procedures performed per year. This number emerged prior to the introduction of widespread micromanipulation techniques that require a longer time to perform. Despite the widespread addition of this time-consuming activity, no changes in staffing norms occurred. During the Survey of 1999 Compensation conducted in 2000,10 staffing norms were estimated although they were not published. The average workload of laboratory personnel in ART laboratories was calculated to be 77.2 cases per laboratory person (average of 286.9 IVF cases per program with an average of 3.72 personnel in each of 110 laboratories in the year 1999). However, this simple ratio is probably not a fair representation of the staffing needs of a program. It is clear that more procedures per employee can be performed in a large program than in a smaller program. There is a fixed volume of work (largely the

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quality control functions) that must be performed in order to maintain an ART laboratory, even with no performance of ART cases. Likewise, ART laboratories generally operate 7 days per week. It is unreasonable and unethical, if not illegal, to expect a technologist to work 7 days per week. For these reasons, it is more reasonable to consider that a fixed number of personnel are required to perform the fixed volume of work. Additional personnel are required to perform the ART cases. Two examples follow.

Example 1

Table 2.3 States

Staffing norms for ART facilities in the United

Average number of laboratory staff Number of IVFs 0 100 250 400 1000

McCulloha

Boone and Higdonb

0.47 1.6 3.3 5.0 11.7

2.92 4.3 6.4 8.5 16.9

a

The number of laboratory personnel was determined using linear regression of the data from the 110 programs represented in the above Survey of 1999 Compensation.10 The relationship of procedures versus personnel (Table 2.3) indicates that roughly 0.5 staff is required without the performance of any IVF procedures. One additional staff person is required for each additional 88.5 embryo transfers. The number of embryo transfers is used as an estimate of the number of IVF treatments performed per year. This estimate could misrepresent the number of IVF treatments performed due to inclusion of embryo transfers from frozen–thawed embryo treatments and due to IVF treatments resulting in no embryo transfer. However, this analysis estimates that the average program will have 1.6 laboratory staff to perform 100 embryo transfers per year. Five laboratory staff are employed to perform 400 embryo transfers per year.

Data assembled in conjunction with McCulloh.10 Average number of laboratory staff in 110 programs responding to the 2000 Survey of 1999 Compensation. Values were determined according to the linear regression equation: #Staff = 0.47 + ([Number of IVFs]/88.5) where [Number of IVFs] was estimated by the number of embryo transfers. The correlation coefficient was 0.697 for the data in this analysis. b Data from Boone and Higdon.11 Average number of laboratory staff in 47 programs responding to their 2002 survey. Values were determined according to the linear regression equation: #Staff = 2.92 + [Number of procedures] × 0.002 where [Number of procedures] was estimated by multiplying the [Number of IVFs] by seven procedures per IVF. (Note that one IVF involves the sum of at least seven procedures: (1) a prior semen analysis, (2) identification of oocytes from follicular aspirates, (3) sperm preparation, (4) in vitro insemination, (5) fertilization scoring, (6) embryo scoring, and (7) preparation of embryos for transfer. The estimates in this table neglect the staffing needs of procedures unrelated to IVF such as sperm preparation for intrauterine inseminations and other diagnostic testing.)

Example 2 Similar analysis has been applied more recently and more directly by counting individual procedures (oocyte retrieval, insemination, ICSI, fertilization check, embryo transfer, embryo cryopreservation)11 instead of counting embryo transfers only. The relationship (Table 2.3) from the preliminary work derived by averaging 47 laboratories in the United States is that staffing requires 2.92 personnel prior to the performance of any andrology or IVF treatments. One additional staff member is required for every 500 procedures (where a procedure is defined as much less than a complete IVF treatment). In Table 2.3, a minimum of roughly seven procedures is performed in one IVF treatment. When seven procedures are involved with an ART treatment, then a staff of roughly 4.3 laboratorians is needed to perform the first 100 IVF cases (700 procedures), assuming that no other andrology activities occur. Discrepancies between these two examples of staffing norms could be due to differences in the surveyed year (199910 vs 200111), differences in the programs that responded, and/or major differences in the emphasis of the surveys. The Survey of Compensation10 was directed at determining an individual’s compensation, whereas the Boone and Higdon survey11 was directed at

assessing all of the tasks performed by personnel in programs, and focused on procedures. The equivalency of all procedures is not clear (e.g. is one determination of sperm count and motility equivalent to the performance of ICSI on all of one patient’s oocytes?). The analysis in conjunction with the Survey of Compensation10 focused on embryo transfers performed, neglecting all the ART procedures that failed to result in embryo transfer and totally neglecting the variability of andrology activities from program to program. Despite these discrepancies, the two examples provide a range of values that are now available for discussion and consideration (Table 2.3).

Quality control of procedures Uniformity of procedures and enforcement of uniform performance will aid in confirming that every procedure is performed consistently. A written protocol for each procedure performed in the facility must be available near the site of performance. Each protocol should be written in a way whereby anyone reading it could perform the procedure. Standards for written laboratory protocols have been assembled by the National Committee for Clinical Laboratory Standards (NCCLS).12 These guidelines are summarized in Table 2.4. The

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Table 2.4

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Preparing a protocola: when preparing a written protocol, include the following information

Principle and/or purpose of the test

Provide a general outline of the point of the procedure and how the procedure is performed

Specimen required for the test

Describe any instructions necessary to be certain that the specimen is collected in a way that will assure correct processing and testing

Reagents, standards, controls, media

List any materials needed to perform the procedure

Instrumentation

List any instruments to be used and any quality control procedures needed to assure that the instrument is functioning correctly

Step-by-step instructions

Carefully describe in narrative form exactly how the procedure is to be performed

Calculations

Describe how to perform any necessary calculations

Frequency and tolerance of controls

Describe any controls that should be run to assure quality of the performance of the procedure

Expected values

List expected values for the results so that the performer will know if the values are within a reasonable range

Limitations

Describe any limitations on the interpretation of the results or on the utility of the procedure

References

List sources of information that the user may wish to consult if questions arise

Effective date and schedule for review

Indicate the date that the procedure will become effective, and date(s) that it is scheduled for review

Distribution

List all persons/locations to which the procedure has been sent

Author

List the person who wrote the procedure

a

From NCCLS GP2-A3.12

protocol should include a clearly understandable, stepby-step description of exactly how to perform the procedure. Since stability is the goal of quality control, consistency of performance is the goal to be achieved by clearly describing the procedure. Procedures will probably be repeated more consistently by different individuals, when the written protocol describes precisely how to perform the procedure. In addition, all materials and equipment that are necessary should be clearly listed in the protocol. Any calculations required should be described simply and directly. Any specific limitations of the technique should be listed. References that have been used in creating the procedure should be listed. It is generally agreed that the laboratory director is responsible for creating and approving all laboratory protocols. The maintenance of a protocol that describes how to write a protocol is often overlooked. It is a simple solution to maintain a copy of NCCLS GP2-A312 or its equivalent (Table 2.4) in the laboratory. All protocols must be reviewed at least annually (and approved by the laboratory director) in order to comply with the College of American Pathologists’ guidelines. Any improvements in methodology should be instituted during this review. This may include any changes brought about following intensive quality assurance/improvement investigations. Each person responsible for performance of the procedure must be familiar with the procedure. If any changes in the procedure are made during review, then the procedure must be reviewed in turn by testing personnel. This important step assures that all

personnel know how the procedure is to be performed. This is often accomplished by requiring staff members to sign a form indicating that they have read the procedure and are aware of any changes.

Quality control of equipment We must be certain that all equipment operates within tolerable limits. This requires periodic testing to confirm proper operation. Tolerable limits should be set prior to use of the equipment, with knowledge of what values are biologically optimal for the specimens and with knowledge of the variability of the instrument. Laboratories generally have many pieces of equipment, including but not necessarily limited to incubators, microscopes, heating surfaces, heating blocks, water baths, refrigerators, freezers, controlled rate freezers, and storage dewars. The single task in the ART laboratory that is often not recognized or acknowledged and that takes the most time is the performance of daily quality control monitoring for equipment. The final publication of CLIA ’881 has amended the requirement for performance of quality control monitoring for equipment. The new standard defers to the manufacturer to define the frequency of quality control monitoring. Although the standard has changed, it is wise to perform equipment quality control monitoring at least daily (some manufacturers will suggest with each shift or with each run, which could be more frequently or less frequently). This means that each piece of equipment should be checked for proper function on at least each day of

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Table 2.5

List of equipment, parameters for QC, and frequency of QC

Equipment

Parameter for QC

Frequency of QC

Comments

Incubator

Temperature CO2 Humidity

Daily Daily Daily

Annual preventive maintenance

Heating surfaces

Temperature

Daily

Heating bath

Temperature Water level

Daily Daily

Heating block

Temperature

Daily

Microscope

Image quality

Daily

Annual preventive maintenance

CASA

Sperm count Motility (%) Motility (velocities) Morphology (%)

Daily Daily Daily Daily

Annual preventive maintenance

Controlled rate freezer

Sufficient refrigerant Start temperature Seeding temperature Final temperature

Each use Each use Each use Each use

Annual preventive maintenance

Storage dewers

Liquid N2 level

Daily

Refrigerator

Temperature

Daily

Freezer

Temperature

Daily

Heating, ventilation, and air conditioning systems

Room temperature Room humidity

Daily Daily

Thermometers

Temperature (accuracy/precision)

Periodically

pH meters

pH (accuracy/precision)

Each use (daily)

Annual preventive maintenance

Osmometers

Osmolality (accuracy/precision)

Each use (daily)

Annual preventive maintenance

Hygrometers

Humidity (accuracy/precision)

Periodically

Timers

Time (accuracy/precision)

Periodically

CO2 monitor

%CO2 (accuracy/precision)

Clean filters/humidifiers periodically

QC equipment

Fyrite should be changed every 300 determinations

QC, quality control; CASA, computerized semen analyzer.

use. Table 2.5 lists some commonly used equipment and the types of testing that should be performed. Any equipment with an ‘out-of-tolerance’ quality control value should have corrective actions performed to rectify the problem.

Daily temperature quality control Gamete/embryo temperature is one of the most important determinants of IVF success that is controlled by the laboratory. Equipment designed to maintain a particular temperature should be monitored daily using a thermometer (external to the unit) that can be traced to a standard thermometer approved by the National Institutes of Standards and Technology (NIST) or some other reliable source. It is standard practice to perform quality control monitoring as the first event in the morning. This time is chosen because most equipment has stabilized overnight. Incubator doors and refrigerator/freezer doors have not been opened, new objects have not been placed on heating surfaces

or in heating blocks or water baths. Temperatures should be determined and recorded. Comparison of the value to the tolerable limits should be made. The use of a reference thermometer of demonstrable accuracy cannot be emphasized enough, especially when calibrating the temperature of incubators. In the United States, the line voltage is generally well regulated, providing a constant source of power to the incubator. However, I have observed incubators under less stable conditions with dire consequences. ‘Backup’ power may not be identical to the power that is typically provided to the incubator. On one occasion, during a sustained, overnight power failure, incubators powered by ‘back-up’ power continued to function, indicating the same temperature with their digitally displayed temperatures. However, independent, external thermometers indicated a drop of chamber temperature by more than 1oC to values less than 36.0oC. Embryos inside the incubators maintained overnight at these low temperatures failed to undergo cell division, despite the presence of multiple

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nuclei in cells. This observation suggests that cytokinesis is more temperature sensitive than is mitosis. On another occasion, a new laboratory had set up a new incubator. The new incubator’s temperature was calibrated using an inaccurate thermometer (the only thermometer available). Several days later, it was discovered that this reference thermometer indicated a temperature of 37°C when the actual temperature inside the incubator was 41°C. This difference of 4°C was extremely detrimental to embryogenesis. On the mornings following ICSI, the incidence of normal fertilization was very low (3/26) and the incidence of oocytes with more than 2 pronuclei was very high (5/26). Expectations based on prior experience in my laboratory are 71.4% (normal fertilization) and 4.4% (more than 2 pronuclei). Further culture at this elevated temperature resulted in no cell divisions (no cytokinesis) for embryos with either 2 pronuclei or more than 2 pronuclei. Immediately after correction of the incubator temperature, normal fertilization and cell divisions occurred as expected for subsequent patients. Therefore, I must conclude that embryos display exquisite temperature sensitivity. Hence, we must calibrate the incubator temperature and maintain it at a value verified by an independent and accurate thermometer. It is important that embryos be cultured at demonstrably permissive temperatures. (It is important to distinguish between long-term developmental exposure to temperatures diverging from body temperature [~37oC] and brief departures of temperature during cryopreservation where development is hoped to be halted and then resumed upon warming. The brief exposure [a few minutes during cryopreservation and a few minutes during warming after cryopreservation] may avoid temperature-sensitive periods that could be critically affected by prolonged developmental exposure.)

Daily quality control of CO2 levels The pH of the medium is another determinant of IVF success that is controlled by the laboratory. The pH of bicarbonate buffered medium is regulated through a complex equilibrium driven by the CO2 content of the atmosphere above the medium. Equipment that controls gas concentrations (incubator with elevated CO2 levels) should be monitored daily using a method that is sensitive to CO2 levels in the range to be maintained. Several methods exist. The Fyrite device uses Bacharach solution (potassium hydroxide solution) that absorbs CO2. The partial volume of gas removed by absorption/dissolution of CO2 is measured, indicating the percentage of the gaseous volume occupied by CO2. An alternative to the Fyrite device is the use of a CO2 monitor that is typically used to monitor CO2 in exhaled gases. Both of these devices should undergo periodic quality control monitoring also, to confirm their accuracy prior to use as quality control monitoring devices. In some laboratories a supply of 5% CO2

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mixed gas is available and may serve as a calibrator for CO2 monitors. A third method of monitoring CO2 levels in the incubator is the determination of pH in culture medium maintained in the incubator. Since most culture media for embryo culture are prepared with bicarbonate as a pH buffer, the pH of the medium is controlled by equilibrium of atmospheric CO2, dissolved CO2, bicarbonate, and hydrogen ions in solution. The major purpose for maintenance of CO2 levels in the incubator is the maintenance of medium pH. Therefore it is sensible to monitor the pH of a small amount of medium that can be maintained in the incubator. This medium should be discarded after its use as a determinant of whether the CO2 levels are appropriate. The pH meter must be calibrated immediately prior to use, and pH measurements must be made rapidly to avoid off-gassing of CO2 after opening the incubator door to insert the pH probe in the medium. Among these three methods, which method is best to achieve our goals? Generally, the partial pressure of CO2 in the incubator atmosphere is maintained near 38 mmHg (5% of standard atmospheric pressure, 1 atm = 760 mmHg) in order to maintain the pH of the culture medium. There are difficulties with each method of performing quality control. The use of a Fyrite analyzer with Bacharach solution (that measures the percentage of the gas that comprises CO2) is generally accepted to have an error of roughly + 0.25– 0.50%. Care must be taken to avoid saturation of the Bacharach solution with CO2 (the solution should be replaced after ~300 determinations). Saturation will lead to underestimates of the actual %CO2. Anesthesia equipment designed to measure the CO2 in exhaled air is expensive and requires calibration. Such an instrument may not permit the display of %CO2 (the value displayed on the incubator display). Rather it may display the partial pressure of CO2 in mmHg. The partial pressure of CO2 in the atmosphere is actually the parameter that determines the dissolved CO2 in the aqueous phase. (Note that in order to maintain a partial pressure of 38 mmHg for CO2, it may be necessary to raise the %CO2 to values exceeding 5%, especially in laboratories at high altitudes, where the atmospheric pressure is reduced.) If the goal of this quality control exercise is to control the pH of the medium, then it may seem logical to perform this exercise by monitoring the pH of the medium maintained in the incubator. However, this can be fraught with problems since pH meters require daily calibration. Calibration must be performed at the incubator temperature (the difference between room temperature and incubator temperature leads to a 5% change in the slope of the voltage vs pH relationship [Nernst equation]). Secondly, an incubator door must be opened to insert the pH probe into medium that has equilibrated with the incubator’s gas mixture. The door opening will affect the very gas

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mixture that controls the pH of the solution. Although this pH measurement attempts to monitor the endpoint of the CO2 control, it is tricky to perform. In addition, the sensitivity of the medium’s pH to the %CO2 in the atmosphere surrounding the medium is rather weak. Near the desired value of roughly 5% CO2, medium pH changes only 0.1 pH unit for each 1% change of the CO2 in the atmosphere. So, given the typical precision of pH measurement (+ 0.1 pH unit) and the difficulty of performing these measurements in a closed incubator, one can only hope to achieve a precision of + 1% in the CO2 control. Therefore, once a pH measurement demonstrates a pH near the level desired, and assuming no changes in medium composition, it is more practical and precise to control the pH by controlling the %CO2 using methods that yield more precise levels of CO2 (Fyrite or calibrated anesthesia monitor), thereby controlling the pH to within roughly 0.025–0.05 pH units.

Daily quality control of computerized semen analyzers Computerized semen analyzers can be used to perform determinations of sperm concentration (sperm count), motility (percentage motile), sperm velocities, and sperm morphology (percentage normal morphology). Proper functioning for these tests should be confirmed on each day of use. Methods of performing quality control can include using a known concentration of latex beads (probably only valuable as quality control material for concentration). Unfortunately, latex spheres are neither the same size nor the same shape as sperm. Therefore, although the spheres can be counted, they are not a test of the algorithm that distinguishes sperm from nonsperm objects in semen. In addition, spherical beads do not exhibit reproducible motility. Therefore, some other mechanism must be used to perform quality control of motility. Videotaped sequences of motile and nonmotile sperm, for which the concentration is known, can be used as standards for quality control checks of concentration and motility. The tapes should be created using the computerized semen analyzer to generate the images to be stored on videotape. An additional possible source would be the production of digital video recordings on digital videodisks. Primary calibration of the material used to create these recorded sequences should be performed prior to their use as quality control standards. Likewise, video records can be generated (with videotape, DVD, or digital still images) using sperm stained for morphology determinations. Such images can be used as quality control images for the computerized semen analyzer. These images should be evaluated as primary standards prior to use as quality control standards. Prestained sperm smear slides are available commercially that are created from samples evaluated by well-known authorities on morphology.

These can serve as primary sources for calibration of computerized semen analyzers, or as controls for periodic quality control of personnel who perform sperm morphology determinations.

Daily quality control of microscopes Microscopes are used daily during diagnostic testing in the andrology laboratory and daily during therapeutic procedures in both the andrology and embryology laboratories. Clarity of images should be confirmed at least daily and should be documented. Procedures for alignment of the optical path and for alignment of any apertures and analyzers should be available near the microscope laboratory bench. Any lack of clarity should be noted and rectified, to be certain that consistency in specimen analysis and procedure performance is maintained. This is generally noticed easily by microscope users.

Periodic review of daily quality control records Daily quality control records should be reviewed periodically by supervisory personnel to confirm that first, data were collected, secondly, corrective actions were taken when needed, and thirdly, corrective actions rectified any problem(s). In addition to daily quality control, equipment should be serviced at regular intervals to prolong its useful lifetime and to prevent any calamitous equipment failures. Records of this preventive maintenance must be kept to demonstrate the performance of these crucial functions. This can be done with the annual preventive maintenance. Automated equipment should be serviced routinely to assure that it is functioning to manufacturers’ standards. Microscopes should be cleaned routinely to eliminate any distortionproducing imperfections within the optical path. There are requirements that the electrical integrity and polarity of electrical equipment be checked at least annually (Occupational Safety and Health Administration). An additional method of confirmation that equipment is functioning correctly is the performance of proficiency testing two or three times per year. Enrollment in a proficiency testing program is available for many commonly performed tests and is a means of performing testing on unknown samples. In the United States, proficiency testing samples are available for sperm count, sperm motility (%), sperm morphology, sperm viability, antisperm antibody detection, culture media toxicity testing, and pH and osmolality determinations. Samples should be treated as they would be for standard patient testing. Results are submitted to the proficiency testing agency and are compared with the results of other laboratories. Proficiency testing exercises provide an opportunity to detect equipment malfunction; but these events are designed to test more than the equipment alone.

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A comparison of your laboratory’s results should be made with the published outcome of other laboratories. This comparison permits you to assess how well your laboratory is performing relative to the other participating laboratories using the same methodology. Documentation of this review is necessary for compliance with accreditation authorities.

Quality control of computers As we move forward and adopt the use of electronic records for patient data and quality control data, we are obliged to perform quality control monitoring on the computers and computer systems used for data entry and storage. Prior to use for data storage, the computer system must be assessed for its ability to handle the task(s) it is planned to perform. Does the system have enough data storage capability? Will it be sufficient to support growth of the practice? Can it function rapidly enough to meet the needs of the personnel using it? Mechanisms for keeping records of problems encountered, maintenance, and upgrades must be implemented. There should be a mechanism to demonstrate that data will not be corrupted through long-term use. Prior to use of the computer system for generation of patient reports using stored data, the ability of the report to present the correct information must be verified and documented. Records of personnel entering data and making changes to any report should be maintained. Any calculations performed must be verified as accurate and must be documented. The accuracy of reports generated from remote locations must be verified and documented. If there are any changes in reported values or with reference ranges, it must be verified that any report should be printed with the reference range used at the time that the test was performed. Patient confidentiality must be protected.13 Access to computer data must be restricted so that no unauthorized user may inspect any patient data. This is most readily accomplished by implementation of restricted access requiring authorized users to enter passwords to gain access to computer operating systems. Further levels of security may be implemented by requiring further password-protected log-ins to sensitive data. Passwords should be changed periodically. Monitors displaying data should be situated so that unauthorized persons cannot inspect the displayed data. In addition, displays that go unused for a brief period of time should be logged off or locked out (requiring password use to re-enter), preventing casual, unauthorized users from gaining access to privileged information. Imagine the person-hours required to enter all the data in your system. Data entry is of such great value that it is clearly reasonable to create back-up copies of the already-entered data at regular intervals so that in the inevitable event that the data is lost or is corrupted, it may be restored. Upon restoration, the data

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will return to its state at the time of back-up. You will need to enter (again) any data that had been entered since the back-up operation. Clearly, less re-entry is necessary when the calamity occurs close in time to the back-up event. Therefore, it is wise to perform back-up operations frequently. Frequent back-up assures that data loss occurs shortly after the most recent back-up. Data back-up varies with the type of computer system. A small laboratory database may fit on one floppy disk. Larger practice databases may require more space for storage and back-up such as streaming tape or compact disks. Some systems perform automatic back-up procedures daily, making copies to several internal magnetic disks and to removable media. Removable media should be removed following each back-up operation and stored in a remote, safe place where the data will not be corrupted by any event that might corrupt the original copy of the data. An additional alternative is to hire a company to perform remote data back-up. These companies can back-up your data automatically over telephone lines or via internet connections that are rapid and secure, but may require encryption of data to assure privacy. Since the companies are not located at your site, they provide added safety of remote storage that is not likely to be affected by a local event that could destroy data. Data restoration times may vary with these different back-up techniques; however, the convenience of having back-up copies may far outweigh the inconvenience of a longer wait prior to data restoration.

Quality control of materials and supplies It should be confirmed that gametes and embryos are never exposed to a substance that will deleteriously impact on their development. Exposure can occur during gamete acquisition, via culture in media, or via plasticware with which the medium has come in contact, or via any airborne volatile or nonvolatile agent that may affect the culture material either directly or indirectly. The effects of electromagnetic radiation are not clearly defined. The purpose of performing quality control for materials and supplies is to determine whether these materials bring some ‘toxic’ substance into the laboratory or into the culture milieu. Any material introduced into the laboratory or culture system that is associated with diminished development should probably be removed from use during human gamete preparation or embryo culture. Gametes and embryos are maintained in culture medium throughout their duration in the ART laboratory. The medium must sustain gametes and support optimal development of embryos. This may be tested using an embryo development assay (usually using mouse embryos – see ‘Process testing’, below). The manufacturer tests commercially available IVF media

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Table 2.6 Contact materials and availability of prior mouse embryo testing

Item Culture medium Culture dishes Embryo transfer catheters Pipettes, serological Pipettes, micropipettes for moving embryos Pipettes, Pasteur Microtools for ICSI, AZH, and holding Centrifuge tubes Culture tubes Pipette tips Gases for maintenance of CO2 levels Filters for gases or medium

Available with prior mouse embryo assay? Yes Yes Yes No, requires testing Yes Yes Yes No, requires testing No, requires testing Yes No, requires testing No, requires testing

ICSI, intracytoplasmic sperm injection; AZH, assisted zona hatching.

and provides the results of their testing with the shipment of media. It may be advisable to test media upon arrival to confirm that nothing occurred during shipping that affected the medium. Most maintenance and culture of human gametes and embryos is performed in plasticware. Great variability exists between different manufacturers of certain plasticware as well as between different lots of the same item.14 However, rinsing the plasticware with medium prior to use can greatly diminish any toxic effects.14 It is now possible to purchase plasticware products that have already tested non-toxic using a mouse embryo assay. Manufacturers are testing more and more contact materials prior to distribution to assure that they are not toxic (Table 2.6). The results of toxicity testing are generally included with the shipment. Gametes and embryos are maintained in plastic containers within incubators during most of their time in the laboratory. The incubator environment is controlled by maintenance of temperature and gas concentrations. Many incubators regulate the partial pressure of CO2 inside the incubator. The gas tanks used to supply gases for use in incubators may have high levels of toxicants, of which the suppliers may be unaware.15,16 Although 4–7% of the incubator environment is CO2, the remaining 93–96% is ambient air from within the laboratory that is circulated into the incubator by a fan. The quality of the ambient air can vary from laboratory to laboratory and within the same laboratory from time to time. There have even been suggestions that success can vary from shelf to shelf within the same incubator and from position to position within the same controlled-rate freezer (Boone, pers comm).

The field of airborne toxicants is beginning to grow. Airborne toxic agents fall into several classes: volatile compounds dissolved in the atmosphere and particulate agents that are suspended in the atmosphere. Paint applied to ceilings or walls in the embryology area or even in a distant location may expose gametes/embryos to toxic fumes. The same is true for construction adhesives.15 Particulate agents can be filtered from the atmosphere; however, volatile agents dissolved in the atmosphere are much more difficult to remove. Carbon filters are capable of absorbing some dissolved agents. Gametes and embryos do not have purification systems such as lungs and livers to detoxify their environments, so we must be very careful what we expose gametes and embryos to. Maintenance of a low content of particulates17 and low concentration of volatile organic compounds15,16 is associated with improved incidence of pregnancy. The use of an oil overlay, covering the medium used for ART procedures, may be an effective way of detoxifying the drops of medium that the oil overlays. Any toxicants with a high oil–water partition coefficient will be more concentrated in the oil overlay phase than in the water (culture medium) phase. This can help to remove lipid-soluble toxicants from the aqueous medium. In addition, the presence of an oil overlay creates an oil barrier between the atmosphere and the aqueous medium phase, thereby decreasing the likelihood that water-soluble or particulate toxicants will ever achieve high concentrations in the aqueous (medium) phase. In addition to the presumed advantage of decreased gas diffusion (decreased loss of CO2), the oil overlay may also be advantageous in slowing toxic agent accumulation in the medium. Although it may be considered a barrier to penetration, the oil overlay may really act only to slow the approach of equilibrium of toxicant between the ambient atmosphere and the culture medium. In the past several years, more and more products for human IVF and embryo culture have become available that have been manufactured to more exacting standards, as required by the FDA.4 Most of the contact materials for the IVF/embryology laboratory can be purchased having already undergone toxicity testing (Table 2.6) (usually by a mouse embryo assay). This may simplify our lives; however, one always wonders if the manufacturers are using as stringent conditions for the assay as we would in our own laboratories. We also question whether there could be exposure or accumulation of toxic substances during shipping (do Styrofoam packing peanuts impart styrene to previously packaged and tested materials?).

Process testing Once we are certain that all personnel are sufficiently educated and trained, all procedures are intact, all equipment is functioning according to predetermined standards, and all contact materials are confirmed to

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be nontoxic, we still would like to be able to confirm that the entire process of gamete handling and embryo culture can be performed in a way that does not harm the gametes/embryos. Although there are no perfect tests of the conditions for culture of human gametes and embryos, various survival/development assays have been used for just this purpose. The three major tests are the hamster sperm survival assay, the human sperm survival assay, and the mouse embryo assay. All of these assays challenge the ensemble of personnel, procedures, equipment, and materials in order to assess the ability of the program to sustain sperm or achieve highly developed embryos. It is assumed that the optimal conditions for these assays mimic the conditions that are optimal for maintenance of human gametes and culture of human embryos. Unfortunately, there is very little standardization of these assays, and results in one laboratory may not be the same as results in another laboratory for a variety of reasons.

Hamster sperm survival An assay using hamster sperm was developed in the late 1980s as a method of testing toxicity of media and contact materials.18,19 The assay was capable of detecting toxicity under some circumstances in which the toxicity was not detected in a mouse embryo assay. Use of this test has not flourished, in part owing to the unavailability of hamster sperm in most andrology/ embryology laboratories.

Human sperm survival An assay utilizing human sperm was developed in the late 1980s as a method of testing toxicity of media and contact materials,20,21 and is still in use.22 Advantages of the assay are that it assesses the ability of the sperm that we use clinically to remain motile. In addition, sperm are readily available in andrology laboratories. Disadvantages include that there is little standardization of the human sperm survival assay. The endpoint can be maintenance of sperm motility or sperm viability (generally assessed using a vital stain). These two endpoints are distinctly different and results may vary dependent upon the endpoint. The duration of exposure varies widely. The exposure to medium or contact material may be in open containers or under oil in the incubator. Oil may act as a sink for lipid-soluble toxicants. Similarly, sperm lipid may act as a sink for lipid-soluble toxicants, resulting in a sensitivity that is inversely dependent upon sperm concentration. However, there may be more fundamental issues with use of human sperm as a toxicity detection system. Human sperm may remain motile under conditions that do not favor fertilization or embryo development. Conditions that favor sperm capacitation (presumed to favor fertilization) may adversely affect the longevity of sperm motility and survival. More directly, optimization of conditions for maintenance of

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sperm survival may not deliver optimization of conditions for sperm that will fertilize oocytes, and most likely will not yield conditions that are optimal for embryo culture. However, use of the human sperm survival assay may be relatively valuable in testing the conditions for sperm treatment and preparation.

Mouse embryo assay The mouse embryo assay (MEA) involves the culture of mouse embryos in medium maintained in an incubator from early stages to stages just prior to implantation. Quantitation is generally in the form of a percentage of the starting embryos that achieve the advanced stage used for scoring. Extreme variability in results is often associated with lack of standardization. In some laboratories, only freshly created embryos are used, while in others, only cryopreserved and thawed embryos are used. In various laboratories, the starting material can be unfertilized oocytes and sperm, 1-cell zygotes, or 2-cell embryos. Further variability results from differing endpoints: presence of a blastocelic cavity, expanding or expanded blastocysts, or hatching or completely hatched blastocysts.23 In addition, the assay may be tailored to different stringencies and sensitivities, through various adjustments, including use of different strains of mouse for gamete sources; use of zona-free versus zona-intact oocytes and embryos;24 use of protein supplementation versus no protein supplementation; use of an oil overlay versus open culture. The use of oil overlay can help to protect embryos from lipid-soluble toxicants,25 and the presence of protein supplementation can also be protective. The mouse embryo assay has been used to detect differences in toxicity of media created using different sources of water (tap water versus type I water); different formulations of medium;26 tests of medium additives such as maternal serum;27 tests of a cultureware coating agent, Matrigel;28 as well as the detection of toxic effects attributable to endotoxin;29 and sterilizing agents (Cidex and ethylene oxide).30 Mouse embryo development in the culture conditions tested using the mouse embryo assay is not necessarily a clear indication of the toxicity or safety of the conditions for culture of human oocytes and embryos. Some investigators have indicated that the results of the MEA do not correlate with the outcome of human embryo culture,31 but may be indicative of the ability to support oocyte fertilization without any predictive value for pregnancy.32 A long-standing discussion within the field is whether mouse embryos are more sensitive or less sensitive than human embryos to toxic or suboptimal conditions.33 One possible approach is to test all contact materials exposed to sperm with a sperm survival assay, and test all contact materials exposed to oocytes and/or embryos with an embryo development assay. There is no single test that can assess the conditions for gamete

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survival, maintenance of fertilizability, and embryogenesis;34 however, the use of a battery of tests35 may help to detect pertinent suboptimal conditions.

Quality control of the assisted reproductive technology Despite demonstration of all aspects of the ART process, by performance of quality control for every employee, every procedure, every piece of equipment, and all materials and supplies, and following the use of survival/development assays, it is necessary to confirm that the clinical treatment procedures as they are applied to patients are resulting in an acceptable incidence of pregnancy for the treated patients. A target for the incidence of pregnancy should be determined prior to the onset of treatment. Treatment of patients should continue only as long as the incidence of pregnancy continues to remain at or above the predetermined target. Any failure to maintain success above the predetermined level requires the institution of quality assurance procedures. It may be considered questionable ethically to use patient material as testing material for quality control exercises; however, it would be equally questionable not to cease treatments when success drops below a predetermined level of success. Further, it is difficult to determine what will result in an improvement without performing patient treatments. Similar standards for several outcome parameters should be monitored to determine that all procedures are succeeding. Among these could be the recovery of sperm during sperm processing for insemination, incidence of fertilization following standard in vitro insemination, incidence of fertilization following intracytoplasmic sperm injection, incidence of embryo development to an acceptable stage on day 3 or day 5, incidence of survival following cryopreservation, or incidence of implantation (per embryo transferred). Once data for success as well as all quality control parameters have been entered as data, quality assurance/improvement analysis of any trends can be conducted.6,9,36

Summary Quality control procedures are performed in order to confirm that all aspects of the program are operating as expected. By careful performance of quality control, the program will continue to operate under stable conditions that should help to result in stable, repeatable results for patients. Critical features that should be performed are listed in this chapter. Experienced practitioners realize that achievement of fertilization and transfer of attractive embryos will not guarantee pregnancy. There are many biological processes in the establishment of pregnancy that remain poorly understood. While improvements of

our treatment paradigms fall into the domain of quality assurance or quality improvement, monitoring success is a quality control task. Monitoring success is necessary to have the data available to attempt improvements. Each quality control parameter measured should be recorded so that it is documented for use when unexpected events occur. The maintenance of this data is necessary to simplify the analytical process that can be used to institute improvements in the program. I believe that quality control will continue to evolve in the future, including more widespread use of quality control monitoring for all tests and procedures, even those that are not considered laboratory procedures, such as ultrasound measurements. More and more programs will store their quality control data electronically, thereby simplifying the analysis process. The recent FDA regulations have led to more widespread availability of materials and supplies that can be purchased after testing for gamete and embryo toxicity. I also expect that in the near future daily quality control will become more automated. Temperatures can be monitored digitally by sensors and be logged into a database remotely. This could also be designed to monitor quality control parameters more frequently, perhaps hourly or every 10 minutes, to assure that quality control values are more verifiably constant. I believe that proficiency testing will improve to the point that it will be available for all tests performed in the andrology/embryology laboratory. In addition, the use of proficiency testing will be more clearly matched to test the appropriate process to be monitored, whether it is a test of the personnel or a test of the conditions, and will be designed to provide proficiency testing for the clinical components in addition to the laboratory component. As the success of assisted reproduction procedures improves, the methods of performing quality control will also evolve. Within the next decade, ART programs will become more uniform in their ability to provide a high incidence of success (pregnancy or delivery). When most programs can offer a high incidence of pregnancy, issues of patient satisfaction other than simple measures of pregnancy outcomes will become much more important in affecting patients’ decisions about which program to chose for treatment. As this evolves, patient satisfaction surveys will become more important in our assessment of the quality of the services that we provide. Until the time when all treated patients can be promised a viable pregnancy, we must continue to perform quality control activities, and careful recording of the data so that we may concentrate on improving the outcome of our procedures.

References 1. Medicare, Medicaid and CLIA Programs; Laboratory requirements relating to quality systems and certain personnel qualifications; Final rule. 42 CFR Part 493, 2003.

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Quality control 2. American Society for Reproductive Medicine. Revised minimum standards for in vitro fertilization, gamete intrafallopian transfer, and related procedures. Fertil Steril 1998; 70(Suppl 2). 3. The Fertility Clinic Success Rate and Certification Act of 1992. 42 U.S.C. 263a-1, 1992. 4. Obstetric and gynecologic devices; Reclassification and classification of medical devices used for in vitro fertilization and related assisted reproduction procedures. 21 CFR Part 884, 1998. 5. Human Cellular and Tissue-Based Products or HCT/Ps. 21 CFR Part 1271, 2001. 6. McCulloh DH. Quality control and quality assurance: record keeping and impact on ART performance and outcome. Infertil Reprod Med Clin North Am 1998; 9: 285–309. 7. Weimer KE, Anderson A, Weikert L. Quality control in the IVF laboratory. In: Gardner DK, Weissman A, Howles CM, Shoham Z, eds. Textbook of Assisted Reproductive Techniques: Laboratory and Clinical Perspectives. London: Taylor & Francis, 2001: 27–33. 8. McCulloh DH. Quality control: maintaining stability in the laboratory. In: Gardner DK, Weissman A, Howles CM, Shoham Z, eds. Textbook of Assisted Reproductive Techniques: Laboratory and Clinical Perspectives, 2nd edn. London: Taylor & Francis, 2004: 25–39. 9. McCulloh DH. Quality assurance in the ART program: can we learn anything from it? Presented at In Vitro Fertilization and Embryo Transfer: A Comprehensive Update – 2001: Minisymposium on the IVF Laboratory, Santa Barbara, UCLA School of Medicine, 2001. 10. McCulloh DH. 2000 Survey of 1999 Compensation. Reproductive Laboratory Technology Professional Group and Reproductive Biology Professional Group of the American Society for Reproductive Medicine, Birmingham, 2000. 11. Boone WR, Higdon HL. Time and staffing issues as they relate to assisted reproductive technology (ART) laboratories in the US. Bull Am Assoc Bioanalysts 2003; 47: 1–7. 12. NCCLS. Clinical Laboratory Technical Procedure Manuals, 3rd edn. Approved guideline GP2-A3, 1996. 13. United States Department of Health and Human Services. OCR Privacy Brief: Summary of the HIPAA Privacy Rule. http://www.hhs.gov/ocr/hipaa, revised May 2003. 14. Boone WR, Shapiro SS. Quality control in the in vitro fertilization laboratory. Theriogenology 1990; 33: 23–50. 15. Cohen J, Gilligan A, Willadsen S. Culture and quality control of embryos. Hum Reprod 1998; 13(Suppl 3): 137–44. 16. Hall J, Gilligan A, Schimmel T, Cecchi M, Cohen J. The origin, effects and control of air pollution in laboratories used for human embryo culture. Hum Reprod 1998; 13(Suppl 4): 146–55. 17. Boone WR, Johnson JE, Locke AJ, Crane MM, Price TM. Control of air quality in an assisted reproductive technology laboratory. Fertil Steril 1999; 71: 150–4. 18. Rinehart JS, Bavister BD, Gerrity M. Quality control in the in vitro fertilization laboratory: comparison of

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bioassay systems for water quality. J In Vitro Fertil Embryo Transf 1988; 5: 335–42. Gorrill MJ, Rinehart JS, Tamhane AC, Gerrity M. Comparison of the hamster sperm motility assay to the mouse one-cell and two-cell embryo bioassays as quality control tests for in vitro fertilization. Fertil Steril 1991; 55: 345–54. Critchlow JD, Matson PL, Newman MC, et al. Quality control in an in vitro fertilization laboratory: use of human sperm survival studies. Hum Reprod 1989; 4: 545–9. Claassens OE, Wehr JB, Harrison KL. Optimizing sensitivity of the human sperm motility assay for embryo toxicity testing. Hum Reprod 2000; 15: 1586–91. DeJonge CJ, Centola GM, Reed ML, et al. Human sperm survival assay as a bioassay for the assisted reproductive technologies laboratory. J Androl 2003; 24: 16–18. Svalander P, Anderson E, Hyllner J, et al. Quality assurance methods for production of culture media and equipment essential for high success rate. In: Maximizing the Potential of Every Embryo to Minimize Multiple Embryo Transfer, Textbook for the Postgraduate Course at the Meeting of the American Society for Reproductive Medicine. American Society for Reproductive Medicine, San Francisco, October 1998: 1–15. Fleetham JA, Pattinson HA, Mortimer D. The mouse embryo culture system: improving the sensitivity for use as a quality control assay for human in vitro fertilization. Fertil Steril 1993; 59: 192–6. Quinn PJ. AAB embryology proficiency testing (PT) surveys as a tool to distinguish variables affecting outcomes. Presented at the AAB College of Reproductive Biology Seventh Annual Symposium, Broomfield, CO, June 2003. Summers MC, Bhatnagar PR, Lawitts JA, Biggers JD. Fertilization in vitro of mouse ova from inbred and outbred strains: complete preimplantation embryo development in glucose-supplemented KSOM. Biol Reprod 1995; 53: 431–7. Deaton JL, Dempsey RA, Miller KA. Serum from women with polycystic ovary syndrome inhibits fertilization and embryonic development in the murine in vitro fertilization model. Fertil Steril 1996; 65: 1224–8. Dawson KM, Baltz JM, Claman P. Culture with Matrigel inhibits development of mouse zygotes. J Assist Reprod Genet 1997; 14: 543–8. Dubin NH, Bornstein DR, Gong Y. Use of endotoxin as a positive (toxic) control in the mouse embryo assay. J Assist Reprod Genet 1995; 12: 147–52. Ackerman SB, Stokes GL, Swanson RJ, Taylor SP, Fenwick L. Toxicity testing for human in vitro fertilization programs. J In Vitro Fertil Embryo Transf 1985; 2: 132-7. Clarke RN, Griffin PM, Biggers JD. Screening of maternal sera using a mouse embryo culture assay is not predictive of human embryo development or IVF outcome. J Assist Reprod Genet 1995; 12: 20–5. van den Bergh M, Baszo I, Diramane J, et al. Quality control in IVF with mouse bioassays: a four years’ experience. J Assist Reprod Genet 1996; 13: 733–8. Quinn P, Horstman FC. Is the mouse a good model for the human with respect to the development of

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the preimplantation embryo in vitro? Hum Reprod 1998; 13: 173–83. 34. Muller CH. The andrology laboratory in an assisted reproductive technologies program. Quality assurance and laboratory methodology. J Androl 1992; 13: 349–60. 35. Scott L, Smith S. Mouse in vitro fertilzation, embryo development and viability, human sperm motility in

substances used for human sperm preparation for assisted reproduction. Fertil Steril 1997; 67: 372–81. 36. McCulloh DH. Quality control and quality assurance: a means of improving and stabilizing IVF results. Presented at In Vitro Fertilization and Embryo Transfer: A Comprehensive Update – 2003: Minisymposium on the IVF Laboratory, Santa Barbara, UCLA School of Medicine, July 2003.

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3 The ART laboratory in the era of ISO 1000 and GLP Cecilia Sjöblom, Christoph Keck

International Standardization Organization (ISO) definition of accreditation: ‘Procedure by which an authoritative body gives formal recognition that a body or person, is competent to carry out specific tasks’ (ISO/IEC Guide 2)

Introduction Quality assurance, quality control, and accreditation are concepts that seem to touch on a wide range of functions in our society. Quality control (QC) systems are especially needed in units for Assisted Reproductive Technologies (ART) to assure reproducibility of all methods and have competence in all duties performed by the personnel. The necessity of a quality control system becomes even clearer when considering the possible risks of ART. Over the years that ART have been practiced, in both large and small clinics, much knowledge has been gained on how to run an ART laboratory, and what methods to use in order to achieve ultimate success. Facing the future, we encounter other variables such as the safety and efficiency of the laboratory and QC becomes a key feature. Professional national and international guidelines on how ART should be performed have been established over the years, and many countries have legislation concerning how ART should be practiced.1–3 Among others, England and the United States have instituted a system whereby the ART clinics have to be authorized to practice these techniques. 4,5 In such systems, the clinic as well as the laboratory can be audited by this third-party authority in order to assure correct practice.6

European and international principles for accreditation The European Union (EU) has a goal to establish harmonized principles for the assessment of laboratories,

certification, and inspection bodies. When the EU principles for conformity assessment were developed, it was clear at an early stage that some sort of quality assurance between the bodies, preferably accreditation bodies, was needed to assess the competence of laboratories and certification and inspection bodies. The European Commission considered that it was very important to create a system that could safeguard the quality assurance of such bodies, active both within the voluntary and mandatory sectors. The European Commission, therefore, took the initiative in bringing together the national accreditation bodies in order to formalize their cooperation. European organizations for cooperation between accreditation bodies and their predecessors have existed in Europe since the mid-1970s, and in November 1997 the European Cooperation for Accreditation of Laboratories (EAL) and the European Accreditation of Certification (EAC) merged to form the EA, or the European Cooperation for Accreditation. The EA covers accreditation in all fields of conformity assessment activities.7 The European Commission states that accreditation must be a transparent, independent, and noncommercial activity. If these requirements can be assured, it would ensure that accreditation would be the last level of control of technical competence. It further states that accreditation should be considered as the most favored technical basis for assessment of the competence of technical actors.8 At present the EA has 35 full members representing 33 European countries. Sixteen non-European accreditation bodies have signed a contract of cooperation with the EA, out of which 9 have entered into a bilateral agreement with the EA, which, as far as recognition and mutual acceptance are concerned, conveys the same rights and duties and benefits as the EA multilateral agreements.9 To make accreditation effective across borders, all EA members must apply the same standard of assessment to the laboratories, certification, and inspection bodies, etc. To ensure that this is happening, EA members can apply for peer-group evaluation of their

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activities by the other members. The EA works mainly within the framework of the two international cooperation organizations for accreditation bodies – the International Laboratory Accreditation Cooperation (ILAC) and the International Accreditation Forum (IAF). A comprehensive program of inter-laboratory comparisons supports mutual confidence in laboratory accreditation. Accreditation, therefore, has a part to play in the mutual recognition agreements on conformity assessment. As a consequence of this, accreditation systems are established in countries throughout the world, and these accreditation bodies cooperate in regional bodies, such as the EA. On the global level, the regional bodies as well as the national accreditation bodies cooperate in international organizations such as ILAC and IAF. This structure ensures harmonized procedures for conformity assessment activities all over the world. Within the framework of IAF, a worldwide multilateral agreement, in the area of certification of quality management systems (ISO 9000), has been signed by the EA as a regional body and national accreditation bodies from all parts of the world.

Standards ISO 9001:200010 is the most widely used standard in ART clinics and covers the demands of the quality system of the whole organization. This standard covers the need for quality management and the provision of resources (both personnel and equipment), and in this standard a substantial section involves customer satisfaction and how to improve service (see Chapter 33).11 During an audit according to ISO 9001, the quality system and the different parts of the clinic such as personnel, finance, and sales is audited. The technical part of the activities is not specifically audited. One could say that the audit is broad, but not specialized. ISO 17025:200512 is the required document used within Europe for laboratory accreditation. An audit of a laboratory covers both the quality system and the layout of the technical part of the activities, including validations of methods and calibration of equipment. The audits are considered specialized. ISO 17025:2005 is based on the European norm (EN) 45001,13 and was originally modeled on the corresponding ISO/International Electrotechnical Commission (IEC) guide.14 Over time, however, these three standards have shown a number of differences. The bodies responsible for developing standards in Europe – Comité Européen de Normalisation (CEN) and the International Standardization Organization (ISO) – realized the danger of developing standards which for a given field are not identical. At the end of 1999 the new standard ISO 17025:1999 was issued,15 which covered both the EN 45001 and ISO/IEC Guide 25, aiming at total harmonization of the existing guides and standards. This standard was further updated in 2005.12

The more recently issued standard for medical laboratories is ISO 15189:2007,16 which is used by government agencies and professional organizations for the accreditation of medical laboratories. ISO 15189:2007 brings together the quality system requirements of ISO 9001, the competency requirements of ISO/IEC 17025:1999, and addresses the specific needs of medical laboratories. Most clinical testing laboratories in Europe are accredited according to ISO 17025:2005 or ISO 15189:2007. There is considerable experience within the European accreditation bodies for auditing according to ISO 17025:2005 in medical disciplines, whereas authorities have struggled with education of lead auditors for the newer ISO 15189. Upon seeking internationally approved accreditation for an in vitro fertilization (IVF) laboratory, both standards can be used; however it is well worth inquiring with the specific national accreditation body what standard they prefer and have experience in auditing to. The success of a medical treatment often depends on the reliability of the result from a medical laboratory. Furthermore, medical laboratories are required to increase the number of analytic procedures and shorten the turnaround time from test request to the reporting of results.17 ISO 15189:2007 addresses the need for equivalency of quality management systems and competency requirements between laboratories. The need for this becomes more obvious at a time when potential and actual patients are increasingly mobile – the systems to collect medical data on these individuals must be standardized independently from their location – and there are similar requirements around the world to improve quality and control costs in healthcare. There are differences between ISO 15189:2007 and ISO 17025:2005. The ISO 15189:2007 focuses on patient outcome without downgrading the need for accuracy, and emphasizes not only the quality of the measurement but also the total service provided by a medical laboratory (e.g. consultation, turnaround time, cost-effectiveness). The language and terms are familiar in the profession, and it highlights important features of pre- and post-investigational issues together with addressing ethics and the information needs of the medical laboratory. Other standards that are less suitable for the IVF laboratory are the Good Manufacturing Practice/Good Laboratory Practice guides (GMP/GLP). These standards apply to research laboratories and the pharmaceutical production industry. They include demands on the laboratory facilities that will be difficult to meet with the limited resources that many IVF clinics face.18,19

The EU Tissues and Cells Directive The increase in use, donation, and storage of human tissue has led to the creation of directives from the

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European Council. In March 2004 the European parliament issued a new version of the directive on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells, usually called the EU Tissues and Cells Directive.20 There have been previous versions of the tissue directive; however, this is the first time gametes and embryos were included. At first, the stringent demands of the tissue directive and its enforcement caused panic within the IVF community. There were general fears over high demands of air quality in the IVF laboratory, incurring costs that would be hard for some small and midsized centers to cover. The panic was followed by confusion, but with the directive and its two technical annexes21,22 now being in full force, many IVF centers in Europe have either already implemented the directive or are well under way in doing so. ESHRE (European Society for Human Reproduction and Embryology) have issued a position paper on the EU tissues and cells directive,23 outlining their standing point with regards to the application of the directive in ART. In it, ESHRE argue that the air quality in the laboratory should be less stringent than what has been recommended (GMP grade A with grade D background), with reference to historical data suggesting that it has been both demonstrated and documented that the environment IVF laboratories have had in the past achieved the quality and safety required for the intended purpose. However, it is important to underline that, regardless of ESHRE’s recommendations, each EU country will interpret the directive differently and in the UK the Human Fertilization and Embryology Authority (HFEA) Code of Practice (CoP) have set the air quality requirements to grade B with a grade D background.24 Further problematic areas of the EU directive identified in the ESHRE position paper include the frequency of screening of patients, insuring certain staff levels, and the training of personnel (clinical embryologists). However, one part of the EU directive is very clear: the demand for a quality system. The directive states that ‘Tissue establishments shall take all necessary measures to ensure that the quality system includes at least the following documentation: standard operating procedures, guidelines training and reference manuals.’ Certainly, by achieving accreditation to either ISO 17025 or ISO 15189, this demand will be fulfilled together with several other demands of the directive.

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quality system requirements of both standards are based on ISO 9001:2000. Consequently, laboratories within ISO 9001 certified clinics seeking accreditation will have major parts of the system requirements of the two laboratory standards already in place. It could be recommended that the first step towards accreditation is to get the clinic certified to ISO 9001 (see Chapter 33). The requirements discussed throughout the continuation of this chapter will be for laboratory accreditation to ISO 17025 and ISO 15189 on top of (over and above) what is already required for certification to ISO 9001. For example, scope, organization, and document control are found in all the standards, and many of the demands are the same, but the requirements discussed in these sections below will be what ISO 17025 and ISO 15189 demand in addition to what has already been implemented through ISO 9001 certification.

The accreditation task force The next step to take towards an accreditation is to make sure that everyone in the organization wants to achieve the same goal. The full understanding of how everyone benefits from an accreditation will make the process easier. A good way to start is to have a staff meeting where the different components of an accreditation are explained together with a brief explanation of the standard itself. The most frequent mistake organizations make when trying to implement a quality control system is not to involve everyone. Divide the project into smaller sections and give out personal responsibilities enabling all staff to be included in the preparation work. This will also make the implementation easier. A suggestion of how to divide the project and put together a ‘task force’ is given in Appendix 3.1. A good way to make sure that all demands in the standard are covered is to make up a table of contents – using the ISO 17025:2005 standard table of contents as a template (Table 3.1). An assessment can then be made of what needs to be added to the quality manual and other documentation. It is important to note that even if all the standards have demands for management structure, internal audit or document control, the laboratory standards have some more specified demands not found in ISO 9001 and these need to be added to the specific procedures.

Scope

The accreditation process for ISO 17025:2005 and ISO 15189:2007 It is important to underline that in no way are all the quality standards independent of each other. ISO 17025:2005 is basically the same standard as ISO 15189:2007, with the major difference being the medical laboratory terminology used in ISO 15189. The

An important feature is to outline the areas covered by the certification and/or accreditation. An accreditation, according to ISO 17025:2005 or ISO 15189:2007, is aimed towards accrediting methods. Not all methods performed in the laboratory have to be accredited for the laboratory to be called accredited, and not all methods can be accredited owing to lack of references or methods for validation. The quality manual needs to clearly outline the scope of the accreditation, listing

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Table 3.1

ISO 17025:2005 standard table of contents

4

Management requirements

4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12 4.13 4.14 4.15

Organization Management system Document control Review of requests, tenders and contracts Subcontracting of tests and calibrations Purchasing services and supplies Service to the customer Complaints Control of nonconforming testing and/or calibration work Improvement Corrective action Preventive action Control of records Internal audits Management reviews

5

Technical requirements

5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10

General Personnel Accommodation and environmental conditions Test and calibration methods and method validation Equipment Measurement traceability Sampling Handling of test and calibration items Assuring the quality of test and calibration results Reporting the results

which methods in the laboratory are covered by the accreditation and an authority-approved accreditation coefficient of variance (CV) that states the percentiles of variations acceptable within the methods.

Management requirements (Chapter 4 ISO 17025:2005 and ISO 15189:2007) Organization (ISO 17025:2005 4.1 and ISO 15189:2007 4.1) The part of organization demands in ISO 17025, not covered by ISO 9001, is the need for a technical manager. Each laboratory, or accredited laboratory process, has to have an appointed technical manager who has the overall responsibility for both laboratory procedures and the resources needed to provide high-quality service. This means that IVF laboratories with a more horizontal management structure, where a group of senior embryologists are managed by a clinical director, will have to appoint one laboratory manager or director who can take on the responsibilities of what the standard calls a technical manager. This person cannot be the clinical director, but has to be an embryologist with full competence to perform and manage all the accredited processes.

The quality system (ISO 17025:2005 4.2 and ISO 15189:2007 4.2) The laws, regulations, and legislation under which the laboratory and clinic work should be listed, with full references. The originals of these should be kept as underlying documentation accessible for all personnel and controlled through the external document procedure.

Document control (ISO 17025:2005 4.3 and ISO 15189:2007 4.3) Each system has to include a full master list of all the documentation included and controlled in the quality system. The list shall clearly state the document name, issue, and current revision status, date of approval, level, and physical location. There is no demand for a master list in ISO 9001, even if it is recommended to have one. In addition to the general demands for document control, the laboratory standards further specifies the word ‘document’ to include instructions or information such as policy statements, memoranda, textbooks, pictures, charts, posters or anything used in the laboratory to support the processes. For example, if the laboratory has a colorful poster on the wall guiding the embryologists on how to score blastocysts, it needs to be a controlled document, even if it is not generated by the laboratory itself. ISO 17025 also has specific demands for identification of changes in documents and, where practicable, all altered text shall be identified in the new issue of the document or in an attachment.

Purchasing services and supplies (ISO 17025:2005 4.6 and ISO 15189:2007 4.6) Demands of the standards All devices used in ART, such as culture media, consumables, and equipment, will affect the outcome of the treatment. The standard therefore demands that the laboratory defines and documents its routines for purchasing equipment, services, devices, etc. First, the laboratory needs to decide what its own requirements are for each type of device: for example, limits in toxicity and results from mouse embryo assays for culture media, oocyte pickup needles or plasticware. It is important to remember that it is not only what requirements you have on the devices but also you have to take into account any national, regional or local regulation which applies. The EU Tissues and Cells directive stipulates that all devices which come into contact with cells, gametes or embryos need to be tested according to the EU devices directives25,26 and be CE marked. The laboratory also has to define requirements for the safe transport of devices from supplier to the laboratory, and also how they will be inspected when they arrive to assure they meet the

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limits specified. Equipment and consumables then have to be verified before being taken into use. Some laboratories choose to culture excess embryos or undertake sperm survival assays in new batches of culture medium; however, this type of verification is not demanded by the standard and it could be argued is not really necessary. If all the devices conform to the EU devices regulation they should already have been stringently tested. ISO 17025 and ISO 15189 only demand that the laboratory actively checks the test reports issued by the manufacturer and confirm that the report complies with their own limits for use. Equipment should be verified by test runs: for example, before a new centrifuge is taken into use in the laboratory a series of mock sperm preparations have to be undertaken and documented. When the devices have been accepted for use in the laboratory, it is crucial that they are stored correctly to insure their continued suitability for use. The laboratory has to safeguard correct storage by defining the exact storage environment. Limits for temperature in refrigerators and freezers are crucial and to store culture medium in a normal kitchen refrigerator is not acceptable; however, an appropriate pharmaceutical refrigerator with temperature monitoring is (see Monitoring of laboratory parameters). The environment in general storage rooms is also important, as plasticware stored at high temperatures will not be suitable for use. All purchased supplies, reagents, and consumables should be included in the laboratory inventory. Information in the inventory shall include LOT number (batch number), date of reception, and date taken into use. The inventory for equipment should include unique identification, date of arrival, date placed in service, last calibration or service, and periodicity of service and calibration. The laboratory is required to keep a list of approved suppliers and critically evaluate all suppliers on an annual basis. The batch or LOT number of any device that comes into contact with a given patient’s gametes or embryos needs to be recorded on that individual patient’s records. It is not appropriate to have a list of batches currently used in the laboratory and draw conclusions from this using date and guesswork of what device was used for what patient.

Specific demands for the IVF laboratory The embryo culture environment is an important factor in a successful in vitro fertilization–embryo transfer (IVF–ET) program. It is the responsibility of the IVF laboratory to establish and maintain a stable, nontoxic, pathogen-free environment providing optimal conditions for fertilization and embryo development in vitro. The gametes and early embryos are extremely sensitive to minor changes in the milieu and small variations in temperature and the physical properties of media such as pH and osmolarity will inevitably affect the outcome of an IVF cycle. Embryotoxic substances in the media

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and in materials used for embryo and gamete handling should be screened for and identified before they are clinically used. There is a lot of debate around the issue of what quality control assay to use when testing devices, and different assays and variations of them to obtain increased sensitivity have been suggested.27 Many clinics today are using the ‘human embryo assay.’ Fluctuations in pregnancy rates despite a constant clinical profile of the patients being treated are the sole indicator of suboptimal culture conditions, and can indicate that there might be a problem in the laboratory. Apart from the ethical dilemma, there are two main limitations to this: namely, the time lag in culturing embryos and diagnosing pregnancy in a substantially large number of patients, and failure to identify the source of the problem. Culture of spare embryos to the blastocyst and hatched blastocyst stages is another frequently used ‘human embryo assay’ method. The problem with this approach is that these poor-quality embryos might suffer from genetic abnormalities. Conclusions from these culture results therefore are hard to draw, and again there is a problem in identifying the source of toxicity. The use of an objective quality control strategy that is independent of patient factors can prove valuable in assessing the media and laboratory ware that come into contact with the patient’s gametes and embryos. A very accurate tool is to use the mouse embryo assay, since this method, if used properly, provides the best means of testing in terms of resembling the human embryo culture system. There are two major parts of the testing system: the assay itself to detect toxicity, and the record of batch numbers to be able to identify the source of problems. Many smaller, privately owned units have neither the economic nor the laboratory resources to facilitate a standardized mouse embryo assay. Nevertheless, there should be strict limits for the acceptance of a test. The test shall be done using an appropriate strain of mice, and ‘fully hatched’ shall be the endpoint of the test. Many manufacturers state in their production sheet that their products have been mouse embryo tested, but we rarely see a certificate from this test and therefore it is important to demand a copy of this certificate before the release of a product into the laboratory. The other essential part of a good-quality system is the recording of batch numbers. A record of the batch numbers of materials used in a treatment cycle should be kept together with the patient’s case file. It is of great advantage to have a computerized case file system whereby each cycle has a batch record page attached. This page includes a full list of culture media and laboratory ware and the batches in use and, with a simple mouse-click, marks what materials were used in every step of the cycle, from culture media down to pipette tips. Together, these two parts create the foundation of an error search system. In the case of a drop in fertilization

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rate, embryo quality, and, subsequently, pregnancy rate, it is easy to obtain computer lists with these variables connected to each batch. There are occasions when batches have been approved with a testing certificate from the producer, and later, in an error search system, have proven to be embryotoxic. The first action to take in this case is to stop the use of that particular batch and then send the item for a mouse embryo assay test. If the result comes back as ‘embryotoxic’, it could indicate that transport conditions and storage times have affected embryo toxicity. So, what should be tested? This is another hot topic for discussion. Culture media and culture dishes are the obvious, but what about the rest? Is the toxicity of pipette tips really important? If automatic pipettes are used to make up dishes and micro drops, those tips are as important as the media and dishes themselves. This ranking of importance could be continued forever; a good rule of thumb is to test all culture media and laboratory ware that come into contact with the patient’s embryos and gametes.

Service to the customer (ISO 17025:2005 4.7 and ISO 15189:2007 4.7) In most IVF clinics the embryologists have none or very little contact with the ‘customer’ (the patient) and also very little input in the exact treatment options. In an accredited laboratory the standards demand that the laboratory actively provide advice on choice of treatment and clarification of any laboratory outcomes. ISO 15189 even demands that the embryologist should take part in the clinical rounds (i.e. meeting with the patients), enabling advice and guidance on embryology in general, and in individual cases.

Audits (ISO 17025:2005 4.14 and ISO 15189:2007 4.14) Audits can be internal or external, vertical or horizontal, or process oriented or system oriented; however, it is easy to get confused and caught up in terminology and miss out on the great opportunity that audits are to improve the system and our service to patients. To find nonconformities at an audit is not bad; it is proof that the system is working and we are capable of recognizing our weaknesses and faults and learn and improve on them. For general internal audit principles, see Chapter 33.

Internal audits The laboratory standards are more precise in what exactly should come out of an audit and what is needed for a correct audit process. When preparing, writing, and implementing internal audit procedures, both ISO 17025 and ISO 15189 are very precise and elaborate on what exactly is needed. The audit chapters in these standards can even be of great

help when implementing audit processes for ISO 9001 systems. In relation to ISO 9001 internal audit demands, the laboratory standards are more stringent, with how often internal audits need to be undertaken, and requires all accredited methods and procedures to be audited on an annual basis. Note also that ISO 17025 demands that additional internal audits need to be undertaken as soon as possible where identified nonconformities cast doubt on the laboratory’s compliance with its own procedures or laws or regulations (4.11.5).

External audits The National Authority for Conformity Assessment performs the external audits for accreditation to ISO 17025 and ISO 15189. When a laboratory is ready to be accredited they need to apply for accreditation and the national authority will assess whether they have the appropriate expertise to perform the audit. If not, they can seek help from other members of the European Cooperation for Accreditation who have the appropriate experienced auditors. Together with the application, the laboratory has to supply evidence of a fully compliant quality system and it is essential that all methods, which accreditation is sought for, have gone through a series of internal audits. Result documentation from these audits are supplement to the application. The accreditation body then arranges a pre-audit to assess the readiness of the laboratory and, pending the outcome of this pre-audit, an accreditation audit will be arranged. When the accreditation audit has been done, the lead auditor or any technical experts can only recommend that the laboratory gets accreditation. This recommendation is then passed on to the board of the accreditation body who will decide if the laboratory is to be awarded accreditation.

Technical requirements (Chapter 5 ISO 17025:2005 and ISO 15189:2007) Personnel (ISO 17025:2005 5.2 and ISO 15189:2007 5.1) There should be defined descriptions of the demands on all personnel groups within the laboratory in respect of education, experience, areas of responsibility, job descriptions, and where they are located in the organization plan. Everyone working in the laboratory has a responsibility to keep up to date with changes in the quality system and to take active part in the improvements. The quality manual should include documentation on how proof of competence is issued and how introduction of new personnel is performed, and the management of the laboratory should formulate goals for each member of staff with respect to further education and training. These goals should be assessed and discussed at annual appraisals, which should be documented but kept confidential. There should be

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clearly documented procedures in place for the introduction and training of new staff and the reintroduction of staff after long periods of absence or leave. In the UK, there is a formal training program for embryologists through ACE (Association of Clinical Embryologists). The program is a minimum of 2 years and has both practical and theoretical components. ACE also provides an online Continual Professional Development (CPD) scheme and has recently made membership of the Royal College of Pathologists available for its members. The HFEA further demands that embryologists who are active in the UK are state registered or working towards state registration. Formal training programs for embryologists like the one in the UK are rare; however, from 2008 ESHRE are offering an ESHRE certification for embryologists.

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inside the laboratory; further storage can be elsewhere. The equipment held in the laboratory should be limited to the absolute necessary; again the laboratory is not a storage room for old equipment.

Access rules The laboratory should have limited access ensured by use of locks, swipe card or other access control. It should also hold documentation verifying who has access to the laboratory. There should be documented and implemented rules for what is required for access to the laboratory, including demands for change of clothes and shoes, the use of hair cover and masks, and washing of hands.

Health and safety Accommodation and equipment (ISO 17025:2005 5.3, 5.5 and ISO 15189:2007 5.2, 5.3) The laboratory shall ensure that the environmental conditions do not invalidate the results or adversely affect the quality of any measurement (ISO 17025:2005 5.3.1). In simple words this means that the IVF laboratory environment has to be designed in such a way that the outcome of any procedures is optimal and not affected by environmental parameters. Live birth results following IVF treatment vary from country to country and from clinic to clinic, and often within a clinic from month to month. A general consensus is that patient demographics such as age and cause of infertility are the main factors affecting outcome. Considering a varying population of patients, it is of great importance that parameters in the laboratory are stable. Defining the environment and setting limits for acceptable working conditions will help to reduce variables and result in the patient being the only factor that varies (see Monitoring). Exactly what this encompasses will always be down to interpretation and international, national or regional regulations; however, the standards have some clear demands and some environmental factors cannot be ignored.

General laboratory layout The theater for oocyte retrieval and embryo transfer should be in close vicinity to the laboratory. The laboratory layout should further assure safe handling of gametes and embryos; small crowded laboratories impose a significant risk for accidents, resulting in loss of gametes and embryos. The laboratory shall not double as an embryologist’s office. There should be a minimal allowance for paper in the laboratory, as it can increase the amount of particles, and therefore only patient records necessary for ongoing treatment should be held in the laboratory. No cardboard should be held in the laboratory, as this imposes a high risk of fungus infections. Furthermore, the laboratory is not a storage room for disposables. Only a weekly stock of disposables should be held

The laboratory is required to ensure the safety of its entire staff. This includes providing an environment that minimizes the risk of transfer of any contagious contaminants through the use of class II biosafety cabinets when handling unscreened patients’ material.

Temperature The optimal IVF laboratory temperature is a matter of great debate; however, it has to be defined to a limited range. Some embryologists argue that an elevated laboratory temperature benefits the embryos through reduced risk of cooling during transport from the incubator to the heated stage. However, high laboratory temperatures will provide a perfect environment for microbes and contaminants. All laboratory equipment is designed to operate at room temperature, usually defined as 22 ± 2°C, and unless the laboratory can show process verification at a different temperature this range will be the one demanded by the standards. A laboratory without temperature control cannot be accredited (see Monitoring).

Air quality Another area of great debate is the demands for clean air in the laboratory and, as previously mentioned, this will also be affected by regional interpretation of the EU Tissues and Cells directive. ISO 17025 5.2.5 requires that attention be paid to sterility and presence of dust and it is highly recommended that laboratories periodically monitor the particle count and presence of volatile organic compounds in the air together with microbial monitoring through settlement plates and swabs.

General cleanliness An IVF laboratory should always be clean and the laboratory standards demand that documented frequent cleaning procedures are implemented and that cleaning is confirmed by active signatures. The use of harsh

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detergents is not recommended and cleaning should be undertaken using 70% alcohol and sterile water. A laboratory should have all the equipment needed to assure the best service. The standards require a documented program for preventive maintenance and it is the responsibility of the laboratory manager to regularly monitor and assure appropriate service, calibration, and function of all equipment. All equipment used in an accredited laboratory has to be clearly labeled with a unique identifier, date of last calibration or service, and date or expiration criteria when recalibration/service is due. Together with this, all equipment used should be included in an equipment record containing information listed in ISO 17025:2005 5.5.5 and ISO 15189:2007 5.3.4. There should be clearly documented processes for validation of equipment function before it is taken into use, as discussed previously. The standard of equipment used in IVF laboratories is generally very high, but even the best equipment can fail and not function optimally if it is not appropriately maintained. All embryologists should have solid knowledge of how to operate all equipment and there should be written implemented procedures in place for action taken if there happens to be an equipment failure. Crucial equipment such as incubators should always be connected to auto-dialers, enabling staff to promptly respond to any faults out of hours.

Sampling, pre- and post-examination procedures (ISO 17025:2005 5.7, 5.8 and ISO 15189:2007 5.4, 5.7) The laboratory standards have specific demands on how the samples, i.e. gametes and embryos, should be collected and stored. The samples have to be correctly and safely identified and any laws regulating the identification of patient samples have to be taken into account. The sample should be accompanied with a written standardized request of what procedure the sample should be used for and it should include information of (minimum) gender and date of birth (gender could be agreed to be not applicable for gametes). Date and time of collection should be noted by the patient and date and time of receipt should be recorded by the laboratory. Usually the procedures for collecting samples, preand post-examination procedures, are documented in the applicable laboratory Standard Operating Procedures (SOPs) for sperm processing and oocyte collection; however it is important to include the specific demands of the standards for these procedures and their documentation.

Methods, processes, and validation (ISO 17025:2005 5.4 and ISO 15189:2007 5.5) The quality manual should include documentation of the methods used in the laboratory and what methods are accredited. The description in the quality manual

should be very brief and refer to underlying documentation such as SOPs, method manuals, or working manuals. According to ISO 17025:2005 5.4, the laboratory should use appropriate methods and procedures for all tests and/or calibrations within its scope. This includes sampling, handling, transport storage, and preparation of the items tested. The methods should include measurement uncertainty as well as statistical techniques for analysis of test and/or calibration data. There should be clear descriptions of how to handle and operate relevant equipment, and all instructions should be available and familiar to the laboratory personnel. At the selection of methods the laboratory are obliged to choose those published as international, regional, or national standards. For IVF laboratories, the only EA (SWEDAC)-approved standard is the minimal guidelines for methods within the field of ART.28 This is a minimal standard for the methods of sperm analysis and preparation, and analysis and handling of oocytes and embryos, including scoring of these. However, this minimal standard is only accessible in Swedish and has not been updated since first formulated. When it comes to methods such as intracytoplasmic sperm injection (ICSI), and freezing and thawing of embryos, there is no existing EAapproved standard. Considering the age and restricted accessibility of the Swedish standards and the lack of any other internationally agreed minimal standards, the accreditation of methods concerning ART should follow the outlined requirements for laboratorydeveloped methods or nonstandardized methods (ISO 17025:2005 5.4.3, 5.4.4). ISO 15189:2007 has a softer approach to the accreditation methods and demands that the procedures used should meet the requirements of the users of the laboratory service, preferably using methods that have been published in established/authoritative textbooks, peer-reviewed texts, or journals. If in-house methods are used, these need to be appropriately validated for the intended use and fully documented by the laboratory. All procedures in the laboratory need to be described and documented in SOPs, method manuals or working manuals. A good SOP should follow a set format and ISO 15189:2007 5.5.3 and ISO 17025:2005 5.4.4 contain very good guides for how all SOPs layout. As with all other documentation, the SOP should be in accordance with the procedures of document control. The document or method title should be followed by a short clinical description of the method. The analytic principles shall include a theoretical description of the method and review of current literature. The SOP should outline the competence demands on personnel performing the test. Sampling and handling of test items should include the sampling procedures and the physical environmental issues such as temperature. Remember that all variables in the SOP, such as those referring to the

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measurement of temperature, have to give a precise range, followed by a description of how the temperature is measured, the accuracy of the thermometer, and how often and how it is calibrated. There should be clear descriptions of how the sample is labeled and, considering the risks associated with the work in an IVF laboratory,29 the marking should be logical and clear, to eliminate completely the risk of mixing of samples. The description of the procedural steps should be written in a noncomplicated way so that they can be easily followed by any new member of staff under supervision. External and internal controls should be applied when applicable for the method and any use of controls should be described in the SOP, together with the periodicity of the controls and descriptions of how they are performed (see Monitoring and traceability and Assuring quality sections). All equipment used for the method should be listed with references to handling instructions and calibration protocols. Any safety routines and occupational hazards involved with the method should be discussed and well known by the personnel involved. References to any textbooks or publications concerning the method should be included last. Validation is the process that confirms that the techniques and methods used in the IVF laboratory are suitable for the production of good embryos, viable pregnancies, and live birth. All methods have to be validated regularly, and the SOP should include information on how often and how validations are done, and should be in accordance with ISO 17025:2005 5.4.5. The EU Tissues and Cells Directive includes demands for validation and in the UK, HFEA CoP24 requires that all processes in the IVF laboratory should be validated. Some methods and techniques used in the laboratory can be difficult to validate, and it is acceptable to use retrospective analysis of fertilization, damage, and pregnancy rates to validate ICSI and IVF. Appropriate validation of new techniques can become very difficult when considering the sample size needed to prove a null hypothesis or small increase in pregnancy rates. An accurate validation of a new culture medium would need hundreds of patients in each study group. Adding to the complexity of validation practice is the fine line between validation and research, and questions are raised regarding the need for ethical approval to undertake validations.30 However, it is highly recommended to regularly validate other practices in the laboratory, such as changes of osmolarity during preparation of dishes, temperature fluctuation during denudation, and temperature distribution in incubators. Validation of temperature in culture medium in different types of dishes at all heated stages in the laboratory should confirm the appropriate range of surface temperature of the heated stage.

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Witnessing As a result of two major incidents with mixed-up embryos and patient identities, the UK HFEA CoP24 outlines that all centers have witnessing protocols in place to double check the identification of samples and patients or donors. The checks should be recorded at each step in the IVF process in order to avoid mistakes. There are certainly huge advantages with the use of double witnessing; however, there is always a slight risk that a procedure like this can cause mistakes, as we cannot double the embryologist workforce. As an alternative to manual double witnessing, clinics now have the option to substitute some witnessing steps with the use of electronic witnessing systems. One major source of incidents in the IVF laboratory is insufficient staffing, and to be interrupted while working with embryos can have disastrous consequences. Many laboratories today have very few embryologists, and with a witnessing routine in place this will not only increase the workload but also add a heightened risk of distraction when an embryologist has to interrupt another embryologist’s work to get a witness for a certain step in the procedure.

Reporting results (ISO 17025:2005 5.10 and ISO 15189:2007 5.8) The reporting of results should always be accurate, clear, unambiguous, and objective. Results documentation should follow the procedure set out by the quality system and include the requirements of the standards. Sources of errors and uncertainty of measurements should be stated and be properly calculated for each method. Apart from the issue of documents and document control, the SOP should define who, in the management, has the method and medical responsibility.

Monitoring and traceability (ISO 17025:2005 5.3, 5.4, 5.5, 5.6 and ISO 15189:2007 5.2, 5.3, 5.5) Monitoring of laboratory parameters All parameters affecting the outcome of the process need to be monitored. An accredited IVF laboratory should have an SOP covering the processes of monitoring in the laboratory. The SOP should clarify what parameters are monitored, together with the range of acceptance. It should also include a plan for action when a measurement is found to be outside the given range. Both laboratory standards require monitoring to be an active action, meaning that the parameter monitored needs to be recorded and approved by an active signature of a member of the laboratory staff. Where continual computerized monitoring is in place, the records have to be approved on a minimum daily basis with an active signature.

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The standards clearly state that the parameters affecting the outcome need to be monitored whenever the process is ongoing, and since most IVF clinics operate a non-stop service it is inadequate to measure temperatures once per week or once per month. An accredited laboratory needs to at least monitor the maximum, minimum, and current temperature of the laboratories, theater, incubators, heated stages and heating blocks, refrigerators, freezers, and cryopreservation machines daily. Monitoring of correct CO2 flow in incubators can be done either through measurements of CO2 in the chamber or through the measurements of the pH in the culture medium. Environmental monitoring such as particle counting, volatile organic compounds (VOC) measurements, and microbe tests using settlement plates and swabs can be done less often; however, validation of practice through frequent monitoring, at the start to confirm stability of the environment, can allow us to decide on a less frequent monitoring schedule, like once every 2–3 months. As important as the monitoring itself, is the use of adequate equipment for monitoring. The acceptable range for embryo culture temperature is 37 ± 0.2°C and this very precise range requires a thermometer with an accuracy of at least 0.2°C. This thermometer has to be calibrated by an accredited calibration laboratory and the certificate of calibration has to state that it has been calibrated at the range of use (37 ± 0.2°C) with an accuracy of 0.2°C. If the same thermometer is used for monitoring of temperatures at other ranges, it has to calibrated for all ranges of use. Monitoring of room temperature, fridges, and freezers, where the range is wider, can be undertaken using normal maximum/minimum digital thermometers. These can be calibrated by the laboratory in-house following documented procedures, including calculations for uncertainty of measurement. The correct monitoring of CO2 concentrations should include calibration of the instrument using a standard gas each time a series measurement is undertaken. To calibrate this type of instrument on a weekly or monthly basis is not considered adequate. Monitoring of pH in culture media requires a pH meter with three-point calibration, calibrated in the range of measurement (pH 7.35 ± 0.05) with temperature compensation to allow measurements of medium at 37°C. The pH probe needs to be adequate for measurements in protein-containing media and have an accuracy of at least 0.05. All monitoring in the laboratory has to be assured through internal audit and be included in the annual audit plan.

Monitoring of key performance indices (KPIs) Most clinics with a quality system in place monitor KPIs. Similar to the monitoring of laboratory environmental parameters, each clinic has to agree on documented limits of performance. Usually, when

monitoring parameters such as live birth, clinical pregnancy, and fertilization, there is no upper limit; however, a lower limit is necessary and documented plans for immediate action whenever a KPI falls under the agreed limit. KPIs essential to monitor in connection with the laboratory include, but are not limited to, fertilization rates for IVF and ICSI, damage rates for ICSI, survival of embryos after thawing, and pregnancy results from embryo transfer. These KPIs should be monitored for the whole laboratory and for each individual. It is important to underline the importance of confidentiality when monitoring individual performance, taking into account the need for training of any embryologist falling under the given limit, but not ignoring the stress and decrease in self-confidence this can lead to. All members of staff need to understand that the monitoring is not a way of punishing people but of assuring that all embryologists perform to the same high standard, minimizing variables. Another important outcome of individual performance monitoring is to identify persons with exceptionally high results so that others can learn more and thereby increase the overall success.

Assuring quality (ISO 17025:2005 5.9 and ISO 15189:2007 5.6) Quality assurance (QA) makes sure that you are doing the right thing in the right way and quality control (QC) makes sure that what you have done is what you expected. In short, quality assurance is process oriented and quality control is product oriented. When discussing QA/QC it is easy to get confused; however, the terminology is not important and what is important is that the laboratory has control mechanisms in place to ensure that they perform according to the SOPs and to the highest standard. ISO 17025:2005 and ISO 15189:2007 demand that a laboratory has QC and QA systems in place for monitoring of the validity of the methods used. This includes the demand for internal and external controls and inter/intra-laboratory comparisons and validations. The laboratory is required to determine the uncertainty of results. This can be difficult with a subjective parameter such as embryo scoring; however, it can easily be done for the assessment of sperm. Through assessment of a series of sperm samples by all laboratory staff involved in the preparation of sperm, a coefficient of variance (CV, %) can be calculated, usually resulting in a 10–15% variance. The standards also demand that all embryologists/ andrologists assess sperm samples and photos or movies of embryos on a regular basis, usually at least every 3 months. It is the responsibility of the laboratory manager to document the results from these comparisons and calculate variations and address any deviance. To collect samples and photos and arrange these types of intra-laboratory comparisons takes time

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and, over and above this, the standards also demand that the laboratory participates in inter-laboratory comparisons. A laboratory can share photos of embryos and samples of sperm with other centers and set up an inter-laboratory comparison scheme; however, the standards clearly state that self-developed programs like this should not be used when organized external schemes are available. In the UK, most laboratories participate in the United Kingdom National External Quality Assessment Service (UK NEQAS) andrology scheme, which uses DVD/video of sperm for motility assessment.31 A web-based inter-laboratory comparison scheme is run by Dr James Stanger and includes schemes for assessment of all stages of human pre-implantation embryos, sperm morphology and concentration, and ultrasound measurement of follicles (www.fertaid.com). The scheme provides monthly assessments of embryos and sperm and allows the laboratory manager to use the information for intra-laboratory comparison. As each of the different schemes has some 200–300 participants around the world, the intra-laboratory comparison scheme provides a solid reference for the laboratory management to implement corrective actions when deviations are found.32 This comparison program is in substantial agreement with the ISO/IEC guide 43-1, which is a requirement by the standard.33

Concluding remarks and future aspects Accreditation is an efficient and effective tool to demonstrate technical competence. Laboratory accreditation is the formal recognition of a laboratory’s technical competence. EA mutual recognition agreements are based on the evidence or assumption of equal technical competence of laboratories across borders. Such evidence is generally provided by the results of a comprehensive program of inter-laboratory comparisons in calibration, although there are fields, such as ART, where harmonization is still needed. In testing and inspection, full evidence of equal technical competence is still missing, and mutual recognition is based rather on the assumption of equivalence. Full equivalence can only be achieved by harmonization of measurement procedures and identical requirements in uncertainty determination and reporting of results. A future aspect that needs to be raised and debated amongst the ART community is that of the costs involved with the process of accreditation. As a proper accreditation can only be carried out by a national accreditation body, approved by the EA, the costs for accreditation and audits are too high to make accreditation an accessible tool for all IVF laboratories. Organizations such as ESHRE therefore have to put pressure on the EA to adjust the costs to the economic reality of IVF. Throughout completing the long and work-intensive process of applying a QC system in an ART laboratory,

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one might have asked what it has meant for the laboratory. There is no doubt that introducing a fully implemented QC system standardizes methods and the way in which embryologists perform their work in the laboratory. The troubleshooting, maintenance of equipment, and the milieu are improved and standardized. This guarantees optimal handling of a couple’s blood samples, gametes, and pre-embryos. Thus, introducing and working according to an accredited QC system in an ART unit is a never-ending project. It is a system that will guarantee constant improvement of the work. Introducing and fully implementing a quality control system in our laboratory has standardized the methods and the way that the embryologists perform their work in the laboratory. It has also optimized the environment in which the patient’s gametes and embryos are handled. The accreditation of more ART laboratories, to the same standards, will bring about alignment through a wide base for external controls. This could lead to an improvement of IVF results worldwide, and we would, for the first time, be able to compare the results between different laboratories.

Appendix 3.1 The accreditation task force Management group Objective To clarify the organization plan and leadership structure. Members The quality manager together with the clinical manager, laboratory manager, and other members in the management group. Specific tasks The management group has as its main objective to sort out the managerial issues of the organization. Documents that this group needs to create are the ones related to the below listed chapters of ISO 17025:2005.

Management and organization plans 4 4.1 4.2 4.3 4.13 4.14 5 5.1 5.2 5.3

Management requirements Organization Quality system Document control Internal audits Management reviews Technical requirements General Personnel Accommodation and environmental conditions

SOP group Objective To organize all activities in the organization and create standard operating procedures (SOPs) for each and every process.

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Members The laboratory manager and senior embryologists need to be in this group. The quality control (QC) manager does not need to be active in the actual task force but needs to supervise the work and be accessible to answer questions and oversee the process.

Purchase

Specific tasks The SOP group should work on creating SOPs. Every process needs to have an SOP and as the embryologists are the owners of the laboratory processes they need to write down what they do, and then sit down and discuss the process and assure that everyone agrees to work according to the written manual. The important issue is conformity – everyone has to do the process in the same way and follow the SOP. Along with the SOP comes the result – how it is established and reported. There are other demands in the standard that this group needs to work out, and they are all involved in laboratory processes such as listing of equipment tests and calibration routines for the equipment. Documents that this group needs to create are the ones related to the below listed chapters of ISO 17025:2005.

Complaints and incident group

Document control and writing and/or coordinating SOPs 4.12 Control of records 5.4 Test and calibration methods and method validation 5.5 Equipment 5.6 Measurement traceability 5.7 Sampling 5.8 Handling of test and calibration items 5.9 Assuring the quality of test and calibration results 5.10 Reporting the results

Purchase group Objective

To organize the purchasing routines.

Members The person responsible for orders and purchases together with the finance or business manager. The QC manager does not need to be active in the actual task force but needs to supervise the work and be accessible to answer questions and oversee the process. Specific tasks The purchase group needs to sort out all routines for purchasing and order of all material entering the laboratory. There is a need for clear routines for how suppliers are evaluated and how the contracts with them are outlined. As many of the disposables and culture media needed in the IVF laboratory have to go through specific testing, a very important job for this task force is to establish the requirements for tests and acceptance limits for these. Documents that this group needs to create are the ones related to the below listed chapters of ISO 17025:2005.

4.4 4.5 4.6

Review of requests, tenders, and contracts Subcontracting of tests and calibrations Purchasing services and supplies

Objectives To create crucial routines on how to handle incidents and complaints. Members The laboratory manager, a senior embryologist, head of nursing, and a member of the administration team. The clinical manager and QC director do not need to be active in the actual task force but need to supervise the work and be accessible to answer questions and oversee the process. Specific tasks To clarify all routines involving incidents and complaints. There have to be documented routines for how the organization control nonconformity and what plans there are for corrective and preventive actions. In short: clarify ways how the organization can learn from mistakes and benefit from the corrective actions.

How to handle complaints 4.7 Service to the client 4.8 Complaints 4.9 Control of nonconforming testing and/or calibration work 4.10 Corrective action 4.11 Preventive action

References 1. ISO/IEC Guide 2. 2004 Standardization and related activities – general vocabulary. 2004. International Organization for Standardization (www.ISO.org), Geneva, Switzerland. 2. Hazekamp JT. Current differences and consequences of legislation on practice of assisted reproductive technology in the Nordic countries. The Nordic Committee on Assisted Reproduction of the Scandinavian Federation of Societies of Obstetrics and Gynecology. Acta Obstet Gynecol Scand 1996; 75: 198–200. 3. Clinical and laboratory guidelines for assisted reproductive technologies in the Nordic Countries: NFOG bulletin supplement. NFOG, 1997: 3. 4. Dawson KJ. Quality control and quality assurance in IVF laboratories in the UK. Hum Reprod 1997; 12: 2590–1. 5. Pool TB. Practices contributing to quality performance in the embryo laboratory and the status of laboratory regulation in the US. Hum Reprod 1997; 12: 2591–3. 6. Lieberman BA, Matson PL, Hamer F. The UK Human Fertilisation and Embryology Act 1990 – how well is it functioning? Hum Reprod 1994; 9: 1779–82.

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ART laboratory: ISO 1000 and GLP 7. Ettarp L. An overview of international conformity assessment systems. Presented at the World Trade Organization Technical Working Group, 1999. 8. The European quality assurance standards EN ISO 9000 and EN 45000. European Commission Doc Certif 97.4, 1997. 9. European Cooperation for Accreditation www. european-accreditation.org. 10. ISO 9001:2000. Quality management systems, 2000. International Organization for Standardization, Geneva, Switzerland. 11. Keck C, Fischer R, Baukloh V, Alper M. Quality management in reproductive medicine. In: Gardner DK, Weissman A, Howles CM, Shoham Z, eds. Textbook of Assisted Reproductive Techniques, 2nd edn. London: Taylor & Francis, 2004: 477–94. 12. EN ISO/IEC 17025:2005. General requirements for the competence of testing and calibration laboratories, 2005. International Organization for Standardization, Geneva, Switzerland. 13. EN 45001. General criteria for the operation of testing laboratories, 1989. International Organization for Standardization, Geneva, Switzerland. 14. ISO/IEC Guide 25. General requirements for the competence of calibration and testing laboratories, 3rd edn, 1990. International Organization for Standardization, Geneva, Switzerland. 15. EN ISO/IEC 17025:1999. General requirements for the competence of testing and calibration laboratories, 1999. International Organization for Standardization, Geneva, Switzerland. 16. ISO 15189:2007. Medical laboratories – particular requirements for quality and competence, 2007. International Organization for Standardization, Geneva, Switzerland. 17. Kenny D. ISO and CEN documents on quality in medical laboratories. Clin Chim Acta 2001; 309: 121–5. 18. The Commission of the European Communities. Commission Directive 2003/94/EC, Laying down the principles and guidelines of good manufacturing practice in respect of medicinal products for human use and investigational medicinal products for human use. Off J Eur Union 2003; 14 October: L262/22–6. 19. European Commission. EC Guide to Good Manufacturing Practice, Revision to Annex 1. Manufacture of Sterile Medicinal Products. Brussels: Enterprise Directorate-General, 30 May 2003. 20. Directive 2003/23/EC of the European Parliament and of the Council of 31 March 2004 on setting standards

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31. 32. 33.

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of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells. Official Journal of the European Union, L 102, 7.4.2004, pp. 48–58. http:// europa.eu.int/eur-lex/en/oj/. Directive 2006/17/EC implementing Directive 2004/ 23/EC as regards traceability requirements, notification of serious adverse reactions and events and certain technical requirements for the donation, procurement and testing, of human tissues and cells annex. Directive 2006/86/EC implementing Directive 2004/23/EC as regards certain technical requirements for the coding, processing, preservation, traceability, storage, distribution of human tissues and cells and adverse events and reactions. ESHRE position paper on the EU Tissues and Cells Directive 2004/23/EC, November 2007, www.eshre.com/ file.asp?filetype=doc/04/010/eshre_position_paper_on_ the_eu_tissues_and_cells_directive_ec_final.pdf. Human Fertilisation and Embryology Authority (HFEA) Code of Practice, 7th edn, 2007, www.HFEA. gov.uk. Council Directive 93/42/EEC of 14 June 1993 concerning medical devices, OJ L 169, 12.7.1993. Directive last amended by Regulation (EC) No 1882/2003 of the European Parliament and of the Council (OJ L 284, 31.10.2003). Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in vitro diagnostic medical devices. OJ L 331, 7.12.1998, Directive as amended by Regulation (EC) No 1882/2003. Ackerman SB, Stokes GL, Swanson RJ, Taylor SP, Fenwick L. Toxicity testing for human in vitro fertilization programs. J In Vitro Fert Embryo Transf 1985; 2: 132–7. SWEDAC document ME 46b. Handling, storing, culture and cryopreservation of fertilized eggs and embryos, 1999. Van Kooij JR, Peeters MF, te Velde ER. Twins of mixed races: consequences for Dutch IVF laboratories. Hum Reprod 1997; 12: 2585–7. Hartshorne GM, Baker H. Fads and foibles in ART; where is the evidence? Hum Fertil (Camb) 2006; 9(1): 27–35. United Kingdom National External Quality Assessment Service (NEQAS), www.ukneqas.org.uk. QAP online FertAid, www.fertaid.com ISO/IEC Guide 43-1:1997. International Organization for Standardization, Geneva, Switzerland.

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4 Evaluation of sperm Kaylen M Silverberg, Tom Turner

Introduction Abnormalities in sperm production or function, alone or in combination with other factors, account for 35– 50% of all cases of infertility. Although a battery of tests and treatments have been described and continue to be used in the evaluation of female infertility, the male has been essentially neglected. It would appear that the majority of programs offering advanced reproductive technologies (ART) employ only a cursory evaluation of the male – rarely extending beyond semen analysis and antisperm antibody detection. Several factors certainly account for this disparity. First, most practitioners of ART are gynecologists or gynecologic subspecialists who have little formal training in the evaluation of the infertile or subfertile male. Second, the urologists, who perhaps theoretically should have taken the lead in this area, have devoted little of their literature or research budgets to the evaluation of the infertile male. Third, and perhaps most important, is the inescapable fact that sperm function testing remains a very controversial area of research. Many tests have been described, yet few have been extensively evaluated in a proper scientific manner. Those that have continue to be weighed down by persistent criticisms of poor sensitivity or specificity, a lack of standardization of methodology, suboptimal study design, problems with outcome assessment, and the lack of long-term follow-up. Although many of these same criticisms could also be leveled against most diagnostic algorithms for female infertility, in that arena, the tests continue to prevail over their critics. Fourth, like female infertility, male infertility is certainly multifactorial. It is improbable that one sperm function test will prove to be a panacea, owing to the multiple steps involved in fertilization. In addition to arriving at the site of fertilization, sperm must undergo capacitation and the acrosome reaction; they must penetrate the cumulus, bind to the zona pellucida, penetrate through the zona, fuse with the oolemma, activate the oocyte, undergo nuclear decondensation, form the male pronucleus, and then fuse with the

female pronucleus. Finally, with the advent and rapid continued development of microassisted fertilization, sperm function testing has assumed a role of even lesser importance. As fertilization and pregnancy rates improve with procedures such as intracytoplasmic sperm injection (ICSI), more and more logical questions are being asked about the proper role for sperm function testing. This chapter reviews the most commonly employed techniques for sperm evaluation, and examines the issues surrounding their utility in the modern ART program.

Patient history A thorough history of the infertile couple at the time of the initial consultation will frequently reveal conditions that could affect semen quality. Some of the important factors to consider are: 1. 2.

3.

4.

Reproductive history, including previous pregnancies with this and other partners. Sexual interaction of the couple, including frequency and timing of intercourse as well as the duration of their attempt to become pregnant. Past medical and surgical history: specific attention should be paid to sexually transmitted diseases, prostatitis, or epididymitis, as well a scrotal trauma or surgery – including varicocele repair, vasectomy, inguinal herniorrhaphy, and vasovasostomy. Exposure to medication, drugs, and toxins, including occupational and leisure activities, either in the past or in the present.1

Semen analysis The hallmark of the evaluation of the male remains the semen analysis. It is well known that the intrapatient variability of semen specimens from fertile men can vary significantly over time.2 This decreases the diagnostic information that can be obtained from a single analysis, often necessitating additional analyses. What is also apparent from literature analyzing

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Table 4.1

World Health Organization (WHO), normal values for semen analysis

Parameter

Normal values

Liquefaction Appearance Consistency Volume pH Concentration Total number Motility

Complete within 60 minutes at room temperature Homogeneous, gray, and opalescent Leaves a pipette as discrete droplets ≥ 2 ml ≥ 7.2 20 million sperm/ml semen or more 40 million sperm per ejaculate or more 50% or more with forward progression, or 25% or more with rapid progression within 60 minutes of collection 30% or more with normal forms; 15% or more with normal forms* 75% or more; 50% or more Fewer than 1 million/ml; fewer than 1 million/ml Fewer than 20% with adherent particles; fewer than 50% motile sperm with adherent particles Fewer than 10% with adherent particles; fewer than 50% motile sperm with adherent particles

Morphology† Vitality† Leukocytes† Immunobead test† MAR test†

*Kruger strict morphology. † First value from 3rd edn; second value from 4th edn. MAR, mixed agglutination reaction.

samples from ‘infertile’ patients is that the deficiencies revealed may not be sufficient to prevent pregnancy from occurring: rather, they may simply lower the probability of pregnancy, resulting in so-called ‘subfertility.’ Clearly, the overall prognosis for a successful pregnancy is dependent on the complex combination of variables in semen quality coupled with the multiple factors inherent in the female reproductive system that must each function flawlessly to enable a pregnancy to occur. The commonly accepted standard for defining the normal semen analysis are the criteria defined by the World Health Organization (WHO). These parameters are listed in Table 4.1.

Collection of the specimen When the semen analysis is scheduled, instructions should be given to the couple to ensure collection of an optimum semen sample. Written instructions are useful, especially if the patient is collecting the specimen outside of the clinical setting. During the initial infertility evaluation, a semen specimen should be obtained following a 2–7 day abstinence from sexual activity.2 A shorter period of time may adversely affect the semen volume and sperm concentration, although it may enhance sperm motility. A longer period of abstinence may reduce the sperm motility. In light of the natural variability in semen quality that all men exhibit, the initial semen collection may not accurately reflect a typical ejaculate for that patient. A second collection, with a 2–7 day abstinence period, can eliminate the tension associated with the initial semen collection, as well as provide a second specimen from which a typical set of semen parameters can be determined. This second collection may also

be used to determine the optimal abstinence period for this particular patient. Masturbation is the preferred method of collection. The use of lubricants is discouraged since most are spermicidal. However, some mineral oils and a few water-based lubricants are acceptable. Since masturbation may present significant difficulty for some men, either in the clinic or at home, an alternative method of collection must be available. The use of certain silastic condoms (seminal collection devices) during intercourse may be an acceptable second choice. Interrupted intercourse should not be considered, as this method tends to lose the sperm-rich initial few drops of semen while transferring many bacteria to the specimen container.2,3

Care of the specimen Appropriate care of the ejaculate between collection and examination is important. Specimens should be collected only in approved, sterile, plastic, disposable cups. Washed containers may contain soap or residue from previous contents, which can kill or contaminate the sperm. Delivery of the semen to the laboratory should occur within 60 minutes of collection, and the specimen should be kept at room temperature during transport. These recommendations are designed to maintain optimal sperm motility through the time of analysis.

Container labeling The information recorded on the specimen container label should include the names of both members of the couple as well as a unique identifying number. Typically, a social-security number, birth date, or a

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clinic-assigned patient number is used. Other helpful information recorded on the label should include the date and time of collection and the number of days since the last ejaculation. When the specimen is received from the patient, it is important to confirm that the information provided on the label is complete and accurate.

Examination of the specimen Liquefaction and viscosity When the semen sample arrives in the laboratory, it is checked for liquefaction and viscosity. Although similar, these factors are distinct from each other.4,5 Liquefaction is a natural change in the consistency of semen from a semi-liquid to a liquid. Before this process is completed, sperm are contained in a gel-like matrix that prevents their homogeneous distribution. Aliquots taken from this uneven distribution of sperm for the purpose of determining concentration, motility, or morphology may not be truly representative of the specimen as a whole. As liquefaction occurs over 15–30 minutes, sperm are released and distributed throughout the semen. Incomplete liquefaction may adversely affect the semen analysis by preventing this even distribution. The coagulum that characterizes freshly ejaculated semen results from secretions from the seminal vesicles. The liquefaction of this coagulum is the result of enzymatic secretions from the prostate. Watery semen, in the absence of a coagulum, may indicate the absence of the ejaculatory duct or seminal vesicles. Inadequate liquefaction, in the presence of a coagulum, may indicate a deficiency of prostatic enzymes.6,7 Viscosity refers to the liquefied specimen’s tendency to form drops from the tip of a pipette. If drops form and fall freely, the specimen has a normal viscosity. If drops will not form or the semen cannot be easily drawn up into a pipette, viscosity is high. Highly viscous semen may also prevent the homogeneous distribution of sperm. Treatment with an enzyme, such as chymotrypsin,8 or aspiration through an 18-gauge needle, may improve the distribution of sperm before an aliquot is removed for counting. Any addition of medium containing enzymes should be recorded, as this affects the actual sperm concentration. The new volume must be factored in when calculating the total sperm count.

Semen volume Semen volume is best measured with a serological pipette that is graduated to 0.1 ml. This volume is recorded and later multiplied by the sperm concentration in order to obtain the total count. A normal seminal volume before dilution is considered to be >2 ml.2

Sperm concentration A variety of counting chambers are available for determining sperm concentration. Those more commonly

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used include the hemocytometer, the Makler counting chamber, and the MicroCell. Regardless of the type of chamber used, an aliquot from a homogeneous, mixed semen sample is placed onto a room temperature chamber. The chamber is covered with a glass coverslip, which allows the sperm to distribute evenly in a very thin layer. Sperm within a grid are counted, and a calculation is made according to the formula for the type of chamber used. Accuracy is improved by including a greater number of rows or squares in the count. Sperm counts should be performed immediately after loading semen onto the chamber. Waiting until the heat from the microscope light increases the speed of the sperm may inaccurately enhance the count. As indicated earlier, a particular patient’s sperm count may vary significantly from one ejaculate to another. This observation holds true for both fertile and infertile males, further complicating the definition of a normal range for sperm concentration. Demographic studies employing historic controls were used to define a sperm concentration of <20 million/ml as abnormal.9,10 Although several investigators observed that significantly fewer pregnancies occurred when men had sperm counts <20 million/ml, the prognosis for pregnancy did not increase proportionately to the sperm concentration above this threshold.

Sperm motility Sperm motility may be affected by many factors, including: • • • • •

the patient’s age and general health the length of time since the last ejaculation the patient’s exposure to outside influences such as excessive heat or toxins the method of collection the length of time and adequacy of handling from collection to analysis.

When the aliquot of semen is placed on the room temperature counting chamber, the count and motility should be determined immediately. As previously stated, this will prevent the influence of the heat from the microscope light source from influencing the results. If a chamber with a grid is used to count the sperm, the motility can be determined at the same time as the concentration by using a multiple-click cell counter to tally motile and nonmotile sperm and then totaling these numbers to arrive at the true sperm concentration. The accuracy improves as more sperm are counted. If a wet-mount slide is used to determine motility, more than one area of the slide should be used, and each count should include at least 100 sperm. Prior to examining the specimen for motility, the slide or counting chamber should be examined for signs of sperm clumping. Sperm clumping to other

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sperm, head to head, head to tail, or tail to tail, may indicate the presence of sperm antibodies in the semen. This should not be confused with clumping of sperm to other cellular debris in the semen, which is not associated with the presence of antibodies. In either case, sperm clumping may affect the accuracy of both the sperm count and the motility.2,3 Motility is one of the most important prerequisites for achieving fertilization and pregnancy. The head of the sperm must be delivered a great distance in vivo through the barriers of the reproductive tract to the site of the egg. Sperm must have sufficient motility in order to penetrate both the layers of coronal cells and the zona pellucida before fusing with the egg’s cell membrane (oolemma). An exact threshold level of motility required to accomplish fertilization and pregnancy, however, has never been described.9 This may be due to variables in the equipment and techniques used in assessing motility.

air-dried. The cells are fixed to the slide and stained using a Wright–Giemsa or Bryan–Leishman stain. When viewed under 400× or 1000×, cell types may be differentiated primarily by their nuclear morphology. Immature sperm have one to three round nuclei within a common cytoplasm. Polymorphonuclear leukocytes may also be multinucleate, but the staining method will typically reveal characteristic nuclear bridges between their irregularly shaped nuclei.2 A peroxidase stain may be used to identify granulocytes and to differentiate them from the immature sperm. The presence of greater than 1 million white blood cells per ml of semen may indicate an infection in the urethra or accessory glands, which provide the majority of the seminal plasma. Such infections could contribute to infertility.1,2 As such, these samples must be cultured so that the offending organism can be identified and appropriate treatment can be instituted.

Progression

Sperm morphology

Whereas sperm motility represents the quantitative parameter of sperm movement expressed as a percentage, sperm progression represents the quality of sperm movement expressed on a subjective scale. A typical scale, such as the one below, attempts to depict the type of movement exhibited by most of the sperm visualized on a chamber grid. With the advent of successful microassisted fertilization, scales such as this have assumed more limited utility. Nevertheless, for those laboratories that quantify motility, a score of 0 means no motility, 1 means motility with vibratory motion without forward progression, 2 means motility with slow, erratic forward progression, 3 means motility with relatively straightforward motion, and 4 means motility with rapid forward progression.3

Sperm morphology can be assessed in several ways. The most common classification systems are the 3rd edn WHO standard and the 4th edn WHO standard that incorporates Kruger strict criteria (Fig 4.1). The third edition WHO method requires either a wet-slide preparation or a fixed, stained slide. A 10–20 µl drop of semen is placed on a slide. After placing a coverslip over the specimen, morphology may be determined at 400× by phase contrast microscopy. Alternatively, the drop of semen may be mixed with an equal volume of fixative plus stain (typically Papanicolaou or a Diff-Quik kit) prior to placing it on the slide. At least 100 sperm must be counted at 400× or 1000× with phase contrast or bright field microscopy. WHO criteria for assessing normal forms include the following:

Sperm vitality When a motility evaluation yields a low proportion of moving sperm (less than 50%), a vitality stain may be beneficial. This is a method used to distinguish nonmotile sperm that are living from those that are dead. This technique will be discussed later in the sperm function section.

Additional cell types While observing sperm in a counting chamber or on a slide, additional cell types may also be seen. These include endothelial cells from the urethra, epithelial cells from the skin, immature sperm cells, and white blood cells. The most common and significant of these cell types is referred to collectively as ‘round cells.’ These include immature sperm cells and white blood cells. In order to distinguish between them, an aliquot of semen can be placed in a thin layer on a slide and

•

• •

Head – oval and smooth heads are normal; round, pyriform, pin, double, and amorphous heads are all abnormal. Mid-piece – a normal mid-piece is straight and slightly thicker than the tail. Tail – single, unbroken, straight tails, without kinks or coils are normal.

A normal semen analysis should contain at least 30% normal sperm using WHO 3rd edn criteria. In order to employ Kruger strict criteria, sperm morphology is evaluated by placing 5 µl of liquefied semen on a slide, making a thin smear, and air-drying it at room temperature. The slide is then fixed and stained (typically with a Papanicolaou stain or a Diff-Quik kit). Slides are read using bright field microscopy under 1000× or higher magnification. At least 200 sperm should be counted for an accurate evaluation. The Kruger criteria for assessing normal forms include the following (Fig 4.2):12,13

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a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

q

r

s

t

43

Fig 4.1 Different types of sperm malformations. Reproduced from reference 11. a, Round head/no acrosome; b, Small acrosome; c, Elongated head; d, Megalo head; e, Small head; f, Pinhead; g, Vacuolated head; h, Amorphous head; i, Bicephalic; j, Loose head; k, Amorphous head; l, Broken neck; m, Coiled tail; n, Double tail; o, Abaxial tail attachment; p, Multiple defects; q, Immature germ cell; r, Elongated spermatid; s, Proximal cytoplasmic droplet; t, Distal cytoplasmic droplet.

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a.

c.1.

b.1.

2.

the 3rd edn WHO method and the Kruger strict method, have been used to predict a patient’s fertility. Several studies have concluded that the Kruger method of strict morphology determination shows the most consistent prediction of fertilization in vitro following conventional insemination.1,17,18 This method of assessing normal sperm morphology, because of its precise, nonsubjective nature, establishes a threshold below which abnormal morphology becomes a contributing factor in infertility.

2.

3.

4.

Computer-assisted semen analysis

Fig 4.2 Diagrammatic representation of quick-stained spermatozoa. a, Normal form; b.1, Slightly amorphous head; b.2, Neck defect; c.1, and 2, Abnormally small acrosome; c.3, No acrosome; c.4, Acrosome >70% of sperm head. Reproduced from reference 12.

•

•

•

Head – smooth; oval configuration; length, 5–6 µm diameter, 2.5–3.5 µm; acrosome, must constitute 40–70% of the sperm head. Mid-piece – slender, axially attached; <1 µm in width and approximately 1.5× head length; no cytoplasmic droplets >50% of the size of the sperm head. Tail – single, unbroken, straight, without kinks or coils approximately 45 µm in length.

As described by Kruger et al, sperm forms that are not clearly normal should be considered abnormal. The presence of ≥15% normal sperm morphology should be interpreted as a normal result. Normal morphology of 4–14% should be considered to be borderline, and normal morphology <4% is abnormal.12,13 Normal sperm morphology has been reported to be directly related to fertilization potential. This may be due to the abnormal sperm’s inability to deliver normal genetic material to the cytoplasm of the egg. From video recordings, it appears that abnormal sperm are more likely to have diminished, aberrant, or absent motility. This reduced or unusual motility may result from hydrodynamic inefficiency due to the head shape, abnormalities in the tail structure which prevent normal motion, and/or deficiencies in energy production necessary for motility.14,15 In addition to compromised motility, abnormal sperm do not appear to bind to the zona of the egg as well as do normal sperm. This has been demonstrated in studies employing the hemizona binding assay.16 In vitro fertilization (IVF) has helped further to elucidate the role that normal sperm morphology plays in the fertilization process and in pregnancy. Both methods of determining normal sperm morphology,

Computer-assisted semen analysis (CASA) was initially developed to improve the accuracy of manual semen analysis. Its goal is to establish a standardized, objective, reproducible test for sperm concentration, motility, and morphology. The technique also attempts, for the first time, to actually characterize sperm movement. The automated sperm movement measurements – known as kinematics – include straight-line velocity, curvilinear velocity, and mean angular displacement (Table 4.2). The use of CASA requires specialized equipment, including a phase contrast microscope, video camera, video recorder, video monitor, computer, and printer. To perform CASA, sperm are placed on either a Makler or a MicroCell chamber and they are then viewed under a microscope. The video camera records the moving images of the sperm cells and the computer digitizes them. The digitized images consist of pixels whose changing locations are recorded frame by frame. Thirty to 200 frames per minute are produced. The changing locations of each sperm are recorded and their trajectories are computed (Fig 4.3).19 In this manner, hyperactive motion can also be detected and recorded. Hyperactive sperm exhibit a whip-like, thrashing movement, which is thought to be associated with sperm that are removed from seminal plasma and ready to fertilize the oocytes.19,20 Persistent questions about the validity and reproducibility of results have kept CASA from becoming a standard procedure in the andrology laboratory. The accuracy of sperm concentration appears to be diminished in the presence of either severe oligospermia or excessive numbers of sperm. In cases of oligospermia, counts may be overestimated due to the machine counting debris as sperm. High concentrations of sperm may be underestimated in the presence of clumping. High sperm concentrations can also cause overestimations in counting due to the manner in which the software handles collisions between motile sperm and nonmotile sperm. In these cases, diluting the sample may improve the accuracy of the count.20,21 Sperm concentration also appears to be closely related to the type of counting chamber employed. Similar to the challenges reported with manual counting, sperm counts may vary whether using a Makler or a MicroCell.

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Table 4.2

Kinematic measurements in computer-assisted semen analysis (CASA)

Symbol

Name

Definition

VSL

Straight-line velocity

VCL VAP LIN WOB STR ALH

Curvilinear velocity Average path velocity Linearity Wobble Straightness Amplitude of lateral head displacement Riser displacement

Time average velocity of the sperm head along a straight line from its first position to its last position Time average velocity of the sperm head along its actual trajectory Time average velocity of the sperm head along its average trajectory Linearity of the curvilinear trajectory (VSL/VCL) Degree of oscillation of the actual sperm-head trajectory around its average path (VAP/VCL) Straightness of the average path (VSL/VAP) Amplitude of variations of the actual sperm-head trajectory about its average trajectory (the average trajectory is computed using a rectangular running average) Point to point distance of the actual sperm-head trajectory to its average path (the average path is computed using an adaptive smoothing algorithm) Time average rate at which the actual sperm trajectory crosses the average path trajectory Fundamental frequency of the oscillation of the curvilinear trajectory around its average path (HAR is computed using the Fourier transformation) Amplitude squared height of the HAR spectral peak (MAG is a measure of the peak to peak dispersion of the raw trajectory about its average path at the fundamental frequency) Area under the fundamental harmonic peak in the magnitude spectrum (VOL is a harmonic measure of the power-bandwidth of the signal) Concentration of sperm cells in a sample in millions of sperm per milliliter of plasma or medium Percentage of sperm cells in a suspension that are motile (in manual analysis, motility is defined by a moving flagellum; in CASA, motility is defined by a minimum VSL for each sperm)

RIS BCF HAR

VOL

Beat-cross frequency Frequency of the fundamental harmonic Magnitude of the fundamental harmonic Area of fundamental harmonic

CON

Specimen concentration

MOT

Percentage motility

MAG

45

Reproduced from reference 19.

θi

BCF VCI

VAP ALH

RIS

the subject of much debate. In summary, persistent questions about results and their interpretation continue to limit the routine use of CASA. As reproducibility improves over all ranges of sperm concentration, CASA may become the standard for semen analysis. The use of fluorescent DNA staining with CASA may also improve its reliability. In addition, as the kinematics of sperm motion becomes better understood, CASA may play an integral role in determining the optimal method of assisted reproductive technology that should be utilized for specific types of male factor patients.

VSL

Sperm antibodies

Fig 4.3 Examples of kinematic measurements involved in a single sperm tracing (see Table 4.2 for explanation of acronyms). Reproduced from reference 19.

Sperm motion parameters identified by CASA have been assessed by several investigators for their ability to predict fertilization potential. Certain types of motion have been determined to be important in achieving specific actions related to fertilization, such as cervical mucus penetration and zona binding. However, the overall value of CASA for predicting pregnancy is still

Because mature spermatozoa are formed after puberty, they can be recognized as foreign protein by the male immune system. In the testicle, the sperm are protected from circulating immunoglobulins by the tight junctions of the Sertoli cells. As long as the sperm are contained within the lumen of the male reproductive tract, they are sequestered from the immune system, and no antibodies form to their surface antigens. If there is a breach in this so-called ‘blood:testis barrier,’ an immune response may be initiated. The most common causes of a breach in the reproductive tract, which could initiate antibody formation, include vasectomy, varicocele repair, testicular biopsy, torsion, trauma, and infection.22,23 Once formed, antibodies are secreted into the fluids of the accessory glands, specifically the prostate and seminal vesicles. At the time of ejaculation, the fluids from these glands

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contribute most of the volume to the seminal plasma. These antibodies can then come into contact with the sperm and may cause them to clump. In women, the atraumatic introduction of sperm into the reproductive tract as a result of intercourse or artificial insemination does not appear to be a factor in the production of sperm antibodies. However, events that induce trauma, or introduce sperm to the mucous membranes outside of the reproductive tract, can induce antibody formation. Proposed examples of such events include trauma to the vaginal mucosa during intercourse or the deposition of sperm into the gastrointestinal tract by way of oral or anal intercourse.23 There are several tests currently employed for detecting the presence of sperm antibodies. The two most common are the mixed agglutination reaction and the immunobead binding test.

The mixed agglutination reaction (MAR) This test is performed by mixing semen, immunoglobulin G (IgG)- or IgA-coated latex beads or red blood cells, and IgG or IgA antiserum on a microscope slide. The slides are incubated and observed at 400×. At least 200 sperm are counted. If antibodies are present, the sperm will form clumps with the coated latex beads or coated red blood cells. If antibodies are absent, the sperm will swim freely. The level of antibody concentration considered to be clinically relevant must be established by each center conducting the test. The WHO considers a level of binding of ≥50% to be clinically significant. This test is used only for detection of direct antibodies in men, and is not specific for the location of bead attachment to the sperm.

To perform an indirect test, known direct antibodynegative sperm are washed free of seminal plasma and resuspended in a small volume of media plus BSA. They are incubated for 1 hour at 37°C with the bodily fluid to be tested. The sperm are then washed free of the bodily fluid, resuspended in media plus BSA, and mixed on a slide with IgG- or IgA-coated latex beads. The test is interpreted by noting the percentage and location of bead attachment. The 3rd edn WHO standard considers a level of binding of ≥20% to represent a positive test, whereas the 4th edn WHO standard considers a level of ≥50% to be positive. Clinical significance is commonly considered to be a level of binding of ≥50%.9,24 The clinical value of antisperm antibody testing is predicated on the observation that the presence of a significant concentration of antibodies may impair fertilization. It has been reported that antibodypositive sperm may have difficulty penetrating cervical mucus. Although, in these cases, intrauterine insemination (IUI) or IVF may improve the prognosis for fertilization, antibody levels >80%, coupled with subpar concentration, motility, or morphology, may necessitate the addition of ICSI in order to achieve the highest percentage of fertilization.25 As suggested by the literature, andrology laboratories may do a significantly better job of preparing sperm if they are aware of the presence of antibodies. Specifically, it has been demonstrated that the use of increased concentrations of protein in the media used for sperm preparation will reduce the adverse effect of antisperm antibodies on sperm motility. In summary, antisperm antibodies have been demonstrated to be a contributing factor in infertility. While their presence alone may not be sufficient to prevent pregnancy, their detection should encourage the andrologist to pursue additional appropriate action.

The immunobead binding test This test is performed by combining IgG- or IgAcoated latex beads and washed sperm on a slide. The sperm must be removed from the seminal plasma by washing the sample with media plus bovine serum albumin (BSA). The presence of human protein on the surface of the sperm interferes with the binding of the immunobeads to the sperm, and thus may mask a positive result. After washing, the sperm are placed on a slide with IgG- or IgA-coated latex beads and read at 200× or 400×. If antibodies are present, the small beads will attach directly to the sperm. This test provides potentially greater information than the mixed agglutination reaction, as results consider the number of sperm bound by beads, the type of antigen involved in binding, and the specific location where the bead is bound to the sperm. If antibodies are absent, the beads will not attach. This test can be used for the detection of direct antibodies in men. However, unlike the MAR test, it may also be used to detect antibodies produced in a woman’s serum, follicular fluid, or cervical mucus by incubating these bodily fluids with washed sperm that have previously tested negative for antibodies.

Sperm vitality An intact plasma membrane is an integral component of, and possibly a biologic/diagnostic indicator for, sperm viability. The underlying principle is that viable sperm contain intact plasma membranes that prevent the passage of certain stains, whereas nonviable sperm have defects within their membranes that allow for staining of the sperm. Several so-called vital stains have been employed for this purpose. They include eosin Y, trypan blue, and/or nigrosin.26 When viewed with either bright field or phase contrast microscopy, these stains allow for the differentiation of viable, nonmotile sperm from dead sperm. This procedure may therefore play a significant role in determining the percentage of immotile sperm that are viable and available for ICSI. Unfortunately, however, dyes such as eosin Y are specific DNA probes that may have toxic effects if they enter a viable sperm or oocyte, which precludes the use of these sperm for ICSI or insemination. Flow cytometry has also been utilized for the determination of sperm viability. Like vital staining, flow cytometry is based on the principle that an intact plasma membrane will prevent the passage of

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nucleic acid-specific stains. Some techniques, such as the one described by Noiles et al, employ dual staining, which can differentiate between an intact membrane and a damaged membrane.27 There are no studies that prospectively evaluate sperm viability staining as a predictor of ART outcome.

Hypo-osmotic swelling test Another means of assessing the sperm plasma membrane is the hypo-osmotic swelling test (HOST). This assay is predicated upon the observation that all living cells are permeable to water, although to different degrees. The human sperm membrane has one of the highest hydraulic conductivity coefficients (2.4 µl/min/atm at 22°C) of any mammalian cell.28 As originally described, the HOST involves placing a sperm specimen into hypotonic conditions of approximately 150 mosmol.29 This environment, while not sufficiently hypotonic to cause cell lysis, will cause swelling of the sperm cells. As the tail wells, the fibers curl, and this change can be detected by phase contrast microscopy, differential interference contrast (DIC), or Hoffman optics. The normal range for a positive test is typically considered to be a score ≥60%, i.e. 60% of the cells demonstrate curling of the tails. A negative test is defined as <50% curling.30 This test generated a significant amount of initial interest, and several investigators compared it to the sperm penetration assay (SPA) as an in vitro surrogate for fertilization, reporting good correlation.31,32 More recently, the test has been employed as a predictor of ART outcome, with conflicting results. Although one group reported a favorable correlation, another found no predictive value for the test.33,34 It has also been suggested that, owing to sperm morphology changes in response to the test, the HOST may facilitate an embryologist’s ability to select sperm appropriate for injection. In our program at the Texas Fertility Center, we use the HOST to identify sperm suitable for use in ICSI cases where all sperm are nonmotile. In summary, the HOST currently lacks sufficient critical evaluation to determine its true role in the assessment and/or treatment of the infertile male.

Assays of the sperm acrosome The acrosome is an intracellular organelle, similar to a lysosome, which forms a cap-like structure over the apical portion of the sperm nucleus.35 The acrosome contains multiple hydrolytic enzymes, including hyaluronidase, neuraminidase, proacrosin, phospholipase, and acid phosphatase, which, when released, are thought to facilitate sperm passage through the cumulus mass, and possibly the zona pellucida as well (Fig 4.4). Once sperm undergo capacitation, they are capable of an acrosome reaction. This reaction is apparently triggered by fusion of the sperm plasma membrane with the outer acrosomal membrane at

47

multiple sites, leading to diffusion of the acrosomal enzymes into the extracellular space. This results in dissolution of the plasma membrane and acrosome, leaving the inner acrosomal membrane exposed over the head of the sperm (Fig 4.5). Although electron microscopy has produced many elegant pictures of acrosome-intact and acrosomereacted sperm, it is not always possible to know if sperm that fail to exhibit an acrosome have truly acrosomereacted, or could possibly be dead. In addition, electron microscopy is not a technique available to all andrologists. This has led to the necessity for the development of biochemical markers for the acrosome reaction. Throughout the 1970s and 1980s, multiple biochemical tests were described using a variety of lectins, antibodies, and stains. Although they apparently correlated well with electron microscopy, the tests were still timeconsuming and difficult to perform.36,37 Contemporary assays for the determination of acrosomal status employ fluorescent plant lectins or monoclonal antibodies, which can be detected much more easily with fluorescence microscopy.38,39 These assays may prove to be of value if they can truly identify males who manifest deficiencies in their ability to undergo the acrosome reaction. Hypothetically, such patients may need to have their sperm specially preincubated – such as with follicular fluid or calcium ionophore – prior to insemination if they fail to acrosome-react on their own. Conversely, this test may help to identify a small subpopulation of males who prematurely acrosome-react. Several studies have reported an association between ejaculated sperm with low percentages of acrosome-intact sperm and poor subsequent fertilization.40 These areas certainly await additional study.

Other biochemical tests As noted above, one of the predominant enzymes present in the acrosome is proacrosin. The enzymatic action of acrosin is not necessarily correlated to the presence of an intact acrosome; therefore, assays for the presence of acrosin have been described.41 Acrosin activity has been reported to be greater in fertile males than in infertile males;42 however, there are no prospective evaluations correlating acrosin activity to fertilization rates in ART patients. Like all other tissues that require energy synthesis and transport, spermatozoa contain measurable levels of creatinine phosphokinase (CPK). Two isomers, CK-M and CK-B, have been described, and differences have been noted in these levels in semen specimens from fertile and infertile males. Specifically, CK-M levels exceed CK-B levels in normospermic males, while CK-B levels are greater in spermatozoa from oligospermic males.43 In this same study, researchers found that semen samples in which CK-M/CK-B ratios exceeded 10% exhibited higher fertilization rates in IVF than specimens with lower ratios. Few other studies have addressed this topic.

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OA

AC

IA

SS

ES

Fig 4.4 Sperm head with intact acrosome. OA, outer acrosomal membrane; AC, acrosomal cap; ES, equatorial segment; SS, subacrosomal space. Reproduced from reference 11.

Sperm penetration assay The sperm penetration assay (SPA) or hamster egg penetration assay (HEPA) was initially described by Yanagimachi et al in 1976.44 It measures the ability of sperm to undergo capacitation and the acrosome reaction, penetrate the oolemma, and then decondense. In this test, oocytes from the golden hamster are first treated in order to remove the zona pellucida. As one of the functions of the zona is to confer species specificity, its presence would preclude performance of this test. However, zona removal obviously prohibits the HEPA from being able to assess sperm for the presence of zona receptors. Following zona removal, human sperm are incubated for 48 hours with the hamster oocytes, and the number of penetrations with nuclear decondensation are calculated. As originally described, it was hoped that the test would correlate with the ability of human sperm to fertilize human oocytes in vitro. Although the test was designed to assess the ability of sperm to fuse to the oolemma, it also indirectly assesses sperm capacitation, the acrosome reaction, and the ability of the sperm to be incorporated into the ooplasm. Unfortunately, however, intrinsic in the design of the test is its inability to assess the sperm’s ability to bind to – and penetrate

Fig 4.5 Acrosome-reacted sperm. IA, inner acrosomal membrane. Reproduced from reference 11.

through – the zona pellucida. This factor continues to be one of the major criticisms that plague this test. Throughout the 1980s, multiple modifications of the SPA were published. These included modifications of the techniques for sperm preparation prior to the performance of the assay, such as inducing the acrosome reaction or incubation with TEST yolk buffer, changes in the protocol methodology itself, and modifications of the scoring system.45,46 Published reports demonstrated widely varying conclusions, such as the finding that the SPA could identify anywhere from 0 to 78% of men whose sperm would fail to fertilize oocytes in ART procedures.47 Most criticisms of the SPA literature center on poor standardization of the assay, poor reproducibility of the test, and lack of a standard normal range. Although some reports suggest a correlation between the SPA and fertility, neither a large literature review47 nor a prospective long-term (5-year) follow-up study demonstrate such a correlation.48 In light of these considerations, support for this test has gradually waned.

Hemizona assay Over the past several years, a growing body of research has demonstrated a significant correlation between tests of sperm:zona pellucida binding and subsequent fertilization in ART. This led the European Society for

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100

Fertilization rate (%)

90 A

80 70 60 50 40 B

30 20 10

C

0 0

10

20

30

40

50

60

70

80

90

100

Hemizona index

Fig 4.6 Cluster analysis of hemizona assay index and fertilization rate. A, good fertilization; B, poor fertilization; C, false-positive hemizona assay index. Reproduced from reference 52.

Human Reproduction and Embryology (ESHRE) Andrology Special Interest Group to recommend inclusion of such tests in the advanced evaluation of the male.49 Like the SPA, the hemizona assay (HZA) employs sperm and nonviable oocytes in an in vitro assessment of fertilization.50 In this test, however, both gametes are human in origin. As described, the HZA assesses the ability of sperm to undergo capacitation, acrosome react, and bind tightly to the zona. Classically, oocytes that failed to fertilize during an ART procedure are bisected, and then sperm from a proven fertile donor (500 000/ml) are added to one hemizona, while sperm from the subject male are added to the other hemizona. Following a 4-hour incubation, each hemizona is removed and pipetted in order to dislodge loosely attached sperm. A comparison or hemizona index (HZI) is then calculated by dividing the number of test sperm tightly bound to the hemizona by the number of control (fertile) sperm bound to the other hemizona: HZI = number of test sperm bound/number of control sperm bound × 100 This test assesses the ability of sperm to bind to the zona itself. Although expensive, labor-intensive, and difficult to perform, there are some data that suggest that the HZA may help to identify individuals with a poor prognosis for success with ART51,52 (Fig 4.6). A more recent prospective study employing receiver operating characteristic (ROC) curve analysis has also suggested that HZA results may be used to predict subsequent fertilization in ART procedures with both high sensitivity and specificity.53

Mannose binding assay Another test has been developed in order to assess the ability of sperm to bind to the zona. This in vitro procedure is based on a series of observations that suggest

Fig 4.7 Mannose-positive (brown) and mannose-negative (clear) sperm. Courtesy of Tammy Dey, Kaylen Silverberg.

that sperm:oocyte interaction involves the recognition by a sperm surface receptor of a specific complementary receptor on the surface of the zona pellucida. This zona receptor appears to be a glycoprotein, the predominant sugar moiety of which is mannose.54 In an elegant series of experiments, Mori et al determined that sperm:zona binding could be curtailed by the addition of a series of sugars to the incubating media. Although many sugars impaired binding, the addition of mannose totally inhibited sperm:oocyte interaction.55 In vitro assays in which labeled probes of mannose conjugated to albumin are co-incubated with semen specimens allow for the differential staining of sperm (Fig 4.7). Those that bind the probe are thought to possess the sperm surface receptor for the mannoserich zona glycoprotein. Several investigators, including our group, have subsequently demonstrated that sperm from fertile populations exhibit greater mannose binding than do sperm from infertile males.56–58 This new area shows promise in the area of sperm function testing, but also invites further study.

Assays of sperm DNA integrity The most current area of investigation into sperm function involves the assessment of sperm DNA integrity. Sperm chromatin has been demonstrated to be packaged very differently than chromatin in somatic cells. Specifically, the DNA is organized in such a manner that it remains very compact and stable.59 As there are many different ways in which either this organization or the sperm chromatin itself can be damaged, several different assays of sperm chromatin assessment have been developed. There are two basic types of assays: direct assays, such as the ‘Comet’ and ‘TUNEL’ assays and indirect assays such as the sperm chromatin structure assay or ‘SCSA.’ The direct assays detect actual breakages in the DNA, while the indirect assays measure the relative proportions of single (abnormal) and double (normal) stranded

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DNA within the sperm following acid treatment. Data from several studies suggest that infertile men have significantly greater amounts of DNA damage than fertile men.59–62 There is also a suggestion that this finding is similarly present in the male partner of couples experiencing recurrent miscarriage. Despite these reports, at the present time, there is no conclusive correlation between the results of sperm DNA integrity testing and pregnancy rates achieved either naturally or with the advanced reproductive technologies. As such, the Practice Committee of the American Society for Reproductive Medicine has recently recommended that the routine testing of sperm DNA integrity should not be included in the evaluation of the infertile couple.63

3. 4.

5.

6.

7.

Conclusion In summary, there have been many recent advances in the diagnostic evaluation of sperm and sperm function. Although many tests of sperm function have been described, there remains a lack of consensus as to both the role of testing and the identification of the appropriate test(s) to perform. Owing to the complicated nature of sperm function, it is improbable that a single test will emerge with sufficient sensitivity, specificity, and positive and negative predictive values required of a firstline diagnostic tool for all affected males. A more likely scenario will be similar to that in female infertility, where a battery of tests – each evaluating a specific function – are employed as needed. In light of profound recent advances in gamete micromanipulation, a more germane issue might be the overall relevance of sperm function testing in the contemporary andrology laboratory. Although this issue is quite controversial, it is likely that sperm function testing will continue to play a role in the evaluation of the infertile male. Just as ART is not the treatment of choice for all infertile females, it is not likely that micromanipulation will become standard treatment for all infertile males. The gold standard of sperm function remains the ability to fertilize an oocyte in vitro. Therefore, in order to continue to address the above questions, it is incumbent upon investigators to design appropriate prospective trials to assess these tests thoroughly. Those tests that demonstrate a statistically significant correlation with fertilization in vitro must then undergo additional evaluation in order to assess clinical significance if we hope to develop an appropriate diagnostic algorithm.

References 1. Gangi CR, Nagler HM. Clinical evaluation of the subfertile man. In: Diamond MP, DeCherney AH, Overstreet JW, eds. Infertility and Reproductive Medicine. Clinics of North America. Philadelphia: WB Saunders, 1992; 3: 299–318. 2. World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and

8.

9.

10.

11.

12.

13.

14. 15.

16.

17.

18.

19.

20.

Sperm–Cervical Mucus Interaction, 4th edn. New York: Cambridge University Press, 1999: 4–33, 60–1. Alexander NJ. Male evaluation and semen analysis. Clin Obstet Gynecol 1982; 25: 463–82. Overstreet JW, Katz DF, Hanson FW, Foseca JR. A simple inexpensive method for objective assessment of human sperm movement characteristics. Fertil Steril 1979; 31: 162–72. Overstreet JW, Davis RO, Katz DF, Overstreet JW, eds. Infertility and Reproductive Medicine. Clinics of North America. Philadelphia: WB Saunders, 1992: 329–40. Koren E, Lukac J. Mechanism of liquefaction of the human ejaculate: I. Changes of the ejaculate proteins. J Reprod Fertil 1979; 56: 493–500. Lukac J, Koren E. Mechanism of liquefaction of the human ejaculate: II. Role of collagenase like peptidase and seminal proteinase. J Reprod Fertil 1979; 56: 501–10. Cohen J, Aafjes JH. Proteolytic enzymes stimulate human spermatozoal motility and in vitro hamster egg penetration. Life Sci 1982; 30: 899–904. Van Voorhis BJ, Sparks A. Semen analysis: what tests are clinically useful? Clin Obstet Gynecol 1999; 42: 957–71. Zuckerman Z, Rodriquez-Rigau IJ, Smith KD, Steinberger E. Frequency distribution of sperm counts in fertile and infertile males. Fertil Steril 1977; 28: 1310–13. Sathananthan AH, ed. Visual Atlas of Human Sperm Structure and Function for Assisted Reproductive Technology. Melbourne: La Trobe and Monash Universities; Singapore: National University, 1996. Kruger TF, Acosta AA, Simmons KF, et al. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 1988; 49: 112–17. Kruger TF, Menkveld R, Stander FS, et al. Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril 1986; 46: 1118–23. Katz DF, Overstreet JW. Sperm motility assessment by videomicrography. Fertil Steril 1981; 35: 188–93. Katz DF, Diel L, Overstreet JW. Differences in the movements of morphologically normal and abnormal human seminal spermatozoa. Biol Reprod 1982; 26: 566–70. Franken DR, Oehninger S, Burkman LJ, et al. The hemizona assay (HZA): a prediction of human sperm fertilizing potential in in vitro fertilization (IVF) treatment. J In Vitro Fert Embryo Transfer 1989; 6: 44–50. Coetzee K, Kruger TF, Lombard CJ. Predictive value of normal sperm morphology: a structured literature review. Hum Reprod Update 1988; 4: 73–82. Enginsu MF, Pieters MGEC, Dumoulin JCM, Evers JLH, Geruedts JPM. Male factor as determinant of in vitro fertilization outcome. Hum Reprod 1992; 7: 1136–40. Davis R. The promise and pitfalls of computer aided sperm analysis. In: Diamond MP, DeCherney AH, Overstreet JW, eds. Infertility and Reproductive Medicine. Clinics of North America. Philadelphia: WB Saunders, 1992; 93: 341–52. Irvine DS. The computer assisted semen analysis systems: sperm motility assessment. Hum Reprod 1995; 10(Suppl 1): 53–9.

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Evaluation of sperm 21. Krause W. Computer assisted semen analysis systems: comparison with routine evaluation and prognostic value in male fertility and assisted reproduction. Hum Reprod 1995; 10(Suppl 4): 60–6. 22. Marshburn PB, Kuttch WH. The role of antisperm antibodies in infertility. Fertil Steril 1994; 61: 799– 811. 23. Golumb J, Vardinon N, Hommonnai ZT, et al. Demonstration of antispermotozoal antibodies in varicocele-related infertility with an enzyme-linked immunosorbent assay (ELISA). Fertil Steril 1986; 45: 397–405. 24. Helmerhost FM, Finken MJJ, Erwich JJ. Detection assays for antisperm antibodies: what do they test? Hum Reprod 1999; 14: 1669–71. 25. Bronson R. Detection of antisperm antibodies: an argument against therapeutic nihilism. Hum Reprod 1999; 14: 1671–3. 26. World Health Organization. Manual for Examination of Human Semen and Semen–Cervical Mucus. Cambridge: Cambridge University Press, 1987: 1–12. 27. Noiles EE, Ruffing NA, Kleinhans FW, et al. Critical tonicity determination of sperm using dual fluorescent staining and flow cytometry. In: Johnson LA, Rath D, eds. Reproduction in Domestic Animals. (Suppl 1) Boar Semen Preservation II. Proceedings of the Second International Conference on Boar Semen Presentation, Beltsville, MD: 1991: 359–64. 28. Noiles EE, Mazur P, Watson PF, et al. Determination of water permeability coefficient for human spermatozoa and its activation energy. Biol Reprod 1993; 48: 99– 109. 29. Jeyendran RS, Van der Ven JJ, Perez-Pelaez M. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 1984; 70: 219–28. 30. Zaneveld LJD, Jeyendran RS. Modern assessment of semen for diagnostic purposes. Semin Reprod Endocrinol 1988; 4: 323–37. 31. Chan SYW, Fox EJ, Chan MMC. The relationship between the human sperm hypoosmotic swelling test, routine semen analysis, and the human sperm zona free hamster ovum penetration test. Fertil Steril 1985; 44: 688–92. 32. Jeyendran RS, Zaneveld LJD. Human sperm hypoosmotic swelling test. Fertil Steril 1986; 46: 151–4. 33. Mladenovic I, Micic S, Genbacev O, et al. The hypoosmotic swelling test for quality control of sperm prepared for assisted reproduction. Arch Androl 1995; 34: 163–9. 34. Joshi N, Kodwany G, Balaiah D, et al. The importance of CASA and sperm function testing in a in vitro fertilization program. Int J Fertil Menopausal Stud 1996; 41(1): 46–52. 35. Critser JK, Noiles EE. Bioassays of sperm function. Semin Reprod Endocrinol 1993; 11(1): 1–16. 36. Talbot P, Chacon RS. A triple stain technique for evaluating acrosome reaction of human sperm. J Exp Zool 1981; 215: 201–8. 37. Wolf DP, Boldt J, Byrd W, et al. Acrosomal status evaluation in human ejaculated sperm with monoclonal antibodies. Biol Reprod 1985; 32: 1157–62.

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38. Cross NL, Morales P, Overstreet JW, et al. Two simple methods for detecting acrosome-reacted sperm. Gamete Res 1986; 15: 213–16. 39. Holden CA, Hyne RV, Sathananthan AH, et al. Assessment of the human sperm acrosome reaction using concanavalin A lectin. Mol Reprod Dev 1990; 25: 247– 57. 40. Chan PJ, Corselli JU, Jacobson JD, et al. Spermac stain analysis of human sperm acrosomes. Fertil Steril 1999; 72: 124–8. 41. Kennedy WP, Kaminski JM, Van der Ven HH, et al. A simple clinical assay to evaluate the acrosin activity of human spermatozoa. J Androl 1989; 10: 221–31. 42. Mohsenian M, Syner FN, Moghissi KS. A study of sperm acrosin in patients with unexplained infertility. Fertil Steril 1982; 37: 223–9. 43. Huszar G, Vigue L, Morshedi M. Sperm creatinine phosphokinase M-isoform ratios and fertilizing potential of men: a blinded study of 84 couples treated with in vitro fertilization. Fertil Steril 1992; 57: 882–8. 44. Yanagimachi R, Yanagimachi H, Rogers BJ. The use of zona-free animal ova as a free system for the assessment of their fertilizing capacity of human spermatozoa. Biol Reprod 1976; 15: 471–6. 45. Aitken RJ, Thatcher S, Glasier AF, et al. Relative ability of modified versions of the hamster oocyte penetration test, incorporating hyperosmotic medium of the ionophore A23187 to predict IVF outcome. Hum Reprod 1987; 2: 227–31. 46. Jacobs BR, Caulfield J, Boldt J. Analysis of TEST (TES and tris) yolk buffer effects on human sperm. Fertil Steril 1995; 63: 1064–70. 47. Mao C, Grimes DA. The sperm penetration assay: can it discriminate between fertile and infertile men? Am J Obstet Gynecol 1988; 159: 279–86. 48. O’Shea DL, Odem RR, Cholewa C, et al. Long-term follow-up of couples after hamster egg penetration testing. Fertil Steril 1993; 60: 1040–5. 49. ESHRE Andrology Special Interest Group. Consensus Workshop on Advanced Diagnostic Andrology Techniques. Hum Reprod 1996; 11: 1463–79. 50. Burkman LJ, Coddington CC, Franken DR, et al. The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil Steril 1988; 49: 688–97. 51. Oehninger S, Acosta AA, Marshedi M, et al. Corrective measures and pregnancy outcome in in vitro fertilization in patients with severe sperm morphology abnormalities. Fertil Steril 1989; 50: 283–7. 52. Oehninger S, Toner J, Muasher S, et al. Prediction of fertilization in vitro with human gametes; is there a litmus test? Am J Obstet Gynecol 1992; 166: 1760–7. 53. Coddington CC, Oehninger SC, Olive DL, et al. Hemizona index (HZI) demonstrates excellent predictability when evaluating sperm fertilizing capacity in in vitro fertilization patients. J Androl 1994; 15: 250–4. 54. Mori K, Daitoh T, Irahara M, et al. Significance of D-mannose as a sperm receptor site on the zona pellucida in human fertilization. Am J Obstet Gynecol 1989; 161: 207–11. 55. Mori K, Daitoh T, Kamada M, et al. Blocking of human fertilization by carbohydrates. Hum Reprod 1993; 8: 1729–32.

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56. Tesarik J, Mendoza C, Carreras R. Expression of D-mannose binding sites on human spermatozoa: comparison of fertile donors and infertile patients. Fertil Steril 1991; 56: 113–18. 57. Benoff S, Cooper GW, Hurley I, et al. Human sperm fertilizing potential in vitro is correlated with differential expression of a head-specific mannose ligand receptor. Fertil Steril 1993; 59: 854–62. 58. Silverberg K, Dey T, Witz C, et al. D-Mannose binding provides a more objective assessment of male fertility than routine semen analysis: correlation with in vitro fertilization. Presented at the 49th Annual Meeting of the American Fertility Society, October 1993. 59. Agarwal A, Said T. Role of sperm chromatin abnormalities and DNA damage in male infertility. Hum Reprod Update 2003; 9: 331–45.

60. Zini A, Bielecki R, Phang D, et al. Correlations between two markers of sperm DNA integrity, DNA denaturation and DNA fragmentation in fertile and infertile men. Fertil Steril 2001; 75: 674–7. 61. Evenson DP, Jost LK, Marshall D, et al. Utility of the sperm chromatin assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod 1999; 14: 1039–49. 62. The Practice Committee of the American Society for Reproductive Medicine. The clinical utility of sperm DNA integrity testing. Fertil Steril 2006: 86(Suppl 4): S35–7. 63. Carrell DT, Liu L, Peterson CM, et al. Sperm DNA fragmentation is increased in couples with unexplained recurrent pregnancy loss. Arch Androl 2003; 49: 49–55.

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5 Sperm preparation techniques Harold Bourne, Janell Archer, David H Edgar, HW Gordon Baker

Overview The aim of sperm preparation for assisted reproductive technologies (ART) is to maximize the chances of fertilization to provide as many normally fertilized oocytes as possible for transfer to the uterus or cryopreservation.1 With normal semen it is easy to obtain motile sperm by a variety of techniques. Abnormal semen, which will not yield adequate sperm for standard in vitro fertilization (IVF), needs to be recognized so that intracytoplasmic sperm injection (ICSI) can be used. Refinements of the preparation procedures are required to obtain spermatozoa or elongated spermatids with the highest potential for normal fertilization from grossly abnormal semen samples or from samples obtained directly from the male genital tract. Sperm characteristics important for fertilization with standard IVF include normal morphology, normal intact acrosomes, straight line velocity (VSL) and linearity (LIN), and ability to bind to the zona pellucida, penetrate the zona pellucida, fuse with the oolemma, activate the oocyte, and form a male pronucleus.1 For ICSI, live sperm with the ability to activate the oocyte and form a pronucleus are necessary, but morphology, motility, and acrosome status are generally not important.1–5 It is probably important to remove seminal plasma, as it contains decapacitation factors and extraneous cells, and degenerating sperm that may produce agents capable of damaging the sperm.6–8 For IVF or gamete intrafallopian transfer (GIFT), the medium should contain protein and buffers which promote sperm capacitation.1 While serum or high-molecularweight fractions from serum appear to be important for sperm motility, more recently relatively pure preparations of human serum albumin, pasteurized to reduce the risk of transmitting infections, have been found to be adequate for sperm preparation for standard IVF and ICSI.9,10 Purified and appropriately tested human serum albumin preparations are now routinely available from the major IVF media suppliers. The inclusion of protein in the culture medium is required to prevent sperm adhering to surfaces. Although the concentration of albumin in human periovulatory oviductal fluid is reported to be of the order of 30 mg/ml, concentrations of around 4 mg/ml will support normal sperm function in IVF. Bicarbonate

ions are required for capacitation of sperm and are normally present at about 25 mmol/l in the medium. Although glucose is utilized as a metabolic substrate by sperm, it is not clear whether it is essential for normal function in vitro. It has been suggested that more recent media formulations, which do not contain glucose, may not be appropriate for fertilization stages of ART procedures. Damage to the sperm from dilution, temperature change, centrifugation, and exposure to potentially toxic material must be minimized. Dilution should be performed slowly, especially with cryopreserved sperm. Temperature changes should be gradual. Preparation of the insemination suspension should be performed at or as close as practicable to 37°C. Centrifugal force should be the lowest possible required to bring down the most motile sperm. Minimizing centrifugation, particularly in the absence of seminal plasma, and separating the live motile sperm from the dead sperm and debris early in the procedure should limit oxidative damage caused by free oxygen radicals released from leukocytes or abnormal sperm.6,7,11 Modifications of sperm preparation may be necessary for the various types of ART. For example, for GIFT or intratubal insemination, suspensions of spermatozoa are to be introduced into the fallopian tubes, so debris and bacteria must be removed and no particulate material added which might damage the female genital tract. If cryopreserved donor sperm are to be used, matching and extra care in preparation of the sample is usually required. If the semen is severely abnormal, sperm are prepared for ICSI. Combinations of gradient centrifugation and swimup may produce higher yields of good-quality sperm.12 In the era of ICSI, the need for special preparation techniques has receded, as simple procedures with swim-up, washing, or allowing sperm to swim to the medium–oil interface from a centrifuged pellet placed in droplets of medium under oil, produce fertilization and pregnancy results as good as those with sperm obtained by more careful and laborious preparation techniques.13 The use of gradient centrifugation may also provide additional safeguards in preparing sperm from men with a chronic viral illness.14,15 The optimal number of sperm for insemination is poorly defined, but several reviews of results of IVF

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Masturbation Coitus interruptus Nontoxic condom Nocturnal emission Urine following retrograde ejaculation

Vibroejaculation

Electroejaculation emission Spermatocele aspiration Percutaneous epididymal aspiration

Prostatic cyst or Seminal vesicle aspiration for ejaculatory duct obstruction

Needle aspiration biopsy of testis

Open surgery Epididymal tubule aspiration Vas aspiration Testicular biopsy

Fig 5.1

Possible sites of collection of sperm or elongated spermatids from the male genital tract for ART.

suggest that there is an increase in fertilization rate with insemination of sperm at between 2000 and 500 000/ml.1 There may be some increase in risks of polyspermy with the higher sperm concentrations, and thus most groups inseminate oocytes with approximately 100 000 sperm/ml for standard IVF or GIFT. This is more than surround the oocyte in vivo and, if better selection of high-quality sperm could be achieved, insemination with lower numbers could be as or more successful. It has been suggested that reduced exposure of the oocyte to sperm may result in improvement in embryo quality and higher implantation rates.16,17 The total volume of sperm suspension added should be minimized to restrict dilution of the oocyte medium.

Seminiferous tubules Seminiferous tubule contents Dissect with needles

Allow to settle and transfer supernatant to new tube

Methods

Suspension of seminiferous tubule contents Incubate 37°C (up to 24 hours)

Procedures for preparation of the culture media and sperm isolation are given in Appendices 5.1–5.8 and shown schematically in Figs 5.1–5.4. Wash and resuspension

CSDG

Collection of semen or sperm While semen is usually collected by masturbation for ART, sperm may be collected by a variety of methods from several sites in the male genital tract (Fig 5.1). The man should collect semen into a sterile disposable plastic jar in a room adjacent to the IVF laboratory. The

ICSI Cryopreserve remaining sperm

Fig 5.2 Procedure for dissection of seminiferous tubules obtained by fine-needle tissue aspiration or open biopsy. CSDG, colloidal silica density gradient; ICSI, intracytoplasmic sperm injection.

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55

Mix well Direc

t swim

Dilution and centrifugation

-up f

rom

Overlay 2 ml medium seme

n

1 ml semen/pellet 37°C 45−60 min 2−3 ml medium or equal volume and

Mix

Direct CSDG

1 ml semen (or all sample for oligozoospermia)

Swim-up

Allow to settle and transfer supernatant to new tube Centrifuge 300 −1800g 10 min up mwi s nd ha as W

Aspirate supernatant Discard supernatant and resuspend pellet

Direct use for ICSI

Mix 2−3 ml of medium

1.0 ml resuspend pellet/semen 1.0 ml 40% density gradient solution 1.0 ml 80% density gradient solution

Wash and swim-up

Centrifuge 300−1800g 10 min

Centrifuge 200−400g 10−15 min

Discard upper layers, resuspend pellet Centrifuge 300−1800g/5 min Repeat wash (for IVF) Resuspend pellet in 0.3−1 ml Assess motile sperm concentration and inseminate oocytes or place in droplets for ICSI

Discard supernatant Resuspend pellet 0.3−1 ml Assess motile sperm concentration and place in droplets for ICSI

Fig 5.3 Methods of sperm preparation for ART. CSDG, colloidal silica density gradient; ICSI, introcytoplasmic sperm injection; IVF, in vitro fertilization.

sperm should be prepared soon after liquefaction of the seminal plasma. If liquefaction is delayed or the specimen is particularly viscous, syringing the sample through a 21-gauge needle or mixing the specimen 1:1 with medium followed by vigorous shaking may help. If the semen sample is unexpectedly poor, a second

sample may provide sufficient sperm. Cryopreserved sperm can also be used, for example, as backup for ICSI for patients with motile sperm present in the semen only intermittently. The timing of semen collection and preparation does not appear to be critical, especially with good

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Semen 40% CSDG layer 80% CSDG layer Pellet

Before centrifugation

After centrifugation

Fig 5.4 Appearance of gradient tubes with overlaid semen prior to and after centrifugation. CSDG, colloidal silica density gradient.

semen samples. In general, the oocytes are inseminated 4–6 hours after collection and the sperm can be prepared during this time. The semen should be placed in a clean area of the laboratory or in a laminar flow hood. The sample must be mixed thoroughly because ejaculation does not result in a homogeneous suspension of sperm in the seminal plasma. The semen sample is examined, any particulate material allowed to settle, and the supernatant transferred to another tube if necessary. Following mixing, a small portion (~10 µl) of the sample is taken to check the sperm concentration and motility. With normal semen samples, usually 1 ml of sample is sufficient for preparation of adequate numbers of motile sperm. If the semen sample is mildly to moderately abnormal but judged adequate for standard IVF, then the whole semen volume should be distributed to several tubes for preparation of as many sperm as possible.

Sperm preparation Initially, IVF involved repeated ‘washing’ of the spermatozoa by dilution of the semen with culture medium supplemented with protein, followed by centrifugation and resuspension of the pellet. This technique has been criticized as it may result in oxidative damage of the sperm by free oxygen radicals.6,7,11,18,19 Sperm for ICSI may be harvested from the oil–medium interface after sperm-containing material is placed in a drop of culture medium under oil (Fig 5.3). Some prepare channels to outlying smaller droplets for this purpose. All plastic, glassware and media should be checked for toxicity to sperm or embryos. Sperm may be immobilized by contact with rubber. A variety of media are suitable for sperm preparation for IVF (see Chapter 2). The medium chosen should be equilibrated with the

gas mixture and the temperature maintained constant at 37°C. If not using a commercially available protein source suitable for human ART but preparing a protein source in-house from serum, then this needs to be checked for sperm antibodies and, if pools are used, the donors must be tested for viral illnesses including human immunodeficiency virus (HIV) and hepatitis. However, the use of pooled serum samples is to be discouraged because of the risk of transmitting both known and unknown diseases. Heat inactivation of the serum should not be relied upon to overcome the risk of transmitting infections.

Swim-up Several variations of the swim-up procedure are possible. The seminal plasma can be overlaid directly with culture medium and the sperm allowed to swim from the seminal plasma into the culture medium. Following this, the sperm suspension should be washed to ensure adequate removal of seminal plasma constituents. Alternatively, the semen sample may be diluted and centrifuged and the pellet loosened and overlaid or the semen sample may be centrifuged without prior dilution of the seminal plasma and the pellet loosened and overlaid with medium for the swim-up procedure. The latter technique may be particularly useful for oligozoospermia as the sperm may be damaged by the dilution procedure. If cryopreserved semen is to be used, dilution of the semen sample should be slow, with dropwise addition of culture medium to the thawed sample. If the thawed semen is overlaid directly, the need for slow dilution is eliminated. After centrifugation, the supernatant is aspirated off the pellet and the pellet gently resuspended in a small volume of liquid. The overlay medium is then gently pipetted onto the surface of the pellet and the tube incubated for 45–60 minutes. Prolonged incubation times may result in a reduced yield of motile sperm from gravitational effects. The use of a conical tube for centrifugation may help maximize yield as the pellet is easier to see and less likely to be disturbed during manipulation. Some recommend that the tubes be placed in the incubator on an angle to increase the surface area of the interface. Following incubation, the upper half to two-thirds of the overlay is aspirated and mixed, and the sperm concentration determined.

Density gradients Various gradient separation procedures have been introduced. The advantage is that the gradient separation techniques are rapid, requiring 20-minute centrifugation compared with an average of 1-hour incubation for swim-up. They are also relatively simple to perform under sterile conditions. The most popular of these is colloidal silica density gradient

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(CSDG) centrifugation, but other agents have also been used.1,12,20 The colloidal silica particles are coated with polyvinylpyrollidone, e.g. PercollΤΜ (Pharmacia AB, Uppsala, Sweden). However, concerns regarding the levels of endotoxins have resulted in the withdrawal of Percoll from use in ART. Other media containing silane-coated silica have become available for clinical use from the major IVF media companies and other specialist suppliers, including IsolateΤΜ (Irvine Scientific, Santa Ana, CA, USA), PureSpermΤΜ (Nidacon Laboratories, AB, Gothenburg, Sweden), SpermGradΤΜ (Vitrolife, Englewood, CO, USA), SupraSpermΤΜ (Medicult, Jyllinge, Denmark), PureCeptionΤΜ (Sage BioPharma, Bedminster, NJ, USA), Sil-SelectΤΜ (FertiPro, Beeman, Belgium), and Sydney IVF density gradient media (William A Cook, Brisbane, QLD, Australia). Discontinuous gradients of two or more steps are used. Sperm and other material form distinct bands at the interfaces on the CSDG (Fig 5.4). It has been claimed that abnormal sperm as well as immotile sperm and debris are largely eliminated and a rapid and efficient isolation of motile human sperm, free from contamination with other seminal constituents, is possible. A number of studies have compared CSDG centrifugation with a swim-up and occasionally other sperm preparation techniques. The endpoints of the studies have been recovery of motile sperm, morphology, chromatin structure assessed by the various techniques, and ultrastructure. Generally, the recovery of motile sperm is greater with the gradient techniques, but the percentage of sperm with progressive motility is usually lower and the proportion of sperm with good morphology lower with gradient centrifugation than with swim-up.1,8,12,21–23 Some studies suggest that the gradient materials may damage the sperm.24,25 Others indicate gradient preparations produce sperm with less mitochondrial and DNA damage than other procedures.26–29 However, while there are some reports of higher fertilization and pregnancy rates, improved results of IVF and ICSI are not consistently found.26,27 CSDG in combination with swim-up has also been reported to reduce the viral load from samples carrying an infectious agent such as HIV.30–34 However, CSDG on its own, or in combination with swim-up, should not be relied upon to minimize the risk of infection to the woman or any resulting pregnancy,31,35–39 although modifications to the procedure may further reduce the chance for viral contamination of the final preparation.39–41

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Sperm selection from immotile samples ICSI with immotile sperm is often associated with low fertilization rates; thus every attempt should be made to ensure that live sperm are injected.1–4,42 A variety of agents have been reported to enhance sperm motility.1 Pentoxifylline (POF) has been used for ART. The maximally effective dose of POF is between 0.3 and 0.6 mmol/l and many groups use 3.6 mmol/l (1 mg/ml). POF has been reported to provide greater stimulation of motility and velocity than caffeine or 2-deoxyadenosine. The use of a laser pulse, applied to the tail tip of an immotile sperm, has been suggested as a useful tool to identify viable sperm for injection.43 However, this approach awaits verification in a wider setting. Appendices 5.10 and 5.11 give methods for stimulating sperm motility with POF and demonstrating membrane integrity by hypoosmotic swelling.

Preparation of semen from HIV-infected men The use of combination antiretroviral (cARV) therapies has markedly improved the prognosis and life expectancy for men infected with HIV. A population of these men in a discordant relationship (i.e. where the man is seropositive and the woman is uninfected) are desiring parenthood using their own gametes. With appropriate medical care and use of ART techniques, safe and effective treatment can now be offered for achieving a pregnancy while minimizing the risk of transmission to either the partner or a resulting baby.14,15,44–49 Most protocols recommend the use of antiretroviral therapies to reduce viral load, subsequent testing of sperm samples for residual viral HIV RNA and DNA using sensitive polymerase chain reaction (PCR) techniques, and the preparation of cleared samples for clinical use via density gradient centrifugation. Normally, cryopreservation of the sperm is required to allow adequate time for the testing regimens to be completed. Samples with undetectable or sufficiently low viral loads are cleared for use and, depending on the quality of the sperm post thaw, an attempt at pregnancy is undertaken using intrauterine insemination (IUI), IVF, or ICSI. A flow diagram for the treatment of discordant couples where the male is seropositive is presented in Fig 5.5.

Results Sperm preparation from surgical aspirates or tissue samples Spermatozoa or elongated spermatids may be obtained for ICSI from the male genital tract by microsurgical epididymal sperm aspiration (MESA), percutaneous epididymal sperm aspiration (PESA), testicular open biopsy or fine needle aspiration biopsy, or other techniques (Fig 5.1) and prepared by the methods outlined in Appendices 5.7 and 5.8.1,2

Comparison of normal fertilization and embryo utilization rates for swim-up and CSDG, categorized according to male indication, are presented in Table 5.1. Apart from the improvement in the normal fertilization rate with CSDG for IVF with oligozoospermic samples, the results are similar. Results with sperm or elongated spermatids obtained from the genital tract, cryopreserved samples, and following the use of hypo-osmotic swelling have been published.4,50,51

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Initial consult Patients on cARV therapy and in good health

Review/alteration of cARV therapy by infectious diseases specialists

Detectable HIV RNA

Pre semen-screening phase Patients to have low or undetectable (<50 copies/ml) HIV RNA in blood for at least 2 months

• •

– ve HIV RNA but + ve HIV DNA

Screening phase At least two successive semen samples with undetectable HIV RNA (<100 copies/ml) to be produced First screening sample to be tested not more than 60 days before subsequent storage phase

•

Storage phase Cryopreserve multiple samples

•

Complete within 60 days of commencing storage

•

An aliquot of semen from every sample is tested for HIV RNA and HIV DNA

+ ve HIV RNA

– ve HIV RNA – ve HIV DNA

Prepare aliquot by CSDG and repeat screen for HIV DNA

– ve HIV DNA

Clearance Samples with undetectable HIV RNA and HIV DNA are suitable for clinical use

+ ve HIV DNA

Discard sample

Discard sample

•

ART treatment Check all tests completed and confirmed

•

Commence ART treatment

•

Prepare cleared samples by CSDG and use for IUI, IVF, or ICSI, depending on post-thaw sperm quality and clinical indications

Fig 5.5 Flow chart for the treatment of human immunodeficeincy virus (HIV) discordant couples (male seropositive and female uninfected) using ART to minimize the risk of HIV transmission. CSDG, colloidal silica density gradient; ICSI, intracytoplasmic sperm injection; IUI, intrauterine insemination; IVF, in vitro fertilization; CARV, combination antiretroviral.

Fertilization results and incidence of poor outcome for different culture conditions and sperm preparation methods are presented in Table 5.2. The normal fertilization rate improved steadily following

the introduction of closed, mini-incubators (William A Cook), and the change from a single-stage medium (human tubal fluid, HTF, Irvine Scientific, supplemented with 4 mg/ml human serum albumin,

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Table 5.1 Comparison of results with swim-up and colloidal silica density gradient (CSDG) preparation of sperm from semen for in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) from men with normal semen (sperm concentration ≥20 × 106/ml, progressive motility ≥40% and abnormal morphology ≤85%), abnormal semen (sperm concentration 1–19 × 106/ml or progressive motility 1–39% or abnormal morphology 86–100%) or oligoasthenoteratozoospermia (sperm concentration 1–19 × 106/ml, progressive motility 1–39%, and abnormal morphology 86–100%) from 1990 to 1999. Men with sperm autoimmunity were excluded. Embryo utilization is the sum of embryos transferred fresh and those frozen for later transfer. Percentages using oocytes collected as the denominator are shown in italics. Asterisks indicate significant differences between results for swim-up and CSDG (p <0.05, χ2 test) IVF Oocytes collected

ICSI

IVF

Normal fertilization

Embryo utilization

Oocytes collected

21031 99 3298 99

12286 58 1833* 55

10520 49 1577* 48

1396

8733 99 5943 97

4236 48 2720* 44

3513 40 2338* 38

5718

Oligoasthenoteratozoospermia Swim-up 360 354 98 CSDG 1183 1158 98

97 27 416* 35

93 26 358 30

1328

Normal semen Swim-up 21255 CSDG

3319

Abnormal semen Swim-up 8826 CSDG

6126

Albumex-20, CSL Ltd, Melbourne, Australia) to a sequential medium formulation containing a different albumin (Quinn’s Advantage, containing 4 mg/ml human albumin, Sage BioPharma). These results were matched by an overall decrease in the incidence of cycles with poor fertilization, indicating that increased fertilization was also achieved in patients with a poor prognosis. Following the routine introduction of density gradient centrifugation, improvement in fertilization rate and a decrease in the incidence of poor fertilization cycles were found for standard IVF inseminations, probably due to a reduced chance for carryover of inhibitory components from seminal plasma following CSDG preparation. No difference in fertilization rate was observed between swim-up and CSDGprepared sperm for ICSI. Thus, improvements in fertilization results can be achieved by optimizing culture conditions. In preparing sperm for ART, CSDG centrifugation appears a more reliable method for standard IVF, while swimup provides a simple alternative approach for intracytoplasmic injection. Results from the treatment of HIV discordant couples are summarized in Table 5.3. Pregnancy rates following the use of standard IVF or ICSI are within expectations. However, only a limited number of pregnancies have been achieved following the use of IUI, which may reflect the effect of illness and cryopreservation on the quality of sperm preparations in

905

6387

2436

ICSI

Normal fertilization

Embryo utilization

1113 80 685 76

665 48 394 44

545 39 322 36

4664 82 5221 82

2804 49 3054 48

2367 41 2567 40

1072 81 2016 83

610 46 1142 47

514 39 941 39

these men. No seroconversions have been identified in any of the women or delivered babies.

Complications Although there is potential for semen- or spermdependent complications of ART, such as infections or allergic reactions, these are very rare. Patients should be tested for serious transmissible infections such as HIV and hepatitis, and standard precautions for handling biological material must be practiced in the embryology laboratory. Transmission of genetic conditions to offspring is possible: suitable counseling and, where possible, screening should be part of the clinical work-up of the couple. Strict laboratory quality control should minimize the risks of loss or errors of identity of gametes or embryos. With ICSI for primary spermatogenic disorders, an increased frequency of sex chromosomal aneuploidies has been noted in the conceptuses.52 In some clinics there appears to be a higher rate of abnormal fertilization with ICSI using testicular sperm.51

Future directions and controversies The main problems to be solved in the future are the accurate identification of patients who are likely to have problems with fertilization and require ICSI, effective treatment of defective sperm production or function, and improved implantation and pregnancy rates with

HTF medium/ open incubators

Sequential medium/ open incubators

Sequential medium/ mini-incubators

1999–2000

2001

2003

No. of cycles

1327 330 465 456

Sperm prep

Swim-up Swim-up CSDG CSDG

Eggs injected

10188 2915 3829 3921

Cycles with <20% fert

106 (8.0%)a,b 32 (9.7%)c,d 24 (5.2%)a,c 17 (3.7%)b,d

2848 (72.6%)e,f

2597 (67.8%)f

1977 (67.8%)e

6605 (64.8%)e,f

Fertilized normally

HTF, human tubal fluid; CSDG, colloidal silica density gradient; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization.

Culture conditions

Time period

477

392

324

1372

No. of cycles

53 (11.1%)h,j

45 (11.5%)g,i

59 (18.2%)i,j

254 (18.5%)g,h

Cycles with <20% fert

4473

3689

3187

12174

Eggs inseminated

Standard IVF

2861 (64.0%)k,l

2152 (58.3%)k,l

1699 (53.3%)l

6455 (53.0%)k

Fertilized normally

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<20% of eggs fertilized normally) for different culture conditions and sperm preparation methTable 5.2 Comparison of fertilization rate and incidence of cycles with poor fertilization (< ods (prep), introduced sequentially over a 3-year period. Results with the same superscripts are significantly different by χ2 test

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Table 5.3 Results from ART cycles in HIV discordant couples (i.e. where the man is seropositive and the women is uninfected) treated since 2002 using cryopreserved semen with an undetectable viral load. No seroconversions have been identified in any women or delivered babies following ART treatment

Couples

Completed cycles

FH pregnancies (% per cycle)

Deliveries to date

HIV transmissions

IUI

13

51

3 (5.9%)

2

Nil

IVF / ICSI (fresh)

12

21

4 (19%)

2

Nil

6

11

4 (36%)

3

Nil

Cycle type

Thaws

ART, assisted reproductive technologies; FH, fetal heart; HIV, human immunodeficiency virus; ICSI, intracytoplasmic sperm injection; IUI, intrauterine insemination; IVF, in vitro fertilization.

ART. Improved prediction of results will come from development of new methods of semen analysis such as automated sperm morphology and simple tests for assessing the ability of sperm to interact with oocytes.1 Effective treatment of most forms of male infertility is only a remote possibility, especially as the pathogenesis remains obscure.53 Further studies should resolve questions about the involvement of free oxygen species in the pathogenesis of sperm defects and whether this may affect the health of the offspring.6,11,18,19,54 New technology may improve the procedures for activation of the oocyte, to allow direct injection of a sperm head or nucleus from spermatids or spermatocytes, although there is rarely a need for this clinically.55 The contribution of the sperm to abnormal embryonic development, failure of implantation, and pregnancy wastage will probably become clear as preimplantation genetic diagnosis and other tests of embryos are more widely used. Practical methods for selection of sperm with normal chromosomes or a desired sex chromosome are likely to be developed.56

•

•

Appendix 5.2 Choice of method •

• •

Conclusion The principles of sperm preparation for IVF and ICSI are outlined and practical methods are given.

Acknowledgments The authors thank Associate Professor GN Clarke for advice about cryopreservation procedures, Dr M Giles, Dr A Mijch, Dr P Foster, Professor S Garland, and L Valent for assistance with the management of HIV discordant couples, and Dr C Garrett and Ms P Sourivong for assistance with the figures.

Appendix 5.1 Preparation of media •

Quinn’s Advantage (QA) sequential culture media (fertilization medium) and QA medium with N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) sodium salt buffer (QA/HEPES) are purchased from Sage BioPharma (Bedminster, USA).

As required, human albumin (ALB) solution (100 mg/ml, Sage BioPharma, pharmaceutical grade) is added (1 part in 25), to give a final concentration of 4 mg/ml. Both QA fertilization medium with albumin (QA fert/ALB) and QA/HEPES/ALB are prepared and stored refrigerated until required (maximum storage time according to the manufacturer’s expiry date: about 6 weeks).

•

Patient and sample identity should be checked with another person and recorded as a quality assurance measure. Examine a drop of undiluted semen (hemocytometer or Makler chamber) For standard IVF: – Prepare sperm by density gradient centrifugation: • If the sample is unexpectedly poor on the day (e.g. concentration <10 × 106/ml, <40% motility and/or poor forward progression), ICSI should be considered. For ICSI: – Prepare sperm by density gradient centrifugation or swim-up: • Even samples with severe oligozoospermia (down to ~10 000/ml) can be prepared using swim-up as long as there are some sperm with good forward progression. • Alternatively, samples may be concentrated by wash and resuspension and used directly or prepared further by density gradient centrifugation or swim-up • Samples with large amounts of debris, extreme oligozoospermia, or severely compromised motility are better prepared by CSDG. • Surgical samples obtained from the testis or epididymis and those collected by electroejaculation typically have a low motile sperm concentration and are more suited to CSDG separation to maximize yield and remove tissue debris. Alternatively, surgical samples can be used directly if there

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is little extraneous cellular material and sufficient progressive motility to allow sperm to migrate to the edge of the drop.

•

Appendix 5.3 Density gradient CSDG stock solutions •

•

‘PureSperm’ (to make 50 ml of solution): – 40% stock solution: 20 ml ‘PureSperm’, 28 ml QA/HEPES, 2 ml of 100 mg/ml human albumin (pharmaceutical grade) – 80% stock solution: 40 ml ‘PureSperm’, 8 ml QA/HEPES, 2 ml of 100 mg/ml human albumin (pharmaceutical grade) ‘Isolate’ (to make 50 ml of solution): – 40% stock solution: 48 ml of 40% ‘Isolate UPPER’, 2 ml of 100 mg/ml human albumin (pharmaceutical grade) – 80% stock solution: 48 ml of 80% ‘Isolate LOWER’, 2 ml of 100 mg/ml human albumin (pharmaceutical grade).

•

•

•

Preparation and use of gradients •

•

• •

•

•

Prepare sufficient tubes for each patient: – dispense 1.0 ml of 40% gradient stock solution into a 15 ml conical tube (Falcon 2095, Becton Dickinson, NJ, USA) – with a clean pasteur pipette, underlay 1.0 ml of 80% gradient stock solution. Carefully overlay ∼1 ml of semen (fresh or thawed) or sperm suspension directly on top of gradient: – ensure gradients are at room temperature before overlaying. Prepare multiple gradients if the sperm concentration is low. To maximize yield (e.g. for severe oligozoospermia or testicular biopsy samples): – samples may be concentrated by wash and resuspension prior to placing onto gradient – centrifugation speed and time may be increased, as described below – additional sperm can also be obtained by wash and resuspension of the upper gradient layers/ supernatant normally discarded after removal of the bottom gradient layer and pellet – a background of fine, flocculent particles (which can occur when processing for maximum yield using ‘PureSperm’ and may interfere with the collection of sperm for injection) can be minimized with the use of an ‘Isolate’ prepared gradient. Centrifuge at 200–300g for 10 minutes (braking may be used during deceleration): – increase centrifugation to 400g for 15 minutes to improve yield of poor ICSI samples. Gently remove all but the bottom 0.3–0.5 ml and place in a discard tube.

•

With a clean pasteur pipette gently aspirate the remaining solution and pellet and transfer to a fresh conical tube containing ∼8 ml of medium, in preparation for a single wash: – avoid contact with the sides of the tube to minimize carryover of seminal plasma and debris – for IVF, the single large volume wash may be replaced by two smaller volume washes (∼3–4 ml each) if there are concerns for carryover of seminal components into the final preparation. Centrifuge at 300g for 5 minutes: – use increased centrifugation (up to 1800g) to maximize yield of poor ICSI samples. Remove supernatant and resuspend in 0.3–1.0 ml of QA/fert medium (for IVF) or QA/HEPES/ALB (for ICSI) Assess sperm quality in the final sample (count, motility, and progression) and calculate volume required for insemination as follows: – place ∼5 µl of prepared sperm onto a counting chamber (hemocytometer or Makler chamber) and allow to settle (>3 minutes) – grade forward progression (FP) as follows: FP0, no movement; FP1, movement but minimal progression; FP2, slow progression; FP3, moderate to rapid progression – count the number of motile sperm in a minimum of 5 squares from the central 25 squares (hemocytometer), or a minimum of 10 squares (Makler chamber), to estimate the motile concentration (to improve accuracy, aim to count at least 50 sperm) – for IVF: calculate volume of final sperm suspension required for insemination (ideally 10–50 µl) to give a total of 100 000 to 200 000 FP3 sperm/ml in the medium containing the oocytes. Ιncubate at 37°C under 5% CO2 (IVF) or room atmosphere (ICSI) until required.

Appendix 5.4 Swim-up •

•

After the semen has liquefied (usually 30 minutes at 37°C), 1 ml aliquots of semen are placed in 5 ml labeled tubes (Falcon 2003) and gently overlaid with 2 ml of medium (QA/HEPES/ALB): – for samples with poor mucolysis, dilute semen with 2–3 volumes of appropriate medium and mix vigorously; allow any particulate matter to settle, transfer supernatant to another test tube and use as described for liquefied semen – alternatively, samples may be prepared as described for wash and resuspension, or the semen may be centrifuged directly and the seminal plasma removed, prior to overlaying the resulting pellet with medium for swim-up. Incubate tubes at 37°C for 45–60 minutes to allow progressively motile sperm to swim into the overlaid medium.

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•

• •

Taking care not to disrupt the interface or collect any seminal plasma, collect the overlaid medium, mix with 2–3 ml of medium and centrifuge at 300g for 5–10 minutes (increase centrifugation up to 1800g to maximize yield of poor ICSI samples). Remove the resulting supernatant and resuspend the pellet in 0.3–1.0 ml of fresh medium. Assess sperm quality (count, motility, and velocity) and incubate at 37°C in room atmosphere until required for ICSI.

Appendix 5.5

Appendix 5.7 MESA/PESA •

• • •

•

Wash and resuspension • • •

• •

Allow semen to liquefy at 37°C for 30 minutes. Mix 1 ml of semen with 2–3 ml of appropriate medium. Centrifuge for 10 minutes at 300g (increase centrifugation up to 1800g to maximize yield of poor ICSI samples). Aspirate supernatant and resuspend pellet in 0.3– 1 ml of appropriate medium. Assess sperm quality in the final sample (count, motility, and progression) and incubate at 37°C until required, as previously described.

63

•

Epididymal sperm are obtained either by microsurgery (microsurgical epididymal sperm aspiration [MESA]) or by percutaneous, fine-needle aspiration (percutaneous epididymal sperm aspiration [PESA]). Expel aspirates into a small Petri dish of warm QA/HEPES/ALB. Pool samples and concentrate if necessary. Depending on concentration, motility, and amount of debris, either use directly, prepare by wash and resuspension, or separate on a density gradient. Leave sperm to incubate to allow sperm to gain motility: – up to 24 hours in QA fert/ALB at 37°C under 5% CO2 – for same day use, prepare plate for ICSI and leave at 37°C in QA/HEPES/ALB (room atmosphere). If extra sperm are available, consider freezing the excess. Samples with >5000 motile sperm/ml should have sufficient yield of live sperm post thaw for subsequent ICSI treatments. A method for cryopreservation of such samples is given in Appendix 5.9.

Appendix 5.8 Appendix 5.6

Testicular biopsy

Frozen sperm

•

• •

• •

• •

•

•

Double check straw/vial code and patient ID. For straws: – Thaw straw in air for 10–20 minutes; check integrity of the straw and discard if damaged. – Soak straw in hypochlorite solution prepared fresh daily (∼0.5% available chlorine; e.g. 1:1 dilution of Milton antibacterial solution, Proctor and Gamble, Australia) for at least 2 minutes to disinfect outside of straw and reduce chance for cross-infection; rinse in fresh water and wipe excess solution from the straw after soaking. Cut one end and aspirate contents. For vials: – loosen cap (to prevent the build-up of pressure during thawing) and thaw at 37°C. Assess sperm concentration, motility, and progression. Prepare sample by density gradient centrifugation as previously described: – alternatively, samples for ICSI may be prepared by swim-up, if sufficient motile sperm are present, or wash and resuspension if there is minimal extraneous cellular material or debris. Assess sperm count, motility, and progression in the final suspension. For IVF samples, calculate the volume required for insemination. Incubate at 37°C under 5% CO2 (IVF) or room atmosphere (ICSI) until required.

• • • •

•

•

Testicular tissue is obtained either by open biopsy (testicular sperm extraction [TESE]) or percutaneous fine-needle aspiration (testicular sperm aspiration [TESA]). Place tissue into a small Petri dish of warm QA/HEPES/ALB. Dissect and squeeze tubules using fine gauge needles (Fig 5.2). Transfer raw suspension to a test tube. Depending on concentration, motility, and amount of debris, either use directly, prepare by wash and resuspension, or separate on a density gradient. Leave sperm to incubate to allow sperm to gain motility: – up to 24 hours in QA fert/ALB at 37°C under 5% CO2 – for same day use, prepare plate for ICSI and leave at 37°C in QA/HEPES/ALB (room atmosphere) If extra sperm are available, consider freezing the excess (Appendix 5.9).

Appendix 5.9 Freezing protocol for oligozoospermia and washed sperm •

•

Semen containing only a few motile sperm and sperm suspensions obtained from the genital tract can be stored for subsequent ICSI. Epididymal, testicular, and oligozoospermic sperm suspensions are routinely processed by

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•

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density gradient centrifugation or wash and resuspension. Sperm excess to that required for treatment can be cryopreserved with glucose–citrate–glycine (GCG)–glycerol cryoprotectant supplemented with human albumin. GCG–glycerol cryoprotectant: – dissolve glucose (1.0 g) and sodium citrate (1.0 g) in 40 ml of sterile deionized water – add glycine (1.0 g) (pH ∼7.5 and osmolality ∼500 mosm/kg) – add 10 ml of glycerol, mix and filter (0.2 µm) – store in 2 ml volumes at −70°C. Albumin stock solution: – dilute albumin (Sage BioPharma, 100 mg/ml) 1:1 with Tyrode buffer to make albumin stock solution (50 mg/ml); filter (0.45 µm) and store at –70°C. As required, thaw a vial of GCG–glycerol. Add equal volume of albumin stock solution to the sperm sample and mix well. Add GCG–glycerol solution to sperm/albumin suspension 1:2 (1 volume of GCG–glycerol to 2 volumes of sperm/albumin) gradually over ~10 minutes with mixing. Package in cryovials (Nunc A/S, Denmark) and freeze: – freeze gradually by suspending in liquid nitrogen vapor and store similarly. Alternatively, samples can be frozen using commercially available freezing media (e.g. QA Sperm Freeze, Sage BioPharma): – add 1 volume of freezing medium, dropwise, to an equal volume of sperm suspension (add slowly over ∼5 minutes and mix well between additions) – place the final sperm/cryoprotectant mixture into cryovials (∼1.5 ml per vial) – freeze and store over liquid nitrogen vapor as described above.

Appendix 5.10 Use of pentoxifylline •

• • • • • •

Prepare a 10 × concentrated solution of pentoxifylline (POF, Sigma) in protein-free QA/HEPES (POF MW = 278.3; 10 × concentrate = 10 mg/ml). Sterilize through a 0.2 µm filter and store at 4°C. Dilute 1:9 with sperm suspension to expose sperm to a final concentration of 1 mg/ml POF (3.6 mmol/l). Spread the treated sperm suspension adjacent to the holding drops in the injection plate. Functional sperm should show motility within 10 minutes of exposure to the stimulant. Move the motile sperm to clean, stimulant-free medium. Expel the treated medium from the injection pipette and rinse with the untreated, clean medium in the holding drop; repeat rinsing of sperm and injection pipette.

• •

Immobilize the selected sperm and perform ICSI as usual. Aim to collect the motile sperm without excessive delay (within ~3 hours) as the treated sperm may lose motility with time.

Appendix 5.11 Use of hypo-osmotic medium • • • • •

• •

•

Prepare a 100–150 mosm/kg solution by diluting QA/HEPES/ALB 1:1 or 1:2 with purified water. Filter and store at 4°C. Add a drop of hypo-osmotic medium adjacent to the holding drops in the injection plate. Transfer sperm using the injection pipette to the hypo-osmotic medium. Immotile sperm with an intact plasma membrane should coil their tails shortly after contacting the hypo-osmotic medium. Move the presumed live sperm to the normo-osmotic oocyte holding drop and leave briefly to equilibrate. Expel the hypo-osmotic medium from the injection pipette and rinse with normo-osmotic medium; repeat rinsing of sperm and injection pipette. Immobilize the selected sperm and perform ICSI as usual.

References 1. Baker G, Liu DY, Bourne H. Assessment of the male and preparation of sperm for ARTs. In: Trounson AO, Gardner DK, eds. Handbook of In Vitro Fertilization. Boca Raton: CRC Press, 1999: 99–126. 2. Nagy Z, Liu J, Cecile J, et al. Using ejaculated, fresh, and frozen–thawed epididymal and testicular spermatozoa gives rise to comparable results after intracytoplasmic sperm injection. Fertil Steril 1995; 63: 808–15. 3. Nagy ZP, Verheyen G, Tournaye H, et al. Special applications of intracytoplasmic sperm injection: the influence of sperm count, motility, morphology, source and sperm antibody on the outcome of ICSI. Hum Reprod 1998; 13(Suppl 1): 143–54. 4. Bourne H, Richings N, Liu DY, et al. Sperm preparation for intracytoplasmic injection: methods and relationship to fertilization results. Reprod Fertil Dev 1995; 7: 177–83. 5. Dozortsev D, Rybouchkin A, De Sutter P, et al. Human oocyte activation following intracytoplasmic injection: the role of the sperm cell. Hum Reprod 1995; 10: 403–7. 6. Aitken RJ, Gordon E, Harkiss D, et al. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 1998; 59: 1037–46. 7. Mortimer D. Sperm preparation techniques and iatrogenic failures of in-vitro fertilization. Hum Reprod 1991; 6: 173–6. 8. Mortimer D. Sperm recovery techniques to maximize fertilizing capacity. Reprod Fertil Dev 1994; 6: 25–31.

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Textbook of Assisted Reproductive Technologies semen by the swim-up method: assisted reproduction technique using spermatozoa free from HIV-1. AIDS 2006; 20: 967–73. Loskutoff NM, Huyser C, Singh R, et al. Use of a novel washing method combining multiple density gradients and trypsin for removing human immunodeficiency virus-1 and hepatitis C virus from semen. Fertil Steril 2005; 84: 1001–10. Casper RF, Meriano JS, Jarvi KA, et al. The hypoosmotic swelling test for selection of viable sperm for intracytoplasmic sperm injection in men with complete asthenozoospermia. Fertil Steril 1996; 65: 972–6. Aktan TM, Montag M, Duman S, et al. Use of a laser to detect viable but immotile spermatozoa. Andrologia 2004; 36: 366–9. Manigart Y, Rozenberg S, Barlow P, et al. ART outcome in HIV-infected patients. Hum Reprod 2006; 21: 2935–40. Gilling-Smith C, Nicopoullos JD, Semprini AE, et al. HIV and reproductive care – a review of current practice. BJOG 2006; 113: 869–78. Terriou P, Auquier P, Chabert-Orsini V, et al. Outcome of ICSI in HIV-1-infected women. Hum Reprod 2005; 20: 2838–43. Semprini AE, Fiore S. HIV and reproduction. Curr Opin Obstet Gynecol 2004; 16: 257–62. Ohl J, Partisani M, Wittemer C, et al. Assisted reproduction techniques for HIV serodiscordant couples: 18 months of experience. Hum Reprod 2003; 18: 1244–9. Pena JE, Thornton MH, Sauer MV. Assessing the clinical utility of in vitro fertilization with intracytoplasmic

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sperm injection in human immunodeficiency virus type 1 serodiscordant couples: report of 113 consecutive cycles. Fertil Steril 2003; 80: 356–62. Harari O, Bourne H, McDonald M, et al. Intracytoplasmic sperm injection: a major advance in the management of severe male subfertility. Fertil Steril 1995; 64: 360–8. Watkins W, Nieto F, Bourne H, et al. Testicular and epididymal sperm in a microinjection program: methods of retrieval and results. Fertil Steril 1997; 67: 527–35. Bonduelle M, Wilikens A, Buysse A, et al. A followup study of children born after intracytoplasmic sperm injection (ICSI) with epididymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI. Hum Reprod 1998; 13(Suppl 1): 196–207. De Kretser DM, Baker HW. Infertility in men: recent advances and continuing controversies. J Clin Endocrinol Metab 1999; 84: 3443–50. Baker HWG. Marvellous ICSI: the viewpoint of a clinician. Int J Androl 1998; 21: 249–52. Antinori S, Versaci C, Dani G, et al. Successful fertilization and pregnancy after injection of frozen– thawed round spermatids into human oocytes. Hum Reprod 1997; 12: 554–6. Fugger EF, Black SH, Keyvanfar K, et al. Births of normal daughters after MicroSort sperm separation and intrauterine insemination, in-vitro fertilization, or intracytoplasmic sperm injection. Hum Reprod 1998; 13: 2367–70.

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6 Sperm chromatin assessment Ashok Agarwal, Juris Erenpreiss, Rakesh Sharma

Introduction Semen analysis is used routinely to evaluate infertile men. Attempts to introduce quality control within and between laboratories have highlighted the subjectivity and variability of traditional semen parameters. A significant overlap in sperm concentration, motility, and morphology between fertile and infertile men has been demonstrated.1 In addition, standard measurements may not reveal subtle sperm defects such as DNA damage, and these defects can affect fertility. New markers are needed to better discriminate infertile men from fertile ones, predict pregnancy outcome in the female partner, and calculate the risk of adverse reproductive events. In this context, sperm chromatin abnormalities have been studied extensively in past decades as a cause of male infertility.2 Focus on the genomic integrity of the male gamete has been intensified by the growing concern about transmission of damaged DNA through assisted reproductive technologies (ART), especially intracytoplasmic sperm injection (ICSI). It is a particular concern if the amount of damage exceeds the DNA repair capacity of oocytes. There are concerns related to potential chromosomal abnormalities, congenital malformations, and developmental abnormalities in ICSI-born progeny.3–6 Accumulating evidence suggests that a negative relationship exists between disturbances in the organization of the genomic material in sperm nuclei and the fertility potential of spermatozoa, whether in vivo or in vitro.2–14 Abnormalities in the male genome characterized by damaged sperm DNA may be indicative of male subfertility, regardless of normal semen parameters.15,16 Sperm chromatin structure evaluation is an independent measure of sperm quality that provides good diagnostic and prognostic capabilities. Therefore, it may be considered a reliable predictor of a couple’s inability to become pregnant.17 This may have an impact on the offspring, resulting in infertility.18 Sperm DNA integrity correlates with pregnancy outcome in in vitro fertilization.17,19–22 high sperm DNA fragmentation can compromise embryo quality

and result in pregnancy loss.10 In addition, sperm DNA fragmentation may also compromise the progression of pregnancy and result in spontaneous miscarriage or biochemical pregnancy following ART. Sperm DNA fragmentation seems to affect embryo postimplantation development in ICSI procedures.10 Therefore, it is recommended that sperm DNA fragmentation analysis should be included in the evaluation of the infertile male.22 Many techniques have been described for evaluation of the chromatin status. In this chapter, we describe the normal sperm chromatin architecture and the causative factors leading to its aberrations. We also provide the rationale for sperm chromatin assessment and discuss the different methods used to analyze sperm DNA integrity.

Human sperm chromatin structure In many mammals, spermatogenesis leads to the production of highly homogeneous spermatozoa. For example, more than 95% of the nucleoprotein in mouse sperm nuclei is composed of protamines.23 This allows mature sperm nuclei to adopt a volume 40 times less than that of normal somatic nuclei.24 The final, highly compact packaging of the primary sperm DNA filament is produced by DNA–protamine complexes. Contrary to nucleosomal organization in somatic cells, which is provided by histones, these DNA–protamine complexes approach the physical limits of molecular compaction.25 Human sperm nuclei, on the other hand, contain considerably fewer protamines (around 85%) than sperm nuclei of the bull, stallion, hamster, and mouse.26,27 Human sperm chromatin is therefore less regularly compacted and frequently contains DNA strand breaks.28,29 To achieve this uniquely condensed state, sperm DNA must be organized in a specific manner that differs substantially from that of somatic cells.24 The fundamental packaging unit of mammalian sperm chromatin is a toroid containing 50–60 kilobases of DNA. Individual toroids represent the DNA

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loop-domains highly condensed by protamines and fixed at the nuclear matrix. Toroids are cross-linked by disulfide bonds formed by oxidation of sulfhydryl groups of cysteine present in the protamines.25,30 Thus, each chromosome represents a garland of toroids, while all 23 chromosomes are clustered by centromeres into a compact chromocenter positioned well inside the nucleus; the telomere ends are united into dimers exposed to the nuclear periphery.31,32 This condensed, insoluble, and highly organized nature of sperm chromatin acts to protect the genetic integrity during transport of the paternal genome through the male and female reproductive tracts. It also ensures that the paternal DNA is delivered in the form that sterically allows the proper fusion of two gametic genomes and enables the developing embryo to correctly express the genetic information.32–34 In comparison with other species,35 human sperm chromatin packaging is exceptionally variable both within and between men. This variability has been mostly attributed to its basic protein component. The retention of 15% histones, which are less basic than protamines, leads to the formation of a less-compact chromatin structure.27 Moreover, in contrast to the bull, cat, boar, and ram – whose spermatozoa contain only one type of protamine (P1) – human and mouse spermatozoa contain a second type of protamine, called P2, which is deficient in cysteine residues.36 Consequently, the disulfide cross-linking responsible for more stable packaging is diminished in human sperm, as compared with species containing P1 alone.37 It is interesting to note that altered P1/P2 ratios and the absence of P2 are associated with male fertility problems.38–42

Origin of sperm chromatin abnormalities The susceptibility of male germ cells to DNA damage stems partly from the down-regulation of DNA repair systems during late spermatogenesis. In addition, the cellular machinery that allows these cells to undergo complete apoptosis is progressively lost during spermatogenesis. As a result, the advanced stages of germ cell differentiation cannot be deleted even though they may have proceeded some way down the apoptotic pathway. As a consequence, the ejaculated gamete may exhibit genetic damage. Such DNA damage will be carried into the zygote by the fertilizing spermatozoon and must be then repaired, preferably prior to the first cleavage division. Several studies have shown that oocytes and early embryos can repair sperm DNA damage.43,44 Consequently, the biological effect of abnormal sperm chromatin structure depends on the combined effects of sperm chromatin damage and the capacity of the oocyte to repair it. Any errors that may occur during this postfertilization period of DNA

repair have the potential to create mutations that can affect fetal development and, ultimately, the health of the child.18,45 The exact mechanisms by which chromatin abnormalities/DNA damage arise in human spermatozoa are not completely understood. Three main theories have been proposed: defective sperm chromatin packaging, abortive apoptosis, and oxidative stress (OS). Deficiencies in recombination may also play a role.

Defective sperm chromatin packaging Stage-specific introduction of transient DNA strand breaks during spermiogenesis has been described.46–48 DNA breaks have been found in round and elongating spermatids. Such breaks are necessary for transient relief of torsional stress. During maturation, the nucleosome histone cores in elongating spermatids are cast off and replaced with transitional proteins and protamines.46,48–50 Thus, chromatin repackaging includes a sensitive step, necessitating endogenous nuclease activity, which is evidently fulfilled by coordinated loosening of the chromatin by histone hyperacetylation and introduction of breaks by topoisomerase II that is able to create and ligate breaks.49,50 Although there is little evidence to suggest that spermatid maturation-associated DNA breaks are fully ligated, unrepaired DNA breaks are not allowed.51 Ligation of DNA breaks is necessary not only to preserve the integrity of the primary DNA structure but also for reassembly of the important unit of genome expression, the DNA loop-domain. However, if these temporary breaks are not repaired because of excessive topoisomerase II activity or a deficiency of topoisomerase II inhibitors,52,53 DNA fragmentation in ejaculated spermatozoa may result. Similarly, if appropriate disulfide bridge formation does not occur because of inadequate oxidation of thiols during epididymal transit, the DNA will be more vulnerable to damage caused by suboptimal compaction.

Abortive apoptosis The incidence of apoptosis in ejaculated sperm is still a contentious issue. Until recently, the inability of a mature spermatozoon to synthesize new proteins was believed to make it impossible for such cells to respond to any of the signals that lead to the programmed death cascade. However, a number of recent observations have raised the possibility that abortive apoptosis may contribute to DNA damage in human spermatozoa: • • •

the detection of Fas on ejaculated spermatozoa54 the high proportion of spermatozoa with potentially apoptotic mitochondria55 the finding that potential mediators of apoptosis, including endonuclease activity, are present in spermatozoa.56

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It has been suggested that an early apoptotic pathway, initiated in spermatogonia and spermatocytes, is mediated by Fas protein. Fas is a type I membrane protein that belongs to the tumor necrosis factor/nerve growth factor receptor family.57,58 It has been shown that Sertoli cells express Fas ligand, which, by binding to Fas, leads to cell death via apoptosis.57,58 This in turn limits the size of the germ cell population to a number that Sertoli cells can support.59 Ligation of Fas ligand to Fas in the cellular membrane triggers the activation of caspases and, therefore, this pathway is also characterized as a caspase-induced apoptosis.60 Men exhibiting deficiencies in their semen profile often possess a large number of spermatozoa that bear Fas, prompting the suggestion that these dysfunctional cells are the product of an incomplete apoptotic cascade.61 However, the contribution of aborted apoptosis to the DNA damage seen in the ejaculated spermatozoa is doubtful in cases where this process is initiated at the early stages of spermatogenesis, because, at the stage of DNA fragmentation, apoptosis is an irreversible process,62 and these cells should be digested by Sertoli cells and removed from the pool of ejaculated sperm. Some studies have not found correlations between DNA damage and Fas expression,63 or, by contrast, have not revealed ultrastructural evidence for the association of apoptosis with DNA damage in sperm.64 Alternatively, if the apoptotic cascade is initiated at the round spermatid phase, where transcription (and mitochondria) is still active, abortive apoptosis might be an origin of the DNA breaks. A Bcl2 antiapoptotic family gene member called Bclw has been shown to suppress apoptosis in elongating spermatids.65 Although many apoptotic biomarkers have been found in the mature male gamete, particularly in infertile men, their definitive association with DNA fragmentation remains elusive.66–75

Oxidative stress Reactive oxygen species (ROS) play an important physiological role, modulating gene and protein activities vital for sperm proliferation, differentiation, and function. In the semen of fertile men, the amount of ROS generation is controlled by seminal antioxidants. The pathogenic effects of ROS occur when they are produced in excess of the antioxidant capabilities of the male reproductive tract or seminal plasma.76 The human spermatozoon is highly susceptible to OS. This process induces peroxidative damage in the sperm plasma membrane and DNA fragmentation. Such stress may arise from a variety of sources. Morphologically abnormal spermatozoa (with residual cytoplasm, in particular) and leukocytes are the main sources of excessive ROS generation in semen.76 Also, a lack of antioxidant protection and the presence of redox cycling xenobiotics may be the cause of OS. Whenever levels of OS in the male germ line are high, the peroxidation of unsaturated fatty acids in

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the sperm plasma membrane leads to the depressed fertilization rates associated with DNA damage.18

Deficiencies in recombination Meiotic crossing-over is associated with the genetically programmed introduction of DNA double-strand breaks (DSBs) by specific nucleases of the SPO11 family.77 These DNA DSBs should be ligated until the end of meiosis I. Normally, a recombination checkpoint in meiotic prophase does not allow meiotic division I to proceed until DNA is fully repaired or defective spermatocytes are ablated.77,78 However, a defective checkpoint may lead to persistent sperm DNA fragmentation in ejaculated spermatozoa. Direct data for this hypothesis in humans are lacking. The processes leading to DNA damage in ejaculated sperm are inter-related. For example, a defective spermatid protamination and disulfide bridge formation caused by inadequate oxidation of thiols during epididymal transit, resulting in diminished sperm chromatin packaging, makes sperm cells more vulnerable to ROS-induced DNA fragmentation.

Contributing factors Advancing age has been associated with an increased percentage of ejaculated spermatozoa with DNA damage.11,79,80 Young men with cancer typically have poor semen quality and sperm DNA damage even before starting therapy. Further damage from radiation or chemotherapy is dependent on both the duration and dose of radiation.81,82 Spermatogenesis may not occur months to years after therapy, but evidence of sperm DNA damage often persists beyond that period.83,84 A recent study on men with testicular cancer showed that radiation therapy induced transient sperm DNA damage and that this damage was present 3–5 years later, but three or more cycles of chemotherapy, in turn, decreased the percentage of sperm with DNA damage.84 Cigarette smoking is associated with a decrease in sperm count and motility and an increase in abnormal sperm forms and sperm DNA damage.85 It is suggested that smoking increases production of leukocytederived ROS; the OS may be the underlying reason why sperm DNA from smokers contain more strand breaks than that from nonsmokers.85,86 Also, genital tract infections and inflammation result in leukocytospermia and have been associated with OS and subsequent sperm DNA damage.87 Exposure to pesticides (organophosphates), persistent organochlorine pollutants, and air pollution have also been associated with sperm DNA damage.11,88–90 Varicoceles have been associated with seminal OS and sperm DNA damage.91–93 Sperm DNA integrity has been shown to improve after varicocele repair.94,95 A deficiency in gonadotropic hormones such as follicle-stimulating hormone (FSH) can cause sperm

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chromatin defects. FSH-receptor knockout mice have been found to have higher levels of sperm DNA damage.96 A febrile illness has been shown to cause an increase in the histone/protamine ratio and DNA damage in ejaculated sperm.97 Direct testicular hyperthermia has also been shown to cause these effects.98,99 Finally, sperm preparation techniques involving repeated high-speed centrifugation and the isolation of spermatozoa from the seminal plasma, which is a protective antioxidant environment, may contribute to increased sperm DNA damage via mechanisms that are mediated by the enhanced generation of ROS.14,100

the SCSA 17,104 or 20% as detected by TUNEL. 111 Thus, sperm DNA integrity may be considered an objective marker of sperm function that serves as a significant prognostic factor for male infertility. 7 Also, a significant increase in SCSA-defined DNA damage in sperm from infertile men with normal sperm parameters has been demonstrated,109 indicating that analysis of sperm DNA damage may reveal a hidden sperm abnormality in infertile men classified with idiopathic infertility based on apparently normal standard semen parameters.

Assisted reproductive technologies

Indications for sperm chromatin assessment Evaluating sperm chromatin can be challenging for several reasons: it can be difficult to link the results of chromatin integrity tests to known physiological mechanisms; the role that sperm chromatin structure assessment plays in clinical practice (especially in ART) is still controversial; and there is no one standardized method for measuring sperm chromatin integrity. On the other hand, sperm chromatin structure is complex, and several methods may be necessary to assess it. In addition, a number of confounding factors can complicate the interpretation of the results, including heterogeneity in the sperm population and the fact that not all DNA damage is lethal (most DNA contains noncoding regions or introns, and oocytes can repair sperm DNA damage). Nevertheless, at the present time, it is clear that sperm chromatin assessment provides good diagnostic and prognostic capabilities for fertility/infertility. It must be stressed that among all methods employed for sperm chromatin assessment, clinical thresholds so far have been demonstrated only for the sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, and these thresholds have been confirmed by different laboratories for SCSA only. Also, the reported biological variability of sperm DNA damage within men over time should be considered, although it is more stable than standard semen parameters.101–103

Diagnosis of male infertility Sperm DNA damage has a significant impact on in vivo fertilization. Many studies have shown, using a variety of techniques, significant differences in sperm DNA damage levels between fertile and infertile men. 104–109 Moreover, spermatozoa from infertile patients are generally more susceptible to the effects of DNA-damaging agents such as H2O2 and radiation.110 The probability of fertilization in vivo seems to be close to zero if the proportion of sperm cells with DNA damage exceeds 30% as detected by

The probability of fertilization by intrauterine insemination (IUI) also seems to be close to zero if the proportion of sperm cells with DNA damage exceeds 30% by means of SCSA,12,19,112,113 or 12% by TUNEL. 19 Whether sperm DNA damage negatively affects the results of in vitro fertilization (IVF) and ICSI is controversial. Although no association between sperm DNA damage and IVF/ICSI outcome has been demonstrated in some studies, 114 most of the studies show a significant negative correlation between sperm DNA damage and embryo quality in IVF cycles, 115 blastocyst development following IVF, 116 and fertilization rates following IVF 117 and ICSI, 118 even though sperm DNA damage may not necessarily preclude fertilization and pronucleus formation during ICSI.119 Two recent meta-analyses concluded that sperm DNA damage is predictive for reduced pregnancy success using routine IVF but has no significant effect on ICSI outcome.9,120 Thus, assessment of sperm chromatin may help predict the success rates of IUI and IVF. It has been also suggested that in patients with a high proportion of DNA-damaged sperm who are seeking ART, ICSI should be the method of choice.12

Embryonal loss Data on miscarriages as a possible consequence of sperm DNA damage are rather scarce. It has been shown that the proportion of sperm with DNA damage is significantly higher in men from couples with recurrent pregnancy loss than in the general population or fertile donors.121 It has also been reported that 39% of miscarriages could be predicted using a combination of selected cut-off values for percentage spermatozoa with denaturated (likely fragmented) DNA and/or abnormal chromatin packaging as assessed by SCSA.17 An increased trend of spontaneous abortions following IVF/ICSI was also demonstrated when sperm from men with a large amount of damaged DNA were used.122,123 Thus, it is possible that the assessment of sperm DNA damage could be a good predictor of possible miscarriage. However, the findings mentioned above need to be supported by additional studies.

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Table 6.1

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Various methods for assessing sperm chromatin abnormalities

Assay

Parameter

Method of analysis

Acidic aniline blue126 Toluidine blue staining131 Chromomycin A3132 DNA breakage detection-fluorescent in situ hybridization136 In situ nick translation138

Nuclear maturity (DNA protein composition) Nuclear maturity (DNA protein composition) Nuclear maturity (DNA protein composition) DNA fragmentation (ssDNA)

Optical microscopy Optical microscopy Fluorescent microscopy Fluorescent microscopy

DNA fragmentation (ssDNA)

Acridine orange144

DNA denaturation (acid)

Sperm chromatin dispersion151 Comet (neutral)157 (alkaline)158 TUNEL64

DNA fragmentation DNA fragmentation (dsDNA) DNA fragmentation (ssDNA/dsDNA) DNA fragmentation

Fluorescent microscopy Flow cytometry Fluorescent microscopy Flow cytometry Fluorescent microscopy Fluorescent microscopy

Sperm chromatin structure assay17 8-OHdG measurement171

DNA denaturation (acid/heat) 8-OHdG

Fluorescent microscopy Flow cytometry Flow cytometry High-performance liquid chromatography

8-OHdG, 8-hydroxy-2-deoxyguanosine; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.

Cancer patients

Acidic aniline blue stain

Patients with cancer are often referred to sperm banks before chemotherapy, radiation therapy, or surgery is initiated. Although pregnancies and births have been reported using cryopreserved sperm from patients with cancer, these semen samples have decreased fertilization potential. The extent of DNA damage may help to determine how semen should be cryopreserved before therapy begins. Specimens with high sperm concentration and motility and low levels of DNA damage should be preserved in relatively large aliquots that are suitable for IUI. If a single specimen of good quality is available, then it should be preserved in multiple small aliquots suitable for IVF or ICSI.124

Principle

Evaluation of sperm nuclear DNA damage Different methods may be used to evaluate the status of the sperm chromatin for the presence of abnormalities or simply immaturity (Table 6.1). These assays include simple staining techniques such as the acidic aniline blue (AAB) and basic toluidine blue (TB) stains, fluorescent staining techniques such as the sperm chromatin dispersion (SCD) test, chromomycin A3 (CMA3), DNA breakage detection–fluorescent in situ hybridization assay (DBD – FISH), in situ nick translation (NT), and flow cytometric-based SCSA. Some assays employ more than one method for the analysis of their results. Examples of these assays include the acridine orange (AO) and TUNEL assays. Other methods less frequently used include measurement of 8-hydroxy-2-deoxyguanosine (8-OHdG) by high-performance liquid chromatography (HPLC).

The AAB stain discriminates between lysine-rich histones and arginine/cysteine-rich protamines. This technique provides a specific positive reaction for lysine and reveals differences in the basic nuclear protein composition of ejaculated human spermatozoa. Histone-rich nuclei of immature spermatozoa are rich in lysine and will consequently take up the blue stain. On the other hand, protamine-rich nuclei of mature spermatozoa are rich in arginine and cysteine and contain relatively low levels of lysine, which means they will not take up the stain.125

Technique Slides are prepared by smearing 5 µl of either raw or washed semen sample. The slides are air-dried and fixed for 30 minutes in 3% glutaraldehyde in phosphate-buffered saline (PBS). The smear is dried and stained for 5 minutes in 5% aqueous aniline blue solution (pH 3.5). Sperm heads containing immature nuclear chromatin stain blue and those with mature nuclei do not. The percentage of spermatozoa stained with aniline blue is determined by counting 200 spermatozoa per slide under bright field microscopy.126

Clinical significance Results of AAB staining have shown a clear association between abnormal sperm chromatin and male infertility.127 However, the correlation between the percentage of aniline blue-stained spermatozoa and other sperm parameters remains controversial. Immature sperm

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(a)

(b)

(c)

Fig 6.1 (a) Human ejaculate stained with toluidine blue: (1) sperm heads with normal chromatin conformation are light blue; (2) sperm heads with abnormal chromatin conformation are violet. (b) DNA breakage detection–fluorescent in situ hybridization (DBD–FISH) labeling with a whole genome probe (red fluorescence), demonstrating extensive DNA breakage in those nuclei that are intensely labeled. (c) Acridine orange (AO) stain to native DNA fluoresces green (1), whereas relaxed/denatured DNA fluoresces red (2).

chromatin may or may not correlate with asthenozoospermic samples and abnormal morphology patterns.125,126 Most important is the finding that chromatin condensation as visualized by aniline blue staining is a good predictor for IVF outcome, although it cannot determine the fertilization potential and the cleavage and pregnancy rates following ICSI.128

Clinical significance TB staining may be considered a fairly reliable method for assessing sperm chromatin. Abnormal nuclei (purple–violet sperm heads) have been shown to be correlated with counts of red–orange sperm heads as revealed by the AO method.129 Also, correlations between the results of the TB, SCSA, and TUNEL tests have been demonstrated.130

Toluidine blue stain Principle

Advantages and limitations

Toluidine blue is a basic nuclear dye used for metachromatic and orthochromatic staining of chromatin. The phosphate residues of sperm DNA in nuclei with loosely packed chromatin and/or impaired DNA become more liable to binding with basic TB, providing a metachromatic shift from light blue to purpleviolet color.129 This stain is a sensitive structural probe for DNA structure and packaging.

In general, the AAB and TB methods are simple and inexpensive and have the advantage of providing permanent preparations for use on an ordinary microscope. The smears stained with the TB method can also be used for morphological assessment of the cells. In this way, the TB stain method is more advantageous. However, these methods may have the inherent limits of repeatability dictated by a limited number of cells that can be reasonably scored.

Technique The protocol of the TB stain includes four steps. The smears are air-dried, fixed in freshly made 96% ethanol–acetone (1:1) at 4°C for at least 30 minutes, hydrolyzed in 0.1 N HCl at 4°C for 5 minutes, and rinsed three times in distilled water for 2 minutes each. Smears are stained with 0.05% TB (Merck, Poole, Dorset, UK) for 5 minutes. The staining buffer consists of 50% citrate phosphate (McIlvaine’s buffer, pH 3.5). Permanent preparations are dehydrated in tertiary butanol twice for 3 minutes each at 37°C, and in xylene (Histoclear RA Lambs Labs, USA) twice for 3 minutes each. Afterwards, the preparations are embedded in DPX. Sperm heads with good chromatin integrity stain light blue and those of diminished integrity stain violet (purple).130 Based on the different optical densities of cells stained by the TB, the image analysis cytometry test has been elaborated131 (Fig 6.1a).

Chromomycin A3 assay Principle Chromomycin A3 is a guanine–cytosine-specific fluorochrome that reveals chromatin that is poorly packaged in human spermatozoa via indirect visualization of protamine-deficient DNA. Chromomycin A3 and protamines compete for the same binding sites in the DNA. Therefore, high CMA3 fluorescence is a strong indicator of the low protamination state of spermatozoa.132

Technique For CMA3 staining, semen smears are first fixed in methanol–glacial acetic acid 3:1 at 4°C for 20 minutes and are then allowed to air-dry at room temperature for 20 minutes. The slides are treated for 20 minutes with 100 µl CMA3 solution. The CMA3 solution consists of

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0.25 mg/ml CMA3 in McIlvaine’s buffer (pH 7.0) supplemented with 10 mmol/l MgCl2. The slides are rinsed in buffer and mounted with 1:1 v/v PBS–glycerol. The slides are then kept at 4°C overnight. Fluorescence is evaluated using a fluorescent microscope. A total of 200 spermatozoa are randomly evaluated on each slide. CMA3 staining is evaluated by distinguishing spermatozoa that stain bright yellow (CMA3 positive) from those that stain a dull yellow (CMA3 negative).132

Clinical significance As a discriminator of IVF success (>50% oocytes fertilized), CMA3 staining has a sensitivity of 73% and specificity of 75%. Therefore, it can distinguish between IVF success and failure.133 In cases of ICSI, Sakkas et al134 reported that the percentage of CMA3 positivity does not indicate failure of fertilization entirely and suggested that poor chromatin packaging contributes to a failure in the decondensation process and probably reduced fertility. It appears that semen samples with high CMA3 positivity (>30%) may have significantly lower fertilization rates if used for ICSI.135

Advantages and limitations The CMA3 assay yields reliable results as it is strongly correlated with other assays used in the evaluation of sperm chromatin.132 In addition, the sensitivity and specificity of the CMA3 stain are comparable with those of the AAB stain (75% and 82%, 60% and 91%, respectively) if used to evaluate the chromatin status in infertile men.136 However, the CMA3 assay is limited by observer subjectivity.

DNA breakage detection – fluorescent in situ hybridization assay Principle Cells embedded within an agarose matrix on a slide are exposed to an alkaline unwinding solution, which transforms DNA-strand breaks into single-stranded DNA (ssDNA) motifs. After neutralization and protein removal, ssDNA is accessible to hybridization with whole genome or specific DNA probes. The probe highlights the chromatin area to be analyzed. As DNA breaks increase, the more ssDNA is produced by the alkaline solution and the more the probe hybridizes, resulting in an increase in the fluorescence intensity and surface area of the FISH signal. Abnormal chromatin packaging in sperm cells greatly increases the accessibility of DNA ligands and the sensitivity of DNA to denaturation by alkali, and this relates to the presence of intense labeling (red fluorescence) by DBD–FISH. Therefore, DBD–FISH allows in situ detection and quantification of DNA breaks and reveals structural features in the sperm chromatin.136,137

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Technique To perform this assay, sperm cells are mixed with low-melting-point agarose to a final concentration of 0.7% at 37°C. A volume of 300 µl of the mixture is pipetted onto polystyrene slides and allowed to solidify at 4°C. The slides are immersed into a freshly prepared alkaline denaturation solution (0.03 mol/l NaOH, 1 mol/l NaCl) for 5 minutes at 22°C in the dark to generate ssDNA from DNA breaks. The denaturation is then stopped, and proteins are removed by transferring the slides to a tray with neutralizing and lysing solution 1 (0.4 mol/l Tris, 0.8 mol/l dithiothreitol (DTT), 1% sodium dodecylsulfate (SDS), and 50 mmol/l ethylenediaminetetra-acetic acid (EDTA), pH 7.5) for 10 minutes at room temperature, which is followed by incubation in neutralizing and lysing solution 2 (0.4 mol/l Tris, 2 mol/l NaCl, and 1% SDS, pH 7.5) for 20 minutes at room temperature. The slides are thoroughly washed in Tris–borate–EDTA buffer (0.09 mol/l Tris–borate and 0.002 mol/l EDTA, pH 7.5) for 15 minutes, dehydrated in sequential 70%, 90%, and 100% ethanol baths (2 minutes each), and air-dried. A human whole genome probe is hybridized overnight (4.3 ng/µl in 50% formamide/2 × standard saline citrate [SSC], 10% dextran sulfate, and 100 mmol/l calcium phosphate, pH 7.0) (1 × SSC is 0.015 mol/l sodium citrate and 0.15 mol/l sodium chloride, pH 7.0). It is then washed twice in 50% formamide/2 × SSC, pH 7.0, for 5 minutes, and twice in 2 × SSC (pH 7.0) for 3 minutes at room temperature. The hybridized probe is detected with streptavidin–indocarbocyamine (1:200) (Sigma Chemical Co., St Louis, MO), and cells are counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1 µg/ml) and visualized using fluorescent microscopy136 (Fig 6.1b).

Advantages and limitations Although the assay reveals chromatin structural features, it is expensive and time-consuming and involves sophisticated procedures. The assay is of less clinical value because the results are not superior to those of other, less cumbersome assays.

In situ nick translation assay Principle The NT assay quantifies the incorporation of biotinylated deoxyuridine triphosphate (dUTP) at singlestrand DNA breaks in a reaction that is catalyzed by the template-dependent enzyme DNA polymerase I. It specifically stains spermatozoa that contain appreciable and variable levels of endogenous DNA damage. The NT assay indicates anomalies that have occurred during remodeling of the nuclear DNA in spermatozoa. In doing so, it is more likely to detect sperm anomalies that are not indicated by morphology.

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Technique To perform the assay, smears containing 500 sperm each should be prepared. The fluorescent staining solution is prepared by mixing 10 µl streptavidin– fluorescein–isothiocyanate, 90 µl Tris buffer, and 900 µl double-distilled water. One hundred microliters of this solution is added to the slides. The slides are incubated in a moist chamber at 37°C for 30 minutes. After incubation, the slides are rinsed in PBS twice, washed with distilled water, and finally mounted with a 1:1 mixture of PBS and glycerol. The slides are examined using fluorescent microscopy. A total of 100–200 spermatozoa should be counted, and those fluorescing and hence incorporating the dye are classified as having endogenous nicks.138

Clinical significance Sperm nuclear integrity as assessed by the NT assay demonstrates a very clear relationship with sperm motility and morphology and, to a lesser extent, sperm concentration.139,140 Results of the assay are supported by the strong positive correlations detected with the sensitivity of CMA3 and TUNEL assays (r = 0.86; p < 0.05 and r = 0.87; p< 0.05, respectively).132 The NT assay can also indicate if there is damage arising from factors such as heat exposure141 or the generation of ROS following exposure to leukocytes within the male reproductive tract.142

Advantages and limitations The advantage of the NT assay is that the reaction is based on direct labeling of the termini of DNA breaks. Thus, the lesions that are measured are identifiable at the molecular level. In addition, if flow cytometry is used to analyze the results, it may be performed on fixed cells, as the time of cell storage in ethanol may vary.138

(methanol:acetic acid, 1:3) for at least 2 hours. The slides are stained for 5 minutes and gently rinsed with deionized water. At least 200 cells should be counted so that the estimate of the numbers of sperm with green and red fluorescence is accurate. For flow cytometry, aliquots of semen (about 25–100 µl, containing 1 million spermatozoa) are suspended in 1 ml of ice-cold PBS (pH 7.4) and centrifuged at 600g for 5 minutes. The pellet is resuspended in ice-cold TNE (0.01 mol/l Tris-HCl, 0.15 mol/l NaCl, and 1 mmol/l EDTA, pH 7.4) and again centrifuged at 600g for 5 minutes. The pellet is then resuspended in 200 µl of ice-cold TNE with 10% glycerol and immediately fixed in 70% ethanol for 30 minutes. The fixed samples are treated for 30 seconds with 400 µl of a solution of 0.1% Triton X-100, 0.15 mol/l NaCl, and 0.08N HCl, pH 1.2. After 30 seconds, 1.2 ml of staining buffer (6 µg/ml AO, 37 mmol/l citric acid, 126 mmol/l Na2HPO4, 1 mmol/l disodium EDTA, 0.15 mol/l NaCl, pH 6.0) is added to the test tube and analyzed by flow cytometry. After excitation by a 488-nm wavelength light source, AO bound to dsDNA fluoresces green (515–530 nm) and AO bound to ssDNA fluoresces red (≥630 nm). A minimum of 5000 cells are analyzed by fluorescent activated cell sorting (FACS).144

Clinical significance Staining with AO shows a significant difference between fertile males and those who are infertile with different andrologic pathologies. The cut-off value set to differentiate between fertile and infertile men varies between 20% and 50%.17,144,145 Studies show that ssDNA that is detected by a low incidence (<50%) of green AO fluorescence negatively affects the fertilization process in a classical IVF program.143,146,147 However, no correlation was found with pregnancy rate and live births achieved by ICSI, except in patients having 0% of spermatozoa with ssDNA, in whom the pregnancy rate was significantly high.146

Acridine orange assay

Advantages and limitations

Principle

The AO assay is a biologically stable measure of sperm quality. The interassay variability is less than 5%, rendering the technique highly reproducible.148 A strong positive correlation exists between the AO assay and other techniques used to evaluate ssDNA, e.g. the TUNEL assay.149 The AO assay still requires expensive instrumentation if flow cytometry is used to interpret the results. Also, observer subjectivity may hinder the results if fluorescent microscopy is used.

The AO assay measures the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ by quantifying the metachromatic shift of AO fluorescence from green (native DNA) to red (denatured DNA). The fluorochrome AO intercalates into double-stranded DNA (dsDNA) as a monomer and binds to ssDNA as an aggregate. The monomeric AO bound to native DNA fluoresces green, whereas the aggregated AO on relaxed or denatured DNA fluoresces red143 (Fig 6.1c).

Sperm chromatin dispersion test Technique

Principle

The AO assay may be used for either fluorescence or flow cytometry. To perform this assay for fluorescent microscopy, thick smears are fixed in Carnoy’s fixative

If spermatozoa with nonfragmented DNA are immersed in an agarose matrix and directly exposed to lysing solutions, the resulting deproteinized nuclei

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Fig 6.2 (a) Spermatozoa embedded in an agarose microgel stained with DAPI (4',6-diamidino-2-phenylindole) staining (blue fluorescence) and showing spermatozoa with different patterns of DNA dispersion: large-sized halo (1); medium-sized halo (2); very smallsized halo (3); no halo (4). (b) Comet images showing damaged (1) and undamaged DNA (2).

(nucleoids) show extended halos of DNA dispersion as monitored by fluorescent microscopy. The presence of DNA breaks promotes the expansion of the halo of the nucleoid.150-155 The SCD test is based on the principle that when sperm are treated with an acid solution prior to lysis buffer, the DNA dispersion halos that are observed in sperm nuclei with nonfragmented DNA after the removal of nuclear proteins are either minimally present or not produced at all in sperm nuclei with fragmented DNA.

Technique Aliquots of either raw or washed semen samples should be adjusted to concentrations ranging between 5 and 10 million/ml. The suspensions are mixed with 1% low-melting-point aqueous agarose (to obtain a 0.7% final agarose concentration) at 37°C. Aliquots of 50 µl of the mixture should be pipetted onto a glass slide precoated with 0.65% standard agarose dried at 80°C, covered with a coverslip, and left to solidify at 4°C for 4 minutes. The coverslips are then carefully removed, and the slides are immediately immersed horizontally in a tray of freshly prepared acid denaturation solution (0.08 N HCl) for 7 minutes at 22°C in the dark, which generates restricted ssDNA motifs from DNA breaks. Denaturation is then stopped, and the proteins are removed by transferring the slides to a tray with neutralizing and lysing solution 1 (0.4 mol/l Tris, 0.8 mol/l DTT, 1% SDS, and 50 mmol/l EDTA, pH 7.5) for 10 minutes at room temperature. The slides are then incubated in neutralizing and lysing solution 2 (0.4 mol/l Tris, 2 mol/l NaCl, and 1% SDS, pH 7.5) for 5 minutes at room temperature. The slides are thoroughly washed in Tris-borate–EDTA buffer (0.09 mol/l Tris-borate and 0.002 mol/l EDTA, pH 7.5) for 2 minutes, dehydrated in sequential 70%, 90%, and 100% ethanol baths (2 minutes each), and air-dried. Cells are stained with DAPI (2 µg/ml) for fluorescence microscopy151 (Fig 6.2a).

Advantages and limitations The major advantage of the SCD test is that it does not require the determination of color or fluorescence intensity. Rather, the percentage of spermatozoa with nondispersed (very small halos or none at all) or dispersed nuclei is determined, which can be easily and reliably accomplished by the naked eye. Furthermore, the test is simple, fast, and reproducible, and its results are comparable to those of the SCSA.152,155 Recent reports suggest that sperm DNA fragmentation as reported by the SCD test are negatively correlated with fertilization rate and embryo quality in IVF/ICSI.153

Comet assay Principle The comet assay, also known as single-cell gel electrophoresis for analysis of DNA damage in an individual cell, was first introduced by Ostling and Johanson in 1984.156 Neutral electrophoresis buffer conditions were used to show that the migration of dsDNA loops from a damaged cell in the form of a tail unwinding from the relaxed supercoiled nucleus was proportional to the extent of damage inflicted on the cell. This finding took on the appearance of a comet with a tail when viewed using the fluorescent microscope and DNA stains. Singh et al modified the comet assay in 1988157 by using alkaline electrophoresis buffers to expose alkali-labile sites on the DNA; this modification increased the sensitivity of the assay to detect both single- and double-stranded DNA breaks.158 The damage is quantified by measuring the displacement between the genetic material of the nucleus ‘comet head’ and the resulting tail. The tail lengths are used as an index for the damage. Also, the ‘tail moment,’ which is the product of the tail length and intensity (fraction of total DNA in the tails), has been used as a measuring parameter. The tail moment can be more precisely defined as being equivalent to the torsional moment of the tail.159

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Technique In this assay, sperm cells are cast into miniature agarose gels on microscope slides and lysed in situ to remove DNA-associated proteins and to allow the compacted DNA in the sperm to relax. The lysis buffer (Tris 10 mmol/l, 0.5 mol/l EDTA, and 2.5 mol/l NaCl, pH 10) contains 1% Triton X-100, 40 mmol/l DTT, and 100 µg/ml proteinase K. Microgels are then electrophoresed (20 minutes at 25 V/0.01 A) in neutral buffer (Tris 10 mmol/l containing 0.08 mol/l boric acid and 0.5 mol/l EDTA, pH 8.2), during which the damaged DNA migrates from the nucleus towards the anode. The DNA is visualized by staining the slides with the fluorescent DNA binding dye SYBR Green I. Comet measurements are performed using fluorescent microscopy. These measurements can be done either manually or with computerized image analysis157 (Fig 6.2b).

Clinical significance The assay has been successfully used to evaluate DNA damage after cryopreservation.160 It may also predict embryo development after IVF and ICSI, especially in couples with unexplained infertility,161,162 although some studies fail to demonstrate such an association.163

temperature. The sample is centrifuged at 10 000g for 4 minutes. After the sperm are washed in PBS (pH 7.4), they are resuspended in 100 µl prewash buffer containing single-strength One-Phor-All buffer (100 mmol/l Tris-acetate, 100 mmol/l magnesium acetate, 500 mmol/l potassium acetate; and 0.1% Triton X-100) for 10 minutes at room temperature. Fixed sperm are spun out of the buffer and resuspended in 50 µl of TdT buffer containing 3 µmol/l biotin-16-dUTP, 12 µmol/l deoxyadenosine triphosphate (dATP), 0.1% Triton X-100, and 10 U of TdT enzyme and incubated at 37°C for 60 minutes. After two washes in PBS, the fixed, permeabilized sperm are resuspended in 100 µl of staining buffer consisting of 0.1% Triton X-100 and 1% streptavidin/Texas red antibiotin and incubated at 4°C in the dark for 30 minutes. The stained cells are washed in PBS/0.1% Triton X-100. To create negative controls, the enzyme terminal transferase may be omitted from the reaction mixture. To create positive controls, the samples are pretreated with 0.1 IU DNAase I for 30 minutes at room temperature and then labeled. Results may be interpreted by assessing 100–500 sperm cells under fluorescent microscopy or by using FACS flow cytometry64 (Fig 6.3 ).

Clinical significance Advantages and limitations The comet assay is a well-standardized assay that correlates significantly with the TUNEL and SCSA assays.164 It is simple to perform, has a low intra-assay coefficient of variation, and is inexpensive. Because it is based on fluorescent microscopy, the assay requires an experienced observer to analyze the slides and interpret the results.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling assay Principle The TUNEL assay quantifies the incorporation of dUTP at double-strand DNA breaks in a reaction catalyzed by the template-independent enzyme terminal deoxynucleotidyl transferase (TdT). This enzyme incorporates biotinlyated deoxyuridine to 3′-OH of DNA to create a signal, which increases with the number of DNA breaks. Sperm with normal DNA therefore have only background staining/fluorescence, whereas those with fragmented DNA (multiple chromatin3′-OH ends) stain/fluoresce brightly.138

The TUNEL assay has been widely used in male infertility research related to sperm chromatin/DNA abnormalities. It provides useful information in many cases of male infertility. A negative correlation was found between the percentage of DNA-fragmented sperm and the motility, morphology, and concentration in the ejaculate. It also appears to be potentially useful as a predictor for IUI pregnancy rate, IVF embryo cleavage rate, and ICSI fertilization rate. In addition, it provides an explanation for recurrent pregnancy loss.18,19,68,121,164 Also a predictive threshold for in vivo fertility has been recently demonstrated between fertile and infertile men (20% of TUNEL-positive cells),165 although it differs from that demonstrated for IUI procedures (12%).19

Advantages and limitations The TUNEL assay is relatively expensive and labor consuming. The flow cytometric method of assessment is generally more accurate and reliable, but it is also more sophisticated and expensive. Fairly goodquality control parameters have been demonstrated for the fluorescent TUNEL assay (the intraobserver variability was found to be <8% and the interobserver variability was <7%),64 although other authors mention high intra-assay and interlab variability.166

Technique Identification of strand breaks can be quantified by flow cytometry or fluorescent microscopy in which DNAdamaged sperm fluoresce intensely.15 To assess the DNA fragmentation by TUNEL, about 2 × 106 sperm are fixed with 1% formaldehyde for 10 minutes at room

Sperm chromatin structure assay Principle The SCSA measures in-situ DNA susceptibility to the acid-induced conformational helix–coil transition by

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Relative cell counts

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Fig 6.3 (a) Terminal deoxynucleotidyl transferase-mediated fluorescein-deoxyuridine triphosphate-nick end labeling (TUNEL) assay fluorescent activated cell sorting (FACS) histograms with markers (M1) for detection of fluorescence set at 650 nm semen sample with low percentage of sperm DNA fragmentation; (b) TUNEL assay FACS histograms with markers (M1) for a semen sample with high percentage of sperm DNA fragmentation.

AO fluorescence staining. The extent of conformational transition in situ following acid or heat treatment is determined by measuring the metachromatic shift of AO fluorescence from green (native DNA) to red (denatured or relaxed DNA). This protocol has been divided into SCSAacid and SCSAheat to distinguish the physical means of inducing conformational transition. The two methods give essentially the same results, but the SCSAacid method is much easier to use. DNA damage that is SCSA-defined is manifested by the DNA Fragmentation Index (DFI).17

that identifies samples compatible with in vivo pregnancy (<30%).12,168 To the best of our knowledge, SCSA is the most successful assay in predicting the various outcomes of ART, including the fertilization and implantation rates.9,12,120,169 Recent reports suggest that DFI can be used as an independent predictor of fertility in couples undergoing IUI.169 It is also proposed that all infertile men should be tested with SCSA as a supplement to the standard semen analysis. Recent data suggest that ICSI should be the method of choice when the DFI exceeds 30%.12

Technique

Advantages and limitations

To perform SCSA, an aliquot of unprocessed semen (about 13–70 µl) is diluted to a concentration of 1–2 × 106 sperm/ml with TNE buffer (0.01 M Tris-HCl, 0.15 M NaCl, and 1mM EDTA, pH 7.4). This cell suspension is treated with an acid detergent solution (pH 1.2) containing 0.1% Triton X-100, 0.15 mol/l NaCl, and 0.08 N HCl for 30 seconds, and then stained with 6 mg/l purified AO in a phosphate–citrate buffer, pH 6.0. The stained sample is placed into the flow cytometer sample chamber.17

The SCSA accurately estimates the percentage of DNA-damaged sperm and has a cut-off point (30% DFI) to differentiate between fertile and infertile samples.168,169 However, it requires the presence of expensive instrumentation (flow cytometer) and highly skilled technicians.

Clinical significance Because the SCSA is more constant over prolonged periods of time than routine World Health Organization (WHO) semen parameters, it may be used effectively in epidemiological studies of male infertility.167 In clinical applications, the SCSA parameters not only distinguish fertile and infertile men but also are able to classify men according to the level of in vivo fertility as high fertility (pregnancy initiated in <3 months), moderate fertility (pregnancy initiated within 4–12 months), and no proven fertility (no pregnancy by 12 months). In addition, a DFI threshold was established

Measurement of 8-hydroxy-2-deoxyguanosine Principle This assay measures levels of 8-OHdG, which is a byproduct of oxidative DNA damage, in the spermatozoa. It is the most commonly studied biomarker for oxidative DNA damage. Among various oxidative DNA adducts, 8-OHdG has been selected as a representative of oxidative DNA damage owing to its high specificity, potent mutagenicity, and relative abundance in DNA.170

Technique Step I DNA extraction is performed with chloroform– isoamyl alcohol (12:1 v/v) after the sperm cells are washed with sperm wash buffer (10 mmol/l Tris-HCl,

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10 mmol/l EDTA, 1 mol/l NaCl, pH 7.0) and lysed at 55°C for 1 hour with 0.9% SDS, 0.5 mg/ml proteinase K, and 0.04 mol/l DTT. After ribonuclease A treatment to remove RNA residue, the extracted DNA is dissolved in 10 mmol/l Tris-HCl (pH 7.0) for DNA digestion. Step II Enzymatic DNA digestion is performed with three enzymes: DNAase I, nuclease P1, and alkaline phosphatase. The final solution is dried under reduced temperature and pressure and is redissolved in distilled and deionized water for HPLC. Step III The third step is HPLC analysis. The HPLC system used for 8-OHdG measurements consists of a pump, a partisphere 5 C18 column, an electrochemical detector, an ultraviolet detector, an autosampler, and an integrator. The mobile phase consists of 20 mmol/l NH4H2PO4, 1 mmol/l EDTA, and 4% methanol (pH 4.7). The calibration curves for 8-OHdG are established with standard 8-OHdG, and the results are expressed as 8-OHdG/104 dG.171

Clinical significance The assay provides the most direct evidence suggesting that oxidative sperm DNA damage is involved in male infertility, based on the finding that levels of 8-OHdG in sperm are significantly higher in infertile patients than in fertile controls and have an inverse relationship with sperm concentration.171 Levels of 8-OHdG in sperm DNA have been reported to be increased in smokers, and they inversely correlate with the intake and seminal plasma concentration of vitamin C, the most important antioxidant in sperm. If not repaired, 8-OHdG modifications in DNA are mutagenic and may cause embryo loss, fetal malformations, or childhood cancer. Moreover, this modification could be a marker of OS in sperm, which may have negative effects on sperm function.172

Advantages and limitations Although 8-OHdG is a potential marker for oxidative DNA damage, artificial oxidation of dG can occur during the analysis, which can lead to inaccurate results. A fixed number of sperm cells should be analyzed as a precaution. However, the DNA yield cannot be excluded as a potential confounder.

Strategies to reduce sperm DNA damage In view of the impact sperm DNA fragmentation has on reproductive outcomes, it is important to develop and implement appropriate treatment methods and strategies to minimize DNA damage in spermatozoa used in assisted reproduction; the following strategies are employed.

Appropriate sperm preparation methods Most of the commonly used methods, such as density gradient centrifugation, swim-up, and glass wool filtration, yield sperm with better DNA integrity than native semen.14 Sperm preparation should be aimed at minimizing damage to the spermatozoa. This may be accomplished by exercising some simple precautions such as (1) slow dilution of the samples, especially when using cryopreserved spermatozoa; (2) gradual change in temperature and tests performed at 37oC; (3) minimal use of centrifugation, and when necessary, this should be carried out at the lowest possible speeds; and (4) controlled exposure to potentially toxic materials. Plastic glassware and media should be checked for potential toxicity to spermatozoa and contact with gloves, as it may immobilize the spermatozoa. In patients who are unable to create a sperm sample by masturbation, use of nontoxic condoms is important and, when necessary, a second sample should be collected a few hours after the first.

Electrophoretic separation of sperm This technique is based on the principle that highquality spermatozoa tend to be viable and morphologically normal and have a low degree of DNA fragmentation as measured by TUNEL assay.173

Antioxidant treatments One of the causes of sperm DNA damage is OS. Studies have investigated the ability of antioxidant treatments to manage male subfertility, both in vivo and in vitro. Significant improvement in clinical pregnancy and implantation rates have been shown in patients with high sperm DNA damage, as assessed by TUNEL assay, when treated with antioxidants before assisted reproduction.174,175 Therefore, in patients in whom OS is the cause of sperm DNA damage, adequate oral antioxidant treatment appears to be a simple strategy to enhance sperm genome integrity and the reproductive outcome. Designing standard and reliable oral antioxidant treatment protocols and alternative treatment strategies for nonresponders is needed.176

Magnetic cell separation Magnetic cell separation is a useful technique to separate apoptotic and nonapoptotic spermatozoa.177 All of these strategies are designed to help select vital, nonapoptotic spermatozoa with minimal DNA damage and positively affect the success rate and safety of ART.

High-magnification ICSI for patients with sperm DNA fragmentation Using inverted microscopes with Nomarski differential interference contrast optics combined with digitally enhanced secondary magnification178,179 allows

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the observation of spermatozoa with apparently normal morphology and shows intranuclear vacuoles that appear to be associated with chromatin packaging.

Conclusion In summary, we emphasize the importance of assessing sperm for chromatin abnormalities as it may provide useful information in cases of male idiopathic infertility and in couples pursuing assisted reproduction. Pathologically increased sperm DNA fragmentation is one other main paternal-derived cause of repeated assisted reproduction failures in the ICSI era. Several studies have demonstrated that sperm DNA integrity correlates with pregnancy outcome in in vitro fertilization. Therefore, sperm DNA fragmentation should be included in the evaluation of the infertile male. Assessment of sperm DNA damage appears to be a potential tool for evaluating semen samples prior to their use in assisted reproduction. It allows the selection of spermatozoa with intact DNA or with the least amount of DNA damage for use in assisted conception. It provides better diagnostic and prognostic capabilities than standard sperm parameters for male fertility potential. There are multiple assays that may be used to evaluate sperm chromatin. Most of these assays have many advantages as well as limitations. Choosing the right assay depends on many factors, such as the expense, the available laboratory facilities, and the presence of experienced technicians. The establishment of a cut-off point between normal levels in the average fertile population and the minimal levels of sperm DNA integrity required for achieving pregnancy still remains to be investigated. Such an average range or value confirmed by different laboratories is still lacking for most of these assays except for the SCSA. Given the importance of sperm DNA integrity, it is important to determine the real cause of DNA damage and provide proper therapeutic treatment. Furthermore, methods for selecting sperm with undamaged DNA should be designed, especially in cases where ICSI is strongly recommended.

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153. Muriel L, Garrido N, Fernandez JL, et al. Value of the sperm deoxyribonucleic acid fragmentation level, as measured by the sperm chromatin dispersion test, in the outcome of in vitro fertilization and intracytoplasmic sperm injection. Fertil Steril 2006; 85: 371–83. 154. Muriel L, Goyanes V, Segrelles E, et al. Increased aneuploidy rate in sperm with fragmented DNA as determined by the sperm chromatin dispersion (SCD) test and FISH analysis. J Androl 2007; 28: 38–49. 155. Fernandez JL, Muriel L, Goyanes V, et al. Halosperm is an easy, available, and cost-effective alternative for determining sperm DNA fragmentation. Fertil Steril 2005; 84: 860. 156. Ostling O, Johanson KJ. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 1984; 123: 291–8. 157. Singh NP, McCoy MT, Tice RR, et al. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988; 175: 184–91. 158. Singh NP, Danner DB, Tice RR, et al. Abundant alkali-sensitive sites in DNA of human and mouse sperm. Exp Cell Res 1989; 184: 461–70. 159. Hellman B, Vaghef H, Bostrom B. The concepts of tail movement and tail inertia in the single cell gel electrophoresis assay. Mutat Res 1995; 336: 123–31. 160. Duty SM, Singh NP, Ryan L, et al. Reliability of the comet assay in cryopreserved human sperm. Hum Reprod 2002; 17: 1274–80. 161. Morris ID, Ilott S, Dixon L, et al. The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development. Hum Reprod 2002; 17: 990–8. 162. Tomsu M, Sharma V, Miller D. Embryo quality and IVF treatment outcomes may correlate with different sperm comet assay parameters. Hum Reprod 2002; 17: 1856–62. 163. Abu-Hassan D, Koester F, Shoepper B, et al. Comet assay of cumulus cells and spermatozoa DNA status, and the relationship to oocyte fertilization and embryo quality following ICSI. Reprod Biomed Online 2006; 12: 447–52. 164. Benchaib M, Braun V, Lornage J, et al. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod 2003; 18: 1023–8. 165. Sergerie M, Laforest G, Bujan L, et al. Sperm DNA fragmentation: threshold value in male fertility. Hum Reprod 2005; 20: 3446–51.

166. Erenpreiss J, Spano M, Erenpreisa J, et al. Sperm chromatin structure and male fertility: biological and clinical aspects. Asian J Androl 2006; 8: 11–29. 167. Spano M, Kolstad AH, Larsen SB, et al. The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Hum Reprod 1998; 13: 2495–505. 168. Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl 2002; 23: 25–43. 169. Evenson D, Wixon R. Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay. Reprod Biomed Online 2006; 12: 466–72. 170. Shen H, Ong C. Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility. Free Radic Biol Med 2000; 28: 529–36. 171. Kodama H, Yamaguchi R, Fukuda J, et al. Increased oxidative deoxyribonucleic acid damage in the spermatozoa of infertile male patients. Fertil Steril 1997; 68: 519–24. 172. Loft S, Kold-Jensen T, Hjollund NH, et al. Oxidative DNA damage in human sperm influences time to pregnancy. Hum Reprod 2003; 18: 1265–72. 173. Ainsworth C, Nixon B, Aitken RJ. Development of a novel electrophoretic system for the isolation of human spermatozoa. Hum Reprod 2005; 20: 2261–70. 174. Agarwal A, Nallella KP, Allamaneni SS, et al. Role of antioxidants in treatment of male infertility: an overview of the literature. Reprod Biomed Online 2004; 8: 616–27. 175. Greco E, Romano S, Iacobelli M, et al. ICSI in cases of sperm DNA damage: beneficial effect of oral antioxidant treatment. Hum Reprod 2005; 20: 2590–4. 176. Rolf C, Cooper TG, Yeung CH, et al. Antioxidant treatment of patients with asthenozoospermia or moderate oligoasthenozoospermia with high-dose vitamin C and vitamin E: a randomized, placebocontrolled, double-blind study. Hum Reprod 1999; 14: 1028–33. 177. Said T, Agarwal A, Grunewald S, et al. Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: an in vitro model. Biol Reprod 2006; 74: 530–7. 178. Berkovitz A, Eltes F, Lederman H, et al. How to improve IVF-ICSI outcome by sperm selection. Reprod Biomed Online 2006; 12: 634–8. 179. Hazout A, Dumont-Hassan M, Junca AM, et al. High-magnification ICSI overcomes paternal effect resistant to conventional ICSI. Reprod Biomed Online 2006; 12: 19–25.

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7 Oocyte retrieval and selection Laura F Rienzi, Filippo M Ubaldi

Introduction Of the several factors that affect oocyte quality, controlled ovarian stimulation protocols comprise one of the most important. To better understand the treatment strategies, their application, and their potential impact on oocyte quality, it is of utmost importance to know the physiology of the ovarian function. The demise of corpus luteum at the end of the luteal phase of the menstrual cycle is responsible for the sudden fall of 17 beta estradiol (E2), inhibin A, and progesterone, which induces an increased frequency of pulsatile gonadotropin-releasing hormone (GnRH) secretion and rising serum follicle-stimulating hormone (FSH) levels.1 When serum FSH concentration reaches a critical ‘threshold’ level for ovarian stimulation, class 5 follicles departing from the resting pool are recruited and start a well-characterized growth trajectory.2,3 In the early follicular phase the increased production of estrogens resulting from the FSH-dependent granulosa cell aromatase activity, together with the increase of inhibin B, are responsible for the falling circulating levels of FSH,4,5 which restricts the time when FSH levels remain above the ‘threshold.’6,7 As a result, one (dominant) follicle continues its growth, probably due to up-regulation by intraovarian factors that may increase sensitivity for FSH stimulation,8,9 whereas other (nondominant) follicles (of the same cohort) enter atresia due to diminished sensitivity to FSH and estrogen biosynthesis (as well as elevated intrafollicular androgen levels).9–11 On the basis of these findings, the ‘FSH window’ concept has been introduced, suggesting the importance of the duration of FSH elevation above the threshold level rather than the height of the elevation of FSH for single dominant follicle selection.6,7,12 The different stimulation protocols used for controlled ovarian hyperstimulation are based on the concept of widening the FSH window with the use of exogenous gonadotropins from the early follicular phase to the day of human chorionic gonadotropin (hCG) administration. Over the last 25 years different stimulation protocols have been proposed. Easier stimulation regimens

such as clomiphene citrate (CC) alone or in combination with human menopausal gonadotropin (hMG) and urinary FSH were gradually abandoned in favor of more complex protocols where GnRH agonists are used in combination with gonadotropins. These lengthy protocols, which are still the most widely used treatments for ovarian stimulation, allow us to manage the activity of in vitro fertilization (IVF) centers more easily, lower cancellation rates, and raise the number of preovulatory follicles, the number of oocytes retrieved, and the number of good quality embryos for transfer, thus leading to better pregnancy rates.13 However, these regimens are not free from complications and costs for the patients. The clinical introduction of GnRH antagonists in IVF,14–16 with their immediate suppression of the pituitary function, allows the administration of low doses of gonadotropins from mid follicular phase, resulting in more ‘patient friendly’ stimulation protocols,17,18 with fewer days of stimulation, lower amounts of gonadotropins administered, and fewer oocytes retrieved. However, if these milder protocols may improve patients’ compliance, reducing the burden of IVF on the couple, the question that remains to be answered is if the reduced number of oocytes obtained after mild protocols may impair the clinical outcome. Whatever stimulation regimen is used for controlled ovarian hyperstimulation, once the correct follicular and hormonal parameters are reached, a bolus of hCG is administered to trigger ovulation. The oocyte meiosis (blocked at the prophase of the first meiotic division) is then reinitiated, going through the germinal vesicle (GV) breakdown, the formation and extrusion of first polar body (IPB). After entering in the second meiotic division, a second arrest occurs at metaphase stage II (MII). Oocyte retrieval is performed 34–36 hours following hCG administration. Once in the laboratory, the oocyte quality is evaluated. This assessment is based on the aspect of the cumulus–corona cells, and if denudation is performed, also on the basis of the morphology of the oocyte cytoplasm and on the aspect of the extracytoplasmic structures (such as zona pellucida [ZP], first

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polar body, perivitelline space [PVS]). Oocyte selection prior to insemination is potentially very important in IVF/ICSI (intracytoplasmic sperm injection) programs because: •

•

•

•

it would give important information with regard to the subsequent developmental ability of the deriving embryo it could help to reduce the number of inseminated oocytes and thus the amount of supernumerary embryos it would help to avoid inseminating ‘bad quality oocytes’ potentially at risk of carrying chromosomal abnormalities it would help to choose the appropriate number of oocytes in egg donation programs.

However, the current literature on oocyte assessment is controversial and the selection methods proposed are still largely ineffective. The presence of cumulus and corona cells makes the morphological oocyte evaluation difficult to perform prior to standard IVF. Moreover, the quality and the degree of expansion of these cells seem to be poor markers of oocyte maturity and mostly depend on the type of ovarian stimulation protocol used.19–22 The oocyte can be easily observed only after cumulus–corona cells removal. The presence of the first polar body is normally considered as a marker of oocyte nuclear maturity. However, recent studies using polarized light microscopy have shown that oocytes displaying an IPB may still be immature.23–25 Moreover, nuclear maturity alone is not enough to determine the quality of an oocyte. In fact, nuclear and cytoplasmic maturation should be completed in a coordinated manner to ensure optimal conditions for subsequent fertilization. However, cytoplasmic maturation assessment is still unclear. It has been suggested that disturbances or asynchrony of these two maturation processes may result in a variety of oocyte morphological abnormalities.26–29 Abnormal ZP, large PVS, vacuoles, refractile bodies, increased cytoplasmic granularity, smooth endoplasmic reticulum clusters, abnormal, fragmented or degenerated first polar body can be observed after oocyte denudation. The correlation between these abnormal morphotypes and oocyte developmental ability will be discussed in this chapter.

Ovarian stimulation protocols Although the first successful pregnancy after IVF and embryo transfer was performed in the natural unstimulated cycle of an infertile woman with a tubal factor,30 it was soon observed that pregnancy rates per IVF attempt increase when more than one embryo is transferred into the uterine cavity.31 Subsequently, natural cycle IVF was replaced by stimulated cycles, allowing significant clinical outcome improvement. Over the last 25 years different stimulation protocols have been proposed. More complex and more demanding protocols have

gradually replaced easier stimulation regimens such as CC alone or in combination with hMG and urinary FSH. At the beginning of the 1990s short-term treatments with GnRH agonists and gonadotropins were abandoned in favor of long-term GnRH agonist stimulation protocols that allowed retrieval of more oocytes selection of one or more embryos for transfer, suggesting that the more oocytes obtained the higher the chance of conception.32,33 Recent evidence, however, suggests that excessive ovarian stimulation may have detrimental effects on oocyte quality34–36 and that there might be an optimal range of oocyte retrieval, below and above which the clinical outcome might be compromised.37 This observation suggests that amongst the cohort of recruited follicles only the most sensitive to stimulation are likely to give better-quality embryos, whereas all the additional oocytes resulting from maximal stimulation might be of impaired quality. Alternatively, the reduction of pregnancy rate observed after maximal stimulation might be due to a direct effect of high serum estradiol concentrations on oocytes, quality18,38 or on endometrial receptivity.38–40 About 10 years ago, GnRH antagonists were clinically introduced in IVF.14–16 These GnRH analogs induce an immediate suppression of the pituitary function, which allows the administration of low doses of gonadotropins from mid follicular phase, resulting in shorter and more ‘patient friendly’ stimulation protocols.17,18 With these milder regimens the number of oocytes retrieved is lower, but a significantly higher proportion of high-quality embryos (according to their morphology) might be obtained, suggesting a better oocyte quality (probably because of less interference with natural follicle selection).18 Although embryo morphology is commonly used to select the best embryo for transfer and is correlated with pregnancy rates,18 it gives limited information regarding the chromosomal constitution of the embryo.41 It has been suggested that ovarian stimulation protocols may affect embryo aneuploidy.42,43 Reducing the duration and intensity of the pharmacological interventions might interfere less with natural follicle selection and result in better oocyte quality, with more physiological chromosome segregation behavior during meiosis and early embryo development.18 To investigate this hypothesis, very recently Baart et al36 designed a prospective randomized study where the chromosomal constitution of the embryos obtained after mild or conventional stimulation was analyzed by preimplantation genetic screening (PGS) in patients younger than 38 years of age. The number of oocytes retrieved and the number of embryos obtained was higher in the conventional stimulation group but, as previously reported,18 the proportion of good morphology embryos was significantly higher in the mild stimulation group. Similarly, a significantly higher proportion of euploid embryos per patient and a lower proportion of mosaic embryos per patient were observed in the mild stimulation group.

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Furthermore, the same authors reported a significant positive correlation between the number of oocytes retrieved and the proportion of abnormal embryos in patients stimulated with milder regimens. On the contrary, no correlation was observed in the conventional stimulation group. These findings suggest that the reduced number of oocytes retrieved in the mild stimulation group is the result of a more physiological oocyte selection, whereas in the conventional stimulation group a low ovarian response is a sign of ovarian aging.36 Although these results seem very interesting, further larger prospective studies are needed to confirm these data. Moreover, with mild protocols a significantly reduced number of oocytes can be retrieved and, unfortunately, so far, no data regarding the cumulative pregnancy rates obtained after mild or standard regimens are available. Whatever stimulation protocol is used, the different pharmaceutical preparations of human gonadotropins used to induce the multiple follicular growth might also influence the oocyte quality. During the last 15 years many studies have compared the efficacy and the safety of different gonadotropin preparations (recombinant FSH vs urinary FSH vs highly purified FSH vs hMG vs highly purified hMG), reporting conflicting data.44–51 Most of these trials focus on clinical parameters such as number of oocytes, dosage and duration of gonadotropin used, fertilization, pregnancy, and implantation rates. The reported conflicting results might be due to either different intrinsic factors, such as cause of infertility, age, ovarian reserve, individual FSH–FSH receptor interaction, and ethnic background, or extrinsic factors, such as stimulation protocols, cigarette smoking, and sample size.52–55 The few studies that have examined the effect of different gonadotropin preparations on oocyte and embryo quality have reported conflicting results. Some56 but not all authors57,58 reported a significantly higher proportion of metaphase II oocytes and fewer oocytes with dark cytoplasm in women receiving highly purified FSH when compared with those treated with hMG. The use of recombinant FSH may induce a better cytoplasmic maturation, which might be negatively influenced by the large amount of urinary proteins (cytokines, growth factors, transferrins, and other proteins) that are found in urinary FSH 51 However, these results were not confirmed in previous59 and in more recent studies.60 Several factors might explain these conflicting data: timing of hCG administration, interval between hCG and oocyte retrieval, and interval between oocyte retrieval and oocyte insemination. These aspects are further discussed in this chapter.

Perifollicular vascularization evaluation Besides stimulation protocols and gonadotropin preparations, oocyte quality might be influenced by

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perifollicular vascularization. It has been suggested that an insufficient perifollicular vascularization measured using color Doppler ultrasonography correlates with intrafollicular hypoxia,61–63 inducing oocyte cytoplasmic defects, disorganized chromosomes, reduced fertilization, and embryos with multinucleated blastomeres.62,64,65 Embryos with high implantation potential originate from well-vascularized and oxygenated follicles.64 According to these data, several studies have also shown higher pregnancy and implantation rates when embryos resulting from the fertilization of oocytes from better perfused follicles are transferred.66–70 Unfortunately, other studies were not able to confirm the clinical value of the association between perifollicular vascularization and oocyte competence to improve the reproductive outcomes in young infertile patients who undergo either intrauterine insemination cycles71 or IVF cycles.72–75 Moreover, it is not technically easy to assess the perifollicular vascularity during the oocyte retrieval procedure and to perform the aspiration and flushing of the selected follicle until the oocytes are retrieved. For these reasons, further prospective randomized studies are needed to verify whether a relationship exists between the perifollicular vascularization of selected follicles measured using color Doppler ultrasonography and their reproductive competence.

Serum and follicular anti-Müllerian hormone measurements Very recent evidence indicates that anti-Müllerian hormone (AMH), a member of the transforming growth factor-beta superfamily produced by the granulosa cells of ovarian follicles mainly from the primary to the preantral and early antral stages of folliculogenesis and independently from FSH,76 is a unique biomarker of ovarian follicular status.77–79 Serum day 3 AMH levels have been strongly correlated with ovarian reserve80,81 and ovarian response to controlled ovarian hyperstimulation,82–84 showing a better cycle-to-cycle reproducibility than serum inhibin B and FSH levels.85 Furthermore, a positive correlation between serum AMH measured around the time of hCG administration and the number of oocytes retrieved, the fertilizaton rates, the embryo score, and the implantation rates was recently reported.86 Similarly, lower serum concentrations of AMH were correlated with reduced fertilization rates and increased miscarriage rates, suggesting serum AMH as a predictor of oocyte quality.87 On the contrary, other recent studies did not confirm these results.88 The observation in the animal model77,89 and in the woman79 that AMH is not expressed by the granulosa cells of atretic follicles suggests a more interesting role of this biomarker measured in the follicular fluid to predict the oocyte competence.89 In a very recent study the AMH levels in follicular fluid

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from women undergoing IVF with fertilized oocytes were statistically significantly higher than in the follicular fluid of patients with fertilization failure, suggesting that high follicular fluid AMH (FF AMH) levels positively correlate with oocyte quality.88 Similarly, FF AMH concentrations observed in natural IVF cycles were strongly and positively correlated with embryo implantation, suggesting FF AMH as a better predictor of oocyte quality than serum AMH.90 According to these results, it can be suggested that FF AMH concentrations may be used for oocyte selection in stimulated cycles. However, although these data are very interesting and promising, further prospective randomized studies are needed to draw any conclusion.

Table 7.1 Oocyte–corona–cumulus complex evaluation scheme Groups

OCCC morphology

Mature

Expanded cumulus Radiant corona Distinct zona pellucida, clear ooplasm Expanded well-aggregated membrana granulosa cells

Approximately mature

Expanded cumulus mass Slightly compact corona radiata Expanded well-aggregated membrana granulosa cells

Immature

Dense compact cumulus if present Adherent compact layer of corona cells Ooplasm if visible with the presence of germinal vesicle Compact and non-aggregated membrana granulosa cells

Post-mature

Expanded cumulus with clumps Radiant corona radiata, yet often clumped, irregular, or incomplete Visible zona, slightly granular or dark ooplasm Small and relatively nonaggregated membrana granulosa cells

Atretic

Rarely with associated cumulus mass Clumped and very irregular corona radiata if present Visible zona, dark and frequently misshapen ooplasm Membrana granulosa cells with very small clumps of cells

Oocyte–corona–cumulus complex evaluation Cumulus cells are Graafian follicular cells that surround and nourish the oocyte during its development in the ovary. The innermost layer of cumulus cells, immediately adjacent to the zona pellucida, is called corona radiata. Cells of the corona radiata extend their cytoplasm towards the oocyte through the ZP. Communications (either paracrine interaction or gapjunction) occur between the oocyte and the cumulus– corona cells. Such interactions allow oocyte nutrition and maturation during its preovulatory growth from the diplotene to the MII stage.91,92 Corona radiata and cumulus cells maintain their contact with the oocyte at the time of ovulation, during a normal menstrual cycle, or after withdrawal by aspiration, in hormonally stimulated assisted reproduction cycles. In mature oocytes, the cumulus–corona mass appears as an expanded and mucified layer, due to the active secretion of hyaluronic acid. This extracellular component interposes among the cells and separates them, conferring to the cumulus–corona mass a fluffy appearance. During unstimulated cycles, the stage of oocyte nuclear maturation is coupled to an increased expansion and mucification of the cumulus layer.19 However, stimulated cycles may be characterized by an asynchrony of these two processes.20 This was suggested to be caused by a different sensitivity of the oocyte and the cumulus– corona mass to the stimulants.20,93 Early studies from Rattanachaiyanont et al,22 performed on oocytes scheduled for denudation and insemination by ICSI, reported no correlation between oocyte–corona–cumulus complex (OCCC) morphology and nuclear maturity, fertilization rate, and embryo cleavage. On the other hand, other authors reported that OCCC scoring related to fertilization and pregnancy rates94 as well as to blastocyst quality and development.95 Recently, Lin and colleagues95 proposed a grading system of OCCCs based on the morphology of the oocyte cytoplasm, cumulus mass, corona cells, and membrana granulosa cells, for oocytes prior to insemination by conventional IVF.

Adapted from Lin et al.95

Five grades (mature group, approximately mature, immature, post-mature, and atretic) were described, as shown in Table 7.1.95 The authors reported higher fertilization rates for the oocytes belonging to the mature group compared to those belonging to the other groups. Moreover, the immature group was characterized by a higher incidence of poor morphology day 3 embryos as compared to the mature group. In support of a positive effect of cumulus–corona cells on oocyte development, it was shown that the partial removal of this layer prior to ICSI improves embryo quality and development.96 It has been suggested that the presence of cumulus–corona cells may help embryonic metabolism, by either stimulating gene expression97 or reducing oxidative stress.98 Although, cumulus–corona mass observation is not sufficient to evaluate oocyte maturity and competence, it is reasonable to hypothesize that ooplasm development is influenced by the action of these cells.

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In accordance with this hypothesis, it has been suggested that cumulus–corona cells play an important role in the in vitro maturation of oocytes that were immature at the time of retrieval.99,100 Therefore, a careful observation of OCCC morphology may be a useful tool for oocyte selection in those circumstances where no direct evaluation of the oocyte is possible. In addition, in our laboratory, where a maximum of 3 oocytes can be inseminated according to the Italian law, we use OCCC evaluation prior to denudation for ICSI. In fact, as suggested by Canipari et al,101 we hypothesize that there is a higher probability of obtaining a better-quality mature oocyte in a normally expanded cumulus than in a nonexpanded one.

Oocyte nuclear maturity evaluation Direct observation of oocyte morphology, including the extracytoplasmic components, is possible only after denudation of its cumulus and corona layers. The use of hyaluronidase enzyme and mechanical pipetting facilitate breaking down of the cumulus– corona extracellular matrix. This method is normally used when insemination by ICSI is going to be performed. A meiotically mature oocyte is blocked at the MII stage. Completion of the meiotic maturation occurs only after sperm entry and consequent oocyte activation. Currently, oocyte nuclear maturity is determined by the presence of an extruded IPB in the perivitelline space and by the absence of a GV. Nonsynchronous oocyte maturation is often observed after ovarian hyperstimulation. Approximately 85% of the denuded oocytes display the IPB and are classified as MII. In about 10% a GV is present in the oocyte cytoplasm; approximately 5% of the oocytes are in metaphase of the first meiotic division (MI) with no visible GV and IPB.102 Immature oocytes (GV and MI) can potentially be matured in vitro.103–105 However, the fertilization rate of such oocytes after ICSI is significantly lower than that which can be obtained with in vivo matured oocytes.104 Although some successful pregnancies are reported with the use of these cells, a high incidence of genetical abnormalities has been observed in the embryos derived from in vitro matured oocytes.106 Therefore, immature oocytes obtained by ovarian hyperstimulation should not be selected for insemination. At MII stage the oocyte chromosomes are aligned at the equatorial region of the meiotic spindle (MS). This structure plays a crucial role in the sequence of events leading to the correct completion of meiosis and fertilization and thus is a key determinant of oocyte developmental potential. However, the MS microtubules, which are responsible for proper chromosomal segregation, are highly sensitive to chemical and physical changes that may occur during oocyte retrieval and handling. It has been shown that the oocyte exposure to slight temperature fluctuations dramatically affects microtubular structure, with

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deleterious consequences on chromosomal organization.107–111 Other parameters, such as increased maternal age112,113 and oocyte in vitro aging114 are also associated with disruption of MS architecture. The most potentially dramatic consequences of MS alteration are the unbalanced disjunction and/or nondisjunction of chromatids, chromosome scattering, and the formation of aneuploid embryos.112,115,116 The introduction of a novel orientation-independent polarized light microscopy system (Spindle View Pol-Scope system, CRI, Woburn, MA, USA), coupled with an image processing software, allows the visualization of MS in living oocytes.117–119 Parallel-aligned MS microtubules are birefringent and able to shift the plane of polarized light, inducing a retardance. These properties enable the system to generate contrast and image the MS structure. Moreover, digital processing enhances signal sensitivity. Unlike conventional methods for MS imaging, the Spindle View system does not require oocyte fixation and staining. In this way the MS can be visualized in a noninvasive way, preserving oocyte viability. With the use of the Spindle View system, new information about human oocyte maturity and developmental potentiality has been recently produced. Several studies120–126 indicate the importance of the presence of a detectable MS in the oocyte cytoplasm prior to ICSI. A clear positive correlation between MS visualization, fertilization rate, and/or embryo development and/or blastocyst progression was described in these studies (Table 7.2). The absence of a detectable MS and the consequent oocyte developmental impairment may be primarily ascribed to oocyte immaturity.116 It has been hypothesized that the lack of MS formation can be the result of aberrant signaling pathways or low energy supply during oocyte growth, resulting in both nuclear and cytoplasmic immaturity.116,125 Moreover, some oocytes were found to be clearly immature at the stage of telophase I (Fig 7.1) when observed with the Spindle View system.23–25 At this stage there is continuity between the ooplasm and the cytoplasm of the forming IPB, and the MS is interposed between the two separating cells. These oocytes would have been classified as ‘mature’ MII with light microscopy, based on the presence of an IPB (Fig 7.1a). Therefore, the use of the Spindle View system allows accurate determination of oocyte nuclear maturity and selection of fully mature eggs. However, it must be underlined that unfavorable culture conditions may also induce MS disassembly.11,120,121 In this case the lack of MS should be ascribed to environmental stress and not to oocyte incompetence to reach maturity. The percentage of oocytes displaying a detectable MS varies between 60 and 90% in the different studies120,122,123,125 (Table 7.2). This difference seems to be related to some important laboratory and clinical parameters: (1) the thermal control during oocytes handling;111,127 (2) the technique of MS visualization;123,125,126 and (3) the time elapsed from hCG

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Table 7.2

Relationship between the presence of a detectable MS in fresh MII oocytes and the ICSI outcome Meiotic spindle presence Yes

No

Wang et al120 Moon et al122 Rienzi et al123 Cohen et al125 Wang et al120 Moon et al122 Rienzi et al123 Cohen et al125 Injected oocytes (%) Fertilized oocytes (%) Good quality embryos (%)

1266 (82.0)

523 (83.5)

484 (91.0)

585 (76.0)

278 (18.0)

103 (16.5)

48 (9.0)

185 (24.0)

879 (69.4)a

430 (82.2)

362 (74.8)b

412 (70.4)c

175 (62.9)d

79 (75.7)

16 (33.3)e

115 (62.2)f

583 (46.0)g

276 (52.8)h

268 (55.4)i

169 (47.2)

97 (34.9)j

28 (27.2)k

9 (18.7)l

32 (35.6)

Adapted from Wang et al,120 Moon et al,122; Rienzi et al,123 and Cohen et al.125 p <0.05; b,ep <0.01; c,fp <0.035; g,jp <0.01; h,kp <0.01; i,lp <0.01.

a,d

(a)

(b)

Fig 7.1 (a) Telophase I oocyte observed with light microscopy (Hoffman contrast) and (b) with polarized light microscopy (Spindle View system). Only with this latter system is it possible to observe the immature MS (arrow) and to assess the maturation stage of this oocyte. (Magnification 400×.)

administration.125 Because of the high sensitivity of the MS to temperature variations, thermal stability is necessary during oocyte observation and manipulation. In addition, oocyte rotation, by means of a micropipette, allows correct orientation of the MS structure, which therefore becomes more favorable to visualization under polarized light.123,124 Finally, Cohen and co-authors125 found that the percentage of oocytes with detectable MS was positively related to the time elapsed from hCG administration. For this reason it was suggested to postpone ICSI to 38–42 hours after hCG injection in order to allow complete oocyte maturation prior to insemination.

Besides its role in chromosome segregation, the MS is also a key organelle in the creation of the IPB. Its position at the very periphery of the cell, attached to the oolemma cortex128 dictates the orientation of the cleavage furrow and thus the IPB extrusion site. However, IPB has been found to be frequently dislocated from the MS location after the denudation procedure.122,123 Artifactual displacement of the IPB from its original extrusion place adjacent to the MS position is believed to be due to the manipulation required for cumulus–corona cells removal.123 No relationship between moderate degree of IPB/MS deviation and ICSI outcomes has been described in

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Fig 7.2 Metaphase II oocyte with an MS located at 3 o’clock (red arrow) in the way of the injection pipette. The three different layers of the zona pellucida (ZP) are clearly visible (white arrows). (Magnification 400×.)

these studies. However, we123 found that mechanical stress that induces IPB dislocation more than 90 degrees from the MS position correlates with lower fertilization ability.123 In addition, MS dislocation is reported to affect embryo development since its position is involved in the correct orientation of the first cleavage plane.124 Another possible drawback of IPB displacement is the potential injury to the MS during microinsemination. In fact, the ICSI procedure is performed with the IPB at 90 degrees from the injection pipette entry site. Displaced IPB may thus expose the MS to the injection pipette passageway during oocyte

(a)

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microinsemination and therefore to mechanical damage (Fig 7.2). The Spindle View system may thus be a useful tool to perform ICSI since it allows the correct orientation of the oocyte with the MS (and not the IPB) as far as possible from the injection needle. The Spindle View system can also produce quantitative information about the MS. The degree of birefringence is in fact directly proportional to the molecular organization of the structure (Fig 7.3). It has been described that increased temperatures cause a decrease in spindle retardance, suggesting a partial loss of MS polymerization.129 A possible correlation between MS birefringence, oocyte quality, and embryo development has also been suggested.25,126,130 Moreover, a negative correlation between female age and MS retardance has been found25,126 (Fig 7.4). These data suggest that older women have a lower MS microtubular density, which could explain their higher risk of producing aneuploid embryos. These results are in agreement with observations by confocal microscopy that MS architecture is strictly related to female age.112

Metaphase II oocyte morphological evaluation It is generally recognized that a ‘normal’ human MII oocyte should have a round, clear ZP, a small PVS containing a single not fragmented IPB, and a pale, moderately granular cytoplasm with no inclusions. 96,131–136 However, the majority of the oocytes retrieved after ovarian hyperstimulation exhibit one or more morphological abnormalities involving the cytoplasm aspect and/or the extracytoplasmic

(b)

Fig 7.3 Metaphase II oocyte observed with the Spindle View system (a). Retardance profile of the meiotic spindle. (b) (Magnification 400×.)

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3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 25–29 ap<0.05

30–35

35–40

>40

female age (years) De Santis et al25 2005

Rama Raju et al126 2007

Fig 7.4 Relationship between MS retardance and female age. (Adapted from De Santis et al25 and Rama Raju et al.126)

structures.96,132–137 The actual negative impact of the different oocyte ‘abnormalities’ on ICSI outcome is unclear (reviewed in Ebner et al96 and Balaban and Urman135). Some authors have suggested that all oocytes could be fertilized by ICSI independently from their morphological appearance.132,134 Furthermore, no impact on embryo quality has been associated with oocyte morphology. Similar clinical pregnancy and implantation rates were also obtained after transferring embryos derived from ‘abnormal’ oocyte as compared with those obtained with embryos derived from ‘normal’appearing oocytes.134,135 On the other hand, different authors have reported a correlation between oocyte morphology and embryo developmental potential. Xia133 showed that oocyte grading based on IPB morphology, size of PVS, and cytoplasmic inclusions was correlated with its developmental potential after ICSI. First polar body morphology has been suggested as a possible predictor of oocyte fertilization and embryo quality after ICSI also by other authors.25,136,138,139 Transferring embryos selected according to IPB morphology leads to an increase in implantation and pregnancy rates.138 Moreover, embryos deriving from oocytes with an intact IPB were more prone to develop into a blastocyst than embryos deriving from oocytes with fragmented IPB.140 Nevertheless, other studies have failed to demonstrate a relationship between IPB fragmentation and embryo development.25,139 Moreover, the aneuploidy rate in MII oocytes is reported to be unrelated to the status of the IPB.141 It must be underlined that frequency of IPB fragmentation is associated with the time elapsed from denudation and ICSI.135,139 This morphological trait seems to be a marker of postovulatory age of the oocyte27 and thus to be a consequence of in vitro aging instead of being a proper marker of oocyte quality. In our experience141a only the presence of degenerated or large IPB (Fig 7.5) (but not fragmented) was

Fig 7.5 Metaphase II oocyte with giant IPB (arrow) and abnormal cytoplasm. (Magnification 400×.)

associated with a reduced fertilization rate after ICSI. The presence of a degenerated IPB may reflect an asynchrony between nuclear and cytoplasmic maturation,27 which would explain the lower developmental potential of the oocyte.25,133,138 On the other hand, the emission of an abnormally large IPB may be ascribed to the inability of the MS to migrate correctly at the very periphery of the cell.25,142 In these cases IPB morphology may be considered as a marker of oocyte maturation disturbance.25,142 Additional information about oocyte quality may be derived by IPB biopsy and chromosomal analysis. This particular aspect is discussed in Chapter 27 by Montag. Another factor that may affect oocyte survival143,144 and fertilization rate145 after insemination by ICSI is the presence of a large perivitelline space. This feature seems to reflect an overmaturity of the oocytes at the time of ICSI.137 However, different studies have failed to find a correlation between size and shape of the PVS and fertilization rate and embryo development.134,135 The negative effect of this morphotype may be related to its degree of extension and to the simultaneous presence of other abnormalities, such as unucleated fragments (Fig 7.6). The aspect of the zona pellucida observed with light microscopy seems not to correlate with normal fertilization and embryo development.28,134,146 However, the thickness and retardance of the ZP observed with polarized light microscopy have been recently proposed as markers of oocyte quality. The ZP is composed of three different layers, each characterized by different molecular arrangement and exhibiting different birefringence patterns (see Fig 7.2). The

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(a)

(b)

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(c)

Fig 7.6 Different degrees of perivitelline space (PVS) abnormalities. (a) Enlarged PVS in two areas and with sets (arrows). (b) Enlarged PVS in one area (arrow). (c) Oval oocyte with enlarged PVS containing fragments (arrows). (Magnification 400×.)

(a)

(b)

(c)

Fig 7.7 Different types of cytoplasmic inclusions: (a) refractile body (arrow), (b) vacuole (arrow), and (c) smooth endoplasmic reticulum cluster (arrow). (Magnification 400×.)

external and central layers seem to be of no predictive value, whereas the retardance of the inner layer has been related to blastocyst formation126 and to implantation.147 It is generally reported that the cytoplasmic texture is a very important characteristic for oocyte selection.29,145,148–151 Cytoplasmic alteration, such as granularity and presence of inclusions (Fig 7.7), may be a sign of cytoplasmic incompetence. Some authors have suggested that despite normal fertilization and early embryo development being achieved in oocytes with abnormal cytoplasmic morphology, the resulting embryos have a lower implantation potential.131,148 Particular cytoplasmic defects (such as centrally located granular area, smooth endoplasmic reticulum clusters, vacuoles) have been associated with poorer fertilization and/or embryo developmental potential.29,141a,145–151 Furthermore, no fertilization has been observed when vacuoles >14 µm were present in the injected oocyte.96,151 Conversely, other studies28,134,148 have

shown that slight deviations from the normal cytoplasmic texture were not associated with unfavorable oocyte development. It may thus be hypothesized that not the type but the severity of cytoplasmic defects correlates with oocyte developmental impairment.28,135 Moreover, the use of different criteria for oocyte evaluation may be responsible for the discrepancies found in different studies. There is one oocyte morphologic characteristic that surely reflects genetical abnormalities of the cell. This is the case of giant oocytes (Fig 7.8) that contain one additional set of chromosomes. These oocytes, when observed under polarized light, display two different MS. Although, the occurrence of these oocytes is relatively rare after ovarian hyperstimulation, the use of these cells for in vitro fertilization is dangerous. It has been described that all embryos generated from giant oocytes are chromosomally abnormal, but they may have a normal cleavage and development to the blastocyst stage.152 The transfer of

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Fig 7.8 Giant oocyte (arrow) and normally sized oocyte. (Magnification 400×.)

these embryos could thus increase the risk of undesired miscarriages.152 Because of the correlation found between specific morphological peculiarities, developmental competence, and genetical constitution, in our opinion, oocytes with severe defects involving cytoplasmic texture, IPB dimension, and oocyte size should not be used for in vitro fertilization.

Conclusions Useful, effective, and noninvasive grading tools are available for pronuclear, embryo, and blastocyst stage scoring.153–157 Combining several different morphological criteria, each of which has been individually shown to be predictive for embryo competence, an accurate embryo selection is possible.23,145,158–160 In addition to morphological assessment, other features of the human embryo may be useful for a more accurate selection. Embryo physiology, evaluated by measurements of metabolic activity and normality, may help to determine embryonic ‘health’. 161–166 Moreover, PGS of blastomeres’ chromosomal constitution may give important information about embryonic developmental potential.167–169 With the current trend towards limiting the number of embryos to be transferred, and thus the occurrence of multiple pregnancies, the ability of the embryologist to identify the embryo with the highest implantation potential is crucial. Nowadays, limited noninvasive tools exist to permit classification of human oocyte quality prior to fertilization. As described above, the stimulation protocols, the different pharmacological preparations, and the perifollicular vascularization might influence human oocyte quality. Very recently, follicular fluid AMH has been positively correlated with oocyte quality, fertilization rate,88 and embryo implantation,90 suggesting that this biomarker is a very promising predictor of oocyte competence. In addition, oocyte

observation under light microscopy provides assessments of several morphological characteristics. It seems, however, that slight deviations from the morphological normality should not be considered as abnormal phenotypes. Only some specific and evident oocyte morphological abnormalities, such as increased cytoplasmic granularity, vacuolization, presence of abnormal IPB, and ZP inner layer low retardance, have been linked to oocyte developmental potentiality. However, the limited predictive power and reliability of these parameters on implantation potential is generally reported. The reason may be ascribed to the subjectivity and inaccuracy in the evaluation. The analysis of MS in living oocytes with the Spindle View system has shown that detectable MS are functionally superior to nondetectable ones.111,120,121,170 Furthermore, embryos derived from oocytes with functionally poor MS have impaired cell development.122,123,125 However, it must be underlined that more than 90% of the MII oocytes, not exposed to unfavorable laboratory conditions, display a detectable MS when observed with the Spindle View system. Therefore, while ascertained that the absence of a signal is an important negative prognostic factor, the presence of MS, which involves the majority of the MII subjected to this evaluation, is of limited value for oocyte selection.25 In our opinion, MS qualitative analysis by measuring polarized light retardance, whose value is directly proportional to microtubule density, is a promising tool for oocyte quality assessment. In addition to the above-mentioned evaluation criteria (such as serum and FF AMH measurements, and screening of intracellular and extracellular morphological features), other methods could be able to provide additional information on oocyte quality. Current research (reviewed in Wang and Sun 171) is focusing on: • • •

•

•

•

assessment of granulosa and cumulus cells apoptosis status172–174 evaluation of oxidative stress in follicular fluid and granulosa cells175–178 follicular cells, gene expression profiles, by realtime polymerase chain reaction (PCR) or DNA microarray97,179 measurement of follicular fluid leptin,179–181 growth factors and related binding proteins, i.e. IGF/IGF-BP (insulin-like growth factor/insulinlike growth factor binding protein); members of the (TGF-β) (transfroming growth factor β superfamily)183–186 quantitation of follicular steroids (such as estradiol, testosterone, progesterone, and prolactin)182,187–192 ultramicrofluorimetric quantitation of oocyte carbohydrate consumption and metabolite release in the culture medium.193

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current trend is toward limiting the creation of supernumerary human embryos, and the identification of efficient parameters for oocyte competence assessment is a priority.

Acknowledgment The authors want to thank Valentina Casciani her help in preparing the manuscript.

PHD

for

References

Fig 7.9 Metaphase II oocytes obtained after ovarian hyperstimulation, all with approximately the same morphological appearance. (Magnification 400×.)

Although potentially useful, further investigation is needed to ensure the consistency, reliability, and sensitivity of these methods. In addition, some of these methods are characterized by time-consuming protocols that imply in vitro oocyte aging. On the basis of the above-mentioned potentialities of oocyte selection tools, future perspectives for the betterment of assisted reproduction technologies reside in creating a direct connection between IVF laboratories and research units. To date, oocyte evaluation taking into account both clinical (ovarian stimulation protocols and follicular vascularization) and biological parameters (AMH measurements, aspect of the cumulus–corona cells, presence and position of the MS, morphology of the oocyte cytoplasm and of the extracytoplasmic structures) gives some important information about the oocyte maturity stage and developmental fate of the deriving embryo. It is important that during laboratory evaluation procedures, the environmental conditions to which the oocytes are exposed should be as stable as possible. In this way these oocytes are preserved from stressful changes that could compromise their developmental potential. To this end, the use of chambers equipped with temperature/gas control is advisable during handling and observation. From observing the egg with light microscopy and with polarized light, it is possible to identify ‘bad’quality oocytes. However, these evaluations are insufficient to select between normal-appearing oocytes the one with the higher developmental potential (Fig 7.9). Thus far, in order to gain reliable information about embryo implantation fate, pronuclear stage and embryo assessment are still essentials in routine clinical applications. More efforts are needed to identify early markers of embryo quality, at the oocyte stage prior to fertilization. In the future, cellular/molecular approaches may help to provide such markers. The

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Oocyte retrieval and selection 99. Goud PT, Goud AP, Qian C, et al. In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium. Hum Reprod 1998; 13: 1638–44. 100. Yamazaki Y, Wakayama T, Yanagimachi R. Contribution of cumulus cells and serum to the maturation of oocyte cytoplasm as revealed by intracytoplasmic sperm injection (ICSI). Zygote 2001; 9: 277–82. 101. Canipari R, Camaioni A, Scarchilli L, Barberi M, Salustri A. Oocyte maturation and ovulation: mechanism of control. 2PN Attual Scient Biol Ripr 2004; 1: 62–8. 102. Ubaldi F, Rienzi L. Micromanipulation techniques in human infertility: PZD, SUZI, ICSI, MESA, PESA, FNA and TESE, in Biotechnology of Human Reproduction Revelli A, Tur-Kaspa I, Holte JG, Massobrio M, Oxford: Parthenon Publishing, pp. 315–36. 103. Nagy ZP, Cecile J, Liu J, et al. Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal-vesicle stage oocytes: case report. Fertil Steril 1996; 65: 1047–50. 104. De Vos A, Van de Velde H, Joris H, Van Steirteghem A. In-vitro matured metaphase-I oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-II oocytes after intracytoplasmic sperm injection. Hum Reprod 1999; 14: 1859–63. 105. Edirisinghe WR, Junk SM, Matson PL, Yovich JL. Birth from cryopreserved embryos following invitro maturation of oocytes and intracytoplasmic sperm injection. Hum Reprod 1997; 12: 1056–8. 106. Nogueira D, Staessen C, Van de Velde H, Van Steirteghem A. Nuclear status and cytogenetics of embryos derived from in vitro-matured oocytes. Fertil Steril 2000; 74: 295–8. 107. Sathananthan AH, Trounson A, Freemann L, Brady T. The effects of cooling human oocytes. Hum Reprod 1988; 3: 968–77. 108. Pickering SJ, Braude PR, Johnson MH, Cant A, Currie J. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990; 54: 102–8. 109. Almeida PA, Bolton VN. The effect of temperature fluctuations on the cytoskeletal organisation and chromosomal constitution of the human oocyte. Zygote 1995; 3: 357–65. 110. Zenzes MT, Bielecki R, Casper RF, Leibo SP. Effects of chilling to 0°C on the morphology of meiotic spindles in human metaphase II oocytes. Fertil Steril 2001; 75: 769–77. 111. Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. Limited recovery of meiotic spindles in living human oocytes after cooling–rewarming observed using polarized light microscopy. Hum Reprod 2001; 16: 2374–8. 112. Battaglia DE, Goodwin P, Klein NA, Soules MR. Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum Reprod 1996; 11: 2217–22. 113. Volarcik K, Sheean L, Goldfarb J, et al. The meiotic competence of in-vitro matured human oocytes is influenced by donor age: evidence that folliculogenesis is compromised in the reproductively aged ovary. Hum Reprod 1998; 13: 154–60.

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114. Eichenlaub-Ritter U, Vogt E, Yin H, Gosden R. Spindles, mitochondria and redox potential in ageing oocytes. Reprod Biomed Online 2004; 8: 45–58. 115. Bernard A, Fuller BJ. Cryopreservation of human oocytes: a review of current problems and perspectives. Hum Reprod Update 1996; 2: 193–207. 116. Eichenlaub-Ritter U, Shen Y, Tinneberg HR. Manipulation of the oocyte: possible damage to the spindle apparatus. Reprod Biomed Online 2002; 5: 117–24. 117. Oldenbourg R, Mei G. New polarized light microscope with precision universal compensator. J Microsc 1995; 180: 140–7. 118. Oldenbourg R. Polarized light microscopy of spindles. Methods Cell Biol 1999; 61: 175–208. 119. Liu L, Trimarchi JR, Oldenbourg R, Keefe DL. Increased birefringence in the meiotic spindle provides a new marker for the onset of activation in living oocytes. Biol Reprod 2000; 63: 251–8. 120. Wang WH, Meng L, Hackett RJ, Keefe DL. Developmental ability of human oocytes with or without birefringent spindles imaged by Polscope before insemination. Hum Reprod 2001; 16: 1464–8. 121. Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. The spindle observation and its relationship with fertilization after intracytoplasmic sperm injection in living human oocytes. Fertil Steril 2001; 75: 348–53. 122. Moon JH, Hyun CS, Lee SW, et al. Visualization of the metaphase II meiotic spindle in living human oocytes using the Polscope enables the prediction of embryonic developmental competence after ICSI. Hum Reprod 2003; 18: 817–20. 123. Rienzi L, Ubaldi F, Martinez F, et al. Relationship between meiotic spindle location with regard to the polar body position and oocyte developmental potential after ICSI. Hum Reprod 2003; 18: 1289–93. 124. Cooke S, Tyler JP, Driscoll GL. Meiotic spindle location and identification and its effect on embryonic cleavage plane and early development. Hum Reprod 2003; 18: 2397–405. 125. Cohen Y, Malcov M, Schwartz T, et al. Spindle imaging: a new marker for optimal timing of ICSI? Hum Reprod 2004; 19: 649–54. 126. Rama Raju GA, Prakash GJ, Krishna KM, Madan K. Meiotic spindle and zona pellucida characteristics as predictors of embryonic development: a preliminary study using PolScope imaging. Reprod Biomed Online 2007; 14: 166–74. 127. Wang WH, Meng L, Hackett RJ, Oldenbourg R, Keefe DL. Rigorous thermal control during intracytoplasmic sperm injection stabilizes the meiotic spindle and improves fertilization and pregnancy rates. Fertil Steril 2002; 77: 1274–7. 128. Maro B, Verlhac MH. Polar body formation: new rules for asymmetric divisions. Nat Cell Biol 2002; 4: E281–3. 129. Sun XF, Wang WH, Keefe DL. Overheating is detrimental to meiotic spindles within in vitro matured human oocytes. Zygote 2004; 12: 65–70. 130. Trimarchi JR, Karin RA, Keefe DL. Average spindle retardance observed using the PolScope predicts cell number in day 3 embryos. Fertil Steril 2004; 82: S268.

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131. Alikani M, Palermo G, Adler A, et al. Intracytoplasmic sperm injection in dysmorphic human oocytes. Zygote 1995; 3: 283–8. 132. De Sutter P, Dozortsev D, Qian C, Dhont M. Oocyte morphology does not correlate with fertilization rate and embryo quality after intracytoplasmic sperm injection. Hum Reprod 1996; 11: 595–7. 133. Xia P. Intracytoplasmic sperm injection: correlation of oocyte grade based on polar body, perivitelline space and cytoplasmic inclusions with fertilization rate and embryo quality. Hum Reprod 1997; 12: 1750–5. 134. Balaban B, Urman B, Sertac A, et al. Oocyte morphology does not affect fertilization rate, embryo quality and implantation rate after intracytoplasmic sperm injection. Hum Reprod 1998; 13: 3431–3. 135. Balaban B, Urman B. Effect of oocyte morphology on embryo development and implantation. Reprod Biomed Online 2006; 12: 608–15. 136. Ebner T, Yaman C, Moser M, et al. Prognostic value of first polar body morphology on fertilization rate and embryo quality in intracytoplasmic sperm injection. Hum Reprod 2000; 15: 427–30. 137. Mikkelsen AL, Lindenberg S. Morphology of in-vitro matured oocytes: impact on fertility potential and embryo quality. Hum Reprod 2001; 16: 1714–18. 138. Ebner T, Moser M, Yaman C, et al. Elective transfer of embryos selected on the basis of first polar body morphology is associated with increased rates of implantation and pregnancy. Fertil Steril 1999; 72: 599–603. 139. Ciotti PM, Notarangelo L, Morselli-Labate AM, et al. First polar body morphology before ICSI is not related to embryo quality or pregnancy rate. Hum Reprod 1996; 19: 2334–9. 140. Ebner T, Moser M, Sommergruber M, et al. First polar body morphology and blastocyst formation rate in ICSI patients. Hum Reprod 2002; 17: 2415–18. 141. Verlinsky Y, Lerner S, Illkevitch N, et al. Is there any predictive value of first polar body morphology for embryo genotype or developmental potential. Reprod Biomed Online 2003; 7: 336–41. 141a. Rienzi L, Ubaldi FM, Iacobelli M, et al. Significance of metaphase II human oocyte morphology on ICSI outcome. Fertil Steril 2008 (in press). 142. Verlhac MH, Lefebvre C, Guillaud P, Rassinier P, Maro B. Asymmetric division in mouse oocytes: with or without Mos. Curr Biol 2000; 10: 1303–6. 143. Ebner T, Yaman C, Moser M, et al. A prospective study on oocyte survival rate after ICSI: influence of injection technique and morphological features. J Assist Reprod Genet 2001; 18: 623–8. 144. Plachot M, Selva J, Wolf JP, Bastit P, de Mouzon J. Consequences of oocyte dysmorphy on the fertilization rate and embryo development after intracytoplasmic sperm injection. A prospective multicenter study. Gynecol Obstet Fertil 2002; 30: 772–9. 145. Rienzi L, Ubaldi FM, Iacobelli M, et al. Significance of metaphase II human oocyte morphology on ICSI outcome, 2008 Feb 4 [Epub ahead of print] 146. Balaban B, Urman B. Embryo culture as a diagnostic tool. Reprod Biomed Online 2003; 7: 671–82. 147. Shen Y, Stalf T, Mehnert C, Eichenlaub-Ritter U, Tinneberg HR. High magnitude of light retardation by the zona pellucida is associated with conception cycles. Hum Reprod 2005; 20: 1596–606.

148. Serhal PF, Ranieri DM, Kinis A, et al. Oocyte morphology predicts outcome of intracytoplasmic sperm injection. Hum Reprod 1997; 12: 1267–70. 149. Meriano JS, Alexis J, Visram-Zaver S, Cruz M, Casper RF. Tracking of oocyte dysmorphisms for ICSI patients may prove relevant to the outcome in subsequent patient cycles. Hum Reprod 2001; 16: 2118–23. 150. Otsuki J, Okada A, Morimoto K, Nagai Y, Kubo H. The relationship between pregnancy outcome and smooth endoplasmic reticulum clusters in MII human oocytes. Hum Reprod 2004; 19: 1591–7. 151. Ebner T, Moser M, Sommergruber M, et al. Occurrence and developmental consequences of vacuoles throughout preimplantation development. Fertil Steril 2005; 83: 1635–40. 152. Balakier H, Bouman D, Sojecki A, Librach C, Squire JA. Morphological and cytogenetic analysis of human giant oocytes and giant embryos. Hum Reprod 2002; 17: 2394–401. 153. Scott LA, Smith S. The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum Reprod 1998; 13: 1003–13. 154. Tesarik J, Greco E. The probability of abnormal preimplantation development can be predicted by a single static observation on pronuclear stage morphology. Hum Reprod 1999; 14: 1318–23. 155. Van Royen E, Mangelschots K, De Neubourg D. Characterization of a top quality embryo, a step towards single-embryo transfer. Hum Reprod 1999; 14: 2345–9. 156. Van Royen E, Mangelschots K, De Neubourg D, et al. Calculating the implantation potential of day 3 embryos in women younger than 38 years of age: a new model. Hum Reprod 2001; 16: 326–32. 157. Gerris J, De Neubourg D, Mangelschots K, et al. Prevention of twin pregnancy after in-vitro fertilization or intracytoplasmic sperm injection based on strict embryo criteria: a prospective randomized clinical trial. Hum Reprod 1999; 14: 2581–7. 158. Gerris J, De Neubourg D, Mangelschots K. Elective single day 3 embryo transfer halves the twinning rate without decrease in the ongoing pregnancy rate of an IVF/ICSI programme. Hum Reprod 2002; 17: 626–31. 159. Scott L. Pronuclear scoring as a predictor of embryo development. Reprod Biomed Online 2003; 6: 201–14. 160. Rienzi L, Ubaldi F, Iacobelli M, et al. Day 3 embryo transfer with combined evaluation at the pronuclear and cleavage stages compares favourably with day 5 blastocyst transfer. Hum Reprod 2002; 17: 1852–5. 161. Lane M, Gardner DK. Selection of viable mouse blastocysts prior to transfer using a metabolic criterion. Hum Reprod 1996; 11: 1975–8. 162. Gardner DK. Changes in requirements and utilization of nutrients during mammalian preimplantation embryo development and their significance in embryo culture. Theriogenology 1998; 49: 83–102. 163. Conaghan J, Hardy K, Handyside AH, Winston RM, Leese HJ. Selection criteria for human embryo transfer: a comparison of pyruvate uptake and morphology. J Assist Reprod Genet 1993; 10: 21–30. 164. Jones GM, Trounson AO, Vella PJ, et al. Glucose metabolism of human morula and blastocyst-stage embryos and its relationship to viability after transfer. Reprod Biomed Online 2001; 3: 124–32.

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Oocyte retrieval and selection 165. Houghton FD, Hawkhead JA, Humpherson PG, et al. Non-invasive amino acid turnover predicts human embryo developmental capacity. Hum Reprod 2002; 17: 999–1005. 166. Sakkas D, Gardner DK. Noninvasive methods to assess embryo quality. Curr Opin Obstet Gynecol 2005; 17: 283–8. 167. Gianaroli L, Magli MC, Ferraretti AP. The in vivo and in vitro efficiency and efficacy of PGD for aneuploidy. Mol Cell Endocrinol 2001; 22: 8–13. 168. Pehlivan T, Rubio C, Rodrigo L. Impact of preimplantation genetic diagnosis on IVF outcome in implantation failure patients. Reprod Biomed Online 2001; 6: 232–7. 169. Hardarson T, Caisander G, Sjögren A, et al. A morphological and chromosomal study of blastocysts developing from morphologically suboptimal human pre-embryos compared with control blastocysts. Hum Reprod 2003; 18: 399–407. 170. Wang WH, Keefe DL. Prediction of chromosome misalignment among in vitro matured human oocytes by spindle imaging with the PolScope. Fertil Steril 2002; 78: 1077–81. 171. Wang Q, Sun QY. Evaluation of oocyte quality: morphological, cellular and molecular predictors. Reprod Fertil Dev 2007; 19: 1–12. 172. Piquette GN, Tilly JL, Prichard LE, Simon C, Polan ML. Detection of apoptosis in human and rat ovarian follicles. J Soc Gynecol Investig 1994; 1: 297–301. 173. Nakahara K, Saito H, Saito T. The incidence of apoptotic bodies in membrana granulosa can predict prognosis of ova from patients participating in in vitro fertilization programs. Fertil Steril 1997; 68: 312–17. 174. Yuan YQ, Van Soom A, Leroy JL, et al. Apoptosis in cumulus cells, but not in oocytes, may influence bovine embryonic developmental competence. Theriogenology 2005; 63: 2147–63. 175. Seino T, Saito H, Kaneko T, et al. Eight-hydroxy-2′deoxyguanosine in granulosa cells is correlated with the quality of oocytes and embryos in an in vitro fertilization–embryo transfer program. Fertil Steril 2002; 77: 1184–90. 176. Bedaiwy MA, Falcone T, Mohamed MS, et al. Differential growth of human embryos in vitro: role of reactive oxygen species. Fertil Steril 2004; 82: 593–600. 177. Kim KH, Oh DS, Jeong JH, et al. Follicular blood flow is a better predictor of the outcome of in vitro fertilization–embryo transfer than follicular fluid vascular endothelial growth factor and nitric oxide concentrations. Fertil Steril 2004; 82: 586–92. 178. Lee TH, Wu MY, Chen MJ, et al. Nitric oxide is associated with poor embryo quality and pregnancy outcome in in vitro fertilization cycles. Fertil Steril 2004; 82: 126–31. 179. Zhang X, Jafari N, Barnes RB, et al. Studies of gene expression in human cumulus cells indicate

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pentraxin 3 as a possible marker for oocyte quality. Fertil Steril 2005; 83: 1169–79. Barroso G, Barrionuevo M, Rao P, et al. Vascular endothelial growth factor, nitric oxide, and leptin follicular fluid levels correlate negatively with embryo quality in IVF patients. Fertil Steril 1999; 72: 1024–6. Tsai EM, Yang CH, Chen SC. Leptin affects pregnancy outcome of in vitro fertilization and steroidogenesis of human granulosa cells. J Assist Reprod Genet 2002; 19: 169–76. Anifandis G, Koutselini E, Louridas K, et al. Estradiol and leptin as conditional prognostic IVF markers. Reproduction 2005; 129: 531–4. Kawano Y, Narahara H, Matsui N, et al. Insulinlike growth factor-binding protein-1 in human follicular fluid: a marker for oocyte maturation. Gynecol Obstet Invest 1997; 44: 145–8. Oosterhuis GJ, Lambalk CB, Michgelsen HW, et al. Follicle-stimulating hormone measured in unextracted urine: a reliable tool for easy assessment of ovarian capacity. Fertil Steril 1998; 70: 544–8. Fried G, Remaeus K, Harlin J, et al. Inhibin B predicts oocyte number and the ratio IGF-I/IGFBP-1 may indicate oocyte quality during ovarian hyperstimulation for in vitro fertilization. J Assist Reprod Genet 2003; 20: 167–76. Chang CL, Wang TH, Horng SG, et al. The concentration of inhibin B in follicular fluid: relation to oocyte maturation and embryo development. Hum Reprod 2002; 17: 1724–8. Xia P, Younglai EV. Relationship between steroid concentrations in ovarian follicular fluid and oocyte morphology in patients undergoing intracytoplasmic sperm injection (ICSI) treatment. J Reprod Fertil 2000; 118: 229–33. Chiu TT, Rogers MS, Law EL, et al. Follicular fluid and serum concentrations of myo-inositol in patients undergoing IVF: relationship with oocyte quality. Hum Reprod 2002; 17: 1591–6. Wunder DM, Mueller MD, Birkhauser MH, Bersinger NA. Steroids and protein markers in the follicular fluid as indicators of oocyte quality in patients with and without endometriosis. J Assist Reprod Genet 2005; 22: 257–64. Wise T, Suss U, Maurer RR. The relationships of oocyte quality and follicular fluid prolactin and progesterone in superovulated beef heifers with and without norgestomet implants. Adv Exp Med Biol 1987; 219: 697–701. Wiswedel K. Granulosa cell metabolism and the assessment of oocyte quality in IVF. Hum Reprod 1987; 2: 589–91. Lindner C, Lichtenberg V, Westhof G, Braendle W, Bettendorf G. Endocrine parameters of human follicular fluid and fertilization capacity of oocytes. Horm Metab Res 1988; 20: 243–6. Preis KA, Seidel G Jr, Gardner DK. Metabolic markers of developmental competence for in vitro-matured mouse oocytes. Reproduction 2005; 130: 475–83.

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8 Preparation and evaluation of oocytes for ICSI Irit Granot, Nava Dekel

Introduction Resumption of meiosis in the oocyte is an essential prelude for successful fertilization. The meiotic division of the mammalian oocyte is initiated during fetal life. It proceeds up to the diplotene stage of the first prophase and arrests at birth. The chromatin in the meiotically arrested oocytes is encapsulated by a nuclear structure known as the germinal vesicle (GV, Fig 8.1a). Meiotic arrest persists throughout infancy until the onset of puberty. In a sexually mature female, at each cycle one or more oocytes, according to the species, reinitiate the meiotic division. Upon reinitiation of meiosis the GV disappears (Fig 8.1b), the condensed chromosomes align on the newly formed meiotic spindle, and the pairs of homologous chromosomes segregate between the oocyte and the first polar body (Fig 8.1c). Emission of the first polar body, which represents the completion of the first round of meiosis, is immediately followed by the formation of the second meiotic spindle with the remaining set of homologous chromosomes aligned on its equatorial plate. The whole series of events, initiated by GV breakdown (GVB) and completed at the metaphase of the second round of meiosis (MII), leads to the production of a mature fertilizable oocyte, also known as an egg. The egg is arrested at MII and will complete the meiotic division only after penetration of the spermatozoon.1 The physiological stimulus for oocyte maturation is provided by the preovulatory surge of luteinizing hormone (LH).2 Once oocyte maturation is completed, LH further induces ovulation, during which the follicle releases the mature oocyte that is picked up by the infundibular fimbria of the oviduct. The egg released from the ovarian follicle is accompanied by the cumulus cells. Prior to ovulation, in concomitance with oocyte maturation, this cumulus undergoes characteristic transformations that are also stimulated by LH. In response to this gonadotropin the cumulus cells produce specific glucosaminoglycans, the secretion of which results in cumulus mucification and its expansion. The major component of the extracellular matrix secreted by the cumulus cells is hyaluronic acid.3–7 The mucified cumulus mass that encapsulates the ovulated egg is penetrated by the spermatozoon that

uses enzymes localized on its surface membrane to accomplish this mission. Sperm membrane protein PH-20 that is present on the plasma membrane of sperms of many species, such as guinea pigs, mice, macaques, and humans exhibits hyaluronidase-like activity that facilitates this action.8–11 Furthermore, a recent study has demonstrated that a plasma membrane-associated hyaluronidase is localized to the posterior acrosomal region of equine sperm.12 Having traversed the cumulus, the spermatozoon undergoes acrosome reaction and binds to the zona pellucida. Sperm-zona binding is mediated by specific sperm surface receptors. ZP3, the primary ligand on the zona pellucida, specifically binds to the plasma membrane of the acrosomal cap of the intact sperm. The secondary zona ligand, ZP2, binds to the inner acrosomal membrane of the spermatozoon.13–15 One of the inner acrosomal membrane sperm receptors was identified as acrosin.16–18 In order to penetrate the zona pellucida the spermatozoon utilizes enzymatic as well as mechanical mechanisms. Specific enzymes that are released by the acrosome-reacted spermatozoon allow the invasion of the zona pellucida by local degradation of its components.19–21 This enzymatic action is assisted by mechanical force generated by vigorous tail beatings that facilitate the penetration of the sharp sperm head.18,22 Having penetrated the zona pellucida the sperm crosses the perivitelline space and its head attaches to the egg’s plasma membrane (oolemma). Sperm head attachment to the oolemma is followed by its incorporation into the egg cytoplasm (ooplasm). Sperm incorporation is initiated by phagocytosis of the anterior region of its head followed by fusion of the head’s posterior region as well as the tail with the egg membrane.23–25 The scientific efforts that have been invested by reproductive biologists in studying the process of gametogenesis and fertilization in animal models laid the groundwork for the design of in vitro procedures for assisted reproduction. These procedures that are successfully practiced at present in human patients essentially attempted to mimic the biological processes in vivo. In vitro fertilization (IVF) regimens of treatment, which are continuously being improved, have allowed the birth of hundreds of thousands of babies all over the world.

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a

b

c

Fig 8.1 Morphological markers characterizing the meiotic status of oocytes. (a) Immature germinal vesicle (GV) oocyte: meiosis has not been reinitiated and the typical nuclear structure is visible. (b) Immature germinal vesicle breakdown (GVB) oocyte (metaphase I, MI): meiosis has been reinitiated, the GV has disappeared, but the first polar body is still absent. (c) Mature oocyte (MII): the GV has disappeared, and the first polar body has been extruded.

One such improvement, which represents a major breakthrough in this area, is intracytoplasmic sperm injection (ICSI). Until 1992, most infertility failures originating from a severe male factor were untreatable. Micromanipulation techniques such as partial zona dissection (PZD)26–29 and subzonal sperm injection (SUZI),28,30–34 designed to overcome the poor performance of sperm cells, did not result in a substantial improvement of the rate of success of in vivo fertilization. However, ICSI, which was established by the team led by Professor Van Steirteghem at The Free University in Brussels, Belgium and initially reported by Palermo et al,34 has generated a dramatic progress.35–38 The ICSI procedure involves the injection of a single sperm cell intracytoplasmatically into an egg. Fertility failures associated with an extremely low sperm count were found to be successfully treated by this technique. Furthermore, as the sperm is microinjected into the ooplasma, it bypasses the passage through the zona pellucida and is not required to interact with the oolemma. Therefore, infertility problems that originate from faulty sperm–egg interaction may also be resolved by this IVF protocol of treatment.

Handling of oocytes Similar to conventional IVF, patients for ICSI undergo programmed induction of superovulation followed by scheduled oocyte retrieval (see Chapter 37). Under all protocols of treatment, identification of the cumulus–oocyte complexes and evaluation of their maturity are carried out immediately after follicle aspiration, as described in Chapter 37. However, unlike conventional IVF, in which intact mature cumulus–oocyte complexes are inseminated, cumulus cells that surround the eggs are removed before microinjection. Denudation of the mature oocytes is an essential prerequisite for ICSI. Cumulus cells may block the

injecting needle, thus interfering with oocyte microinjection. Furthermore, in the presence of the cumulus, visualization of the egg is very limited. Since only mature oocytes that have reached metaphase II (MII) are suitable for ICSI, optimal optical conditions that allow the assessment of the meiotic status of the oocytes are required. Oocyte maturation is determined morphologically, by the absence of the GV and the presence of the first polar body. Good optical conditions are also necessary for the positioning of the mature oocyte in the right orientation for injection (see Chapter 11). Preparation of the retrieved mature oocytes for ICSI should be carried out under conditions of constant pH of 7.3 and stable temperature of 37oC. In order to maintain the appropriate pH, 4-(2-hydroxyethyl)-1piperazineethane sulfonic acid (HEPES)-buffered culture media are used. The correct temperature is maintained during egg handling by the use of a microscope equipped with a heated stage. Most of the procedures are performed under Earle’s balanced salts solution (EBSS)-treated and CO2-equilibrated paraffin/mineral oil that prevents evaporation of the medium and minimizes the fluctuations of both the pH and the temperature. Temperature fluctuations that are likely to accompany the handling of eggs have been shown to be specifically detrimental for the microtubular system. Changes in spindle organization were observed in human mature oocytes cooled to room temperature for only 10 minutes. These changes included a reduction in spindle size, disorganization of microtubules within the spindle, and, in some cases, even a complete absence of microtubules.39,40 The susceptibility of the microtubules to temperature variations has been also shown in mature mouse oocytes.41 Interference with spindle organization can disturb the orderly segregation of the chromosomes, resulting in uneuploidity.

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Laboratory procedures Removal of the surrounding cumulus cells is accomplished by a combined enzymatic and mechanical treatment carried out under a stereoscopic dissecting microscope. A preincubation period of at least 3 hours between oocyte retrieval and removal of the cumulus cells was recommended by one study.42 This recommendation was challenged by other studies, which did not demonstrate differences in ICSI outcomes that correlate with the time interval between egg aspiration and microinjection.43,44 On the other hand, preincubation time that exceeded 9 hours resulted in embryos of lower quality.43 Since oocyte denudation cannot be carried out before some preliminary laboratory preparations that are described below are completed, a preincubation period of at least 1 hour is unavoidable. During this period the retrieved mature cumulus–oocyte complexes are kept in the incubator at 37oC with 5% CO2.

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aspirated through a Pasteur pipette for up to 30–40 seconds. At this time dissociation of the cells is initially observed. Further mechanical denudation is carried out in the enzyme-free HEPES-buffered medium droplets by repeated aspiration through commercially prepared stripper tips with decreasing inner diameters of 150 and 135 µm. The oocytes are then transferred through the droplets of medium, until all coronal cells have been finally removed and all traces of enzyme have been washed off. This procedure is carried out very gently in order to avoid mechanical damage to the oocytes. Pricking of the oocyte has been shown to induce parthenogenetic egg activation.47,48 Finally, the denuded oocytes are placed in the droplets of the injecting dish and their morphology and meiotic status are evaluated. The procedures described above are performed on the heated area in the hood.

Preliminary preparations for oocyte denudation

Evaluation of denuded oocytes for ICSI

Injecting dish A special shallow Falcon dish (type 1006) is used for placing the denuded eggs. Nine small droplets of HEPES-buffered culture media, 5 µl each, are arranged in a square of 3 × 3 within this dish. An additional 10th droplet serves for orientation. The middle droplet, in which the sperm will be placed, contains 10% polyvinylpyrrolidone (PVP). The droplets are then covered with paraffin or mineral oil, and the dish is placed on the heated area in the hood, to warm up before removal of the cumulus cells.

Oocytes are assessed for their maturation and for their morphology under an inverted microscope equipped with Nomarski differential interference contrast (DIC) optics, at 200× magnification. It is commonly accepted that only mature oocytes that resumed their first meiotic division reaching MII are appropriate for ICSI. Evaluation of the meiotic status of the oocyte is based on morphological markers. In mature oocytes, the GV has disappeared and the first polar body is present and localized in the perivitelline space (Fig 8.1c). Several studies have reported that 10–12% of the retrieved oocytes have not resumed their meiotic division.49–52 These oocytes can be divided into two categories: first, GV oocytes in which meiosis has not been reinitiated and the typical nuclear structure is visible (Fig 8.1a), and secondly, GVB oocytes in which meiosis has been reinitiated but did not proceed beyond the first metaphase (MI). In these oocytes the GV has disappeared but the first polar body has not been extruded (Fig 8.1b). Oocytes of both these categories are separated from the MII oocytes and further incubated until the first polar body is extruded. It has been reported that 74% of the MI oocytes completed meiosis in vitro within 20 hours after retrieval. This report did not find differences in the rates of fertilization and embryo development between these oocytes and other oocytes retrieved at MII. However, only one pregnancy was achieved following the transfer of embryos obtained from fertilized MI oocytes that had matured in vitro.53 Another study demonstrated that 26.7% of MI oocytes extruded the first polar body in vitro within 4 hours. These oocytes were injected on the day of follicle aspiration in parallel to the oocytes retrieved at MII. In this study, however, the MI oocytes that completed their maturation in vitro exhibited a lower fertilization rate, but again no differences were observed in embryo quality between oocytes that underwent maturation in vitro

Enzymatic solution Since hyaluronic acid is a major component of the mucified cumulus mass that surrounds the mature oocyte, hyaluronidase is employed for enzymatic removal of these cells. Hyaluronidase (type III, specific activity 327 IU/mg solid, Sigma Chemical Co., St Louis, MO, USA) is dissolved in HEPES-buffered Earle’s medium. The high concentration of 760 IU/ml of hyaluronidase that was used initially (1991) was found to induce parthenogenetic activation of the mature oocytes. Lower concentrations of the enzyme such as 80 IU/ml, which is being commonly used, significantly decrease the rate of parthenogenesis.45 A concentration as low as 10 IU/ml has also been shown to denude mature oocytes efficiently.46 Denuding dish A droplet of 100 µl of hyaluronidase solution and five droplets of HEPES-buffered medium covered with oil are placed in a large culture dish and placed on the heated area in the hood to warm up for 10 minutes. In order to keep the droplets at 37oC, the temperature in the working areas (hood and microscope) are calibrated to around 38oC.

Removal of the cumulus cells Cumulus–oocyte complexes are transferred into the droplet of hyaluronidase solution and repeatedly

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a

b

c

d

e

f

Fig 8.2 Various morphological abnormalities exhibited by oocytes. (a) Granulated perivitelline space; (b) a fragmented polar body; (c) thickened and dark-colored zona pellucida; (d) cytoplasmic inclusions; (e) enlarged and granulated perivitelline space; (f) a large cytoplasmic vacuole.

and those retrieved at MII. Similar to the previous study, only one pregnancy was obtained following transfer of embryos developed from MI oocytes that had matured in vitro.54 More recent studies support these observations, showing that although in vitro matured (IVM) MI oocytes can be normally fertilized, the embryos derived from these oocytes rarely provide pregnancies,55,56 suggesting that rescue of MI oocytes of patients with few MII oocytes may increase the number of embryos for transfer; however, the chance to improve pregnancy rates by this procedure is minimal. GV oocytes require an overnight (30 hours) incubation in order to reach the MII stage. Only sporadic pregnancies were reported from oocytes that were retrieved at the GV stage although standard IVF treatment with controlled ovarian hyperstimulation was performed.55,57 Because of the poor results, these GV oocytes are usually discarded. Only in cases in which very few or no MII oocytes were retrieved are the GV oocytes rescued for fertilization, provided that they have completed their maturation. Immature GV oocytes can also be retrieved from the small (3–13 mm) ovarian follicles present in nonstimulated patients.58–61 These oocytes, which were not exposed to LH in vivo, apparently do not exhibit meiotic incompetence and can be expected to mature spontaneously in vitro and produce normal eggs. In 1998 Goud et al showed a fertilization rate of 46% by ICSI of such IVM GV oocytes,61 with only a few

pregnancies. Later studies have shown that, as more experience is gained in handling immature oocytes, success rates are increasing worldwide.62,63 In addition to the meiotic status, the morphology of the oocytes is also evaluated before ICSI. The various morphological defects may be manifested by an amorphic shape of the oocyte, enlargement of or granularity in the perivitelline space, inclusions, vacuolization, granularity and dark color of the cytoplasm, changes in the color and construction of the zona pellucida, and changes in the shape and size of the polar body (Fig 8.2). Most defective oocytes exhibit more than one of the above-mentioned abnormalities. All these observations should be recorded and may help in later analysis of the fertilization rate, embryo development, and pregnancy outcomes after ICSI. The correlation between egg morphology and the rates of fertilization, embryo quality, and pregnancy after ICSI has been extensively studied. Most of the studies reported that abnormal egg morphology of patients undergoing ICSI is associated with a lower fertilization rate, embryos of poor quality, and, consequently, a lower pregnancy rate.64–66 Other studies demonstrated successful fertilization and normal early embryo development in microinjected eggs with defective morphology, such as large perivitelline space, cytoplasmic vacuoles, or a fragmented polar body.67–70 However, the transfer of these seemingly normal embryos resulted in a poor implantation rate66

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b

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c

Fig 8.3 Microtubule images in metaphase II (MII) human oocytes. (a) Microtubules of the midbody extending from the cytoplasm into the first polar body (PB). (b) Microtubules of the second meiotic spindle located adjacent to the first PB. (c) Microtubules of the second meiotic spindle at a distal location from the PB.

and a high incidence of early pregnancy loss.68 This controversy may be partially attributed to the absence of standard criteria for evaluation of oocyte morphology. To overcome this confusion, the use of triple markers for human oocyte grading that include polar body, size of perivitelline space, and cytoplasmic inclusions has been suggested by Xia.65 This laboratory reported that evaluation of oocyte quality based on these criteria correlated well with the rate of fertilization and with embryo quality after ICSI. As mentioned previously in this chapter, the integrity of the meiotic spindle in MII oocytes is crucial for normal fertilization and embryo development. Therefore, in addition to the above-mentioned features of the oocyte, the morphology of the spindle may serve as a reliable marker for predicting its potential for normal fertilization. A modification of the polarized light microscope ‘Polscope’, equipped with novel imageprocessing software,71 has emerged as a noninvasive tool to view the meiotic spindle in living oocytes and is being used in several IVF units worldwide.40,72,73 The image of the spindle is based on the highly birefringent characteristic of the microtubule filaments under a polarization microscope. The obvious advantage of the Polscope over conventional techniques such as immunocytochemistry and electron microscopy is the ability to view the spindle in a living oocyte. Use of the Polscope for examination of human oocytes has indeed demonstrated that the absence of, or abnormal morphology of, the spindle is highly correlated with lower fertilization rates and impaired embryonic development.72–74 In most MII oocytes, the second meiotic spindle is adjacent to the first polar body (Fig 8.3b), making the first polar body a marker for appropriate orientation of the ICSI micropipette to avoid interference with chromosome alignment. However, observations by Silva et al72 and ourselves that the meiotic spindle is not always located near the polar body (Fig 8.3c) has made use of the Polscope even more valuable. Furthermore, in those oocytes

that have not yet completed formation of the first polar body, the Polscope can detect the presence of microtubules in the midbody, suggesting that the second meiotic spindle has not yet been fully organized (Fig 8.3a). These oocytes are considered suitable for ICSI, having high potential for developing into an embryo. Appropriate ovarian stimulation protocols normally provide functional fertilizable mature oocytes, while oocytes of poor quality may represent a disturbed hormonal balance. For example, exposure to high dosage of human menopausal gonadotropin (hMG) has been shown to be associated with granularity of the perivitelline space.51 Moreover, an extended exposure to high dosage of this hormone may lead to the senescence of the mature oocyte before retrieval. As previously mentioned, oocyte maturation and ovulation are both stimulated by LH. However, studies have shown that the ovulatory response is less sensitive to this gonadotropin, requiring higher concentrations of the hormone.75 Therefore, the relatively high concentration of LH in hMG effectively promotes oocyte maturation, but is insufficient to stimulate ovulation. Delayed administration of human chorionic gonadotropin (hCG) in these patients entraps the mature oocytes in the follicle, leading to egg aging. One notable morphological marker in this case is the fragmentation of the first polar body.76 The presence of aged eggs can also explain the decreased quality of oocytes and lower fertilization rate in polycystic ovarian syndrome (PCOS) patients77 who exhibit relatively high serum concentrations of LH throughout their menstrual cycle.78

Epilogue A baby girl is born with her ovaries containing about two million oocytes, all of them arrested at the prophase of the first meiotic division. This pool of oocytes remains dormant throughout infancy until

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the onset of puberty. In sexually mature females, at each cycle, one such ‘sleeping beauty’ is being kissed by the LH ‘prince’ and awakened to continue its meiotic division. Once maturation has been completed the oocyte is released from the ovarian follicle into the fallopian tube, a site at which it will eventually meet the spermatozoon and undergo fertilization. Hormonal stimulation protocols are designed to mimic the natural events that lead to production of mature oocytes. In IVF patients, these oocytes are aspirated from the ovarian follicles prior to ovulation and allowed to meet the sperm cells in the Petri dish. A higher scale of assistance, designed to overcome poor performance of spermatozoa, is offered by ICSI. The information regarding oocyte handling for this relatively novel protocol has been summarized in this chapter.

2.

3.

Microscopic evaluation 1.

2.

Appendix Laboratory protocol The following protocol is used in our laboratory.

Preliminary preparations for oocyte denudation 1.

2.

3.

4.

Injecting dish. Place nine, 5 µl each, droplets of HEPES-buffered human tubal fluid medium containing 10% synthetic serum, arranged in a 3 × 3 square within a shallow Falcon dish (type 1006). Place one additional droplet for orientation. Cover with oil. Replace 4 µl of the middle droplet, with a solution of 10% polyvinylpyrrolidone (PVP). Place the dish on the heated area in the hood to warm up. Enzymatic solution. Dissolve 10 mg of hyaluronidase (type III, specific activity 327 IU/mg solid, Sigma Chemical Company, St Louis, MO, USA) in 5.5 ml of HEPES-buffered human tubal fluid medium containing 10% serum, to obtain a final concentration of 600 IU/ml. After passing through 0.2 µm filters, divide into aliquots of 0.15 ml and store in −20oC. When needed, thaw one aliquot and add 1.35 ml HEPES-buffered medium containing 10% serum to obtain a final concentration of 60 IU/ml, and warm to 37oC. Denuding dish. Place a droplet of 100 µl of the above hyaluronidase solution and five droplets of enzyme-free HEPES-buffered medium in a large culture dish. Cover with oil and place on the heated area in the hood to warm up. Prepare two stripper tips with inner diameters of 150 and 135 µm.

Removal of the cumulus cells 1.

Place the cumulus–oocyte complexes into droplet of hyaluronidase solution (up to five complexes at a time) and aspirate repeatedly through a Pasteur pipette for up to 40 seconds.

Transfer the cumulus–oocyte complexes to a droplet of enzyme-free HEPES-buffered medium and aspirate repeatedly through a 150 µm-diameter stripper tip. Continue aspirating with a 135-µm tip while passing the oocytes through the other four droplets of the medium, until all coronal cells have been totally removed. Transfer the denuded oocytes to the droplets of HEPES-buffered medium in the injecting dish, one in each droplet.

Place the injecting dish containing the oocytes on the heated stage of an inverted microscope equipped with DIC. Evaluate oocyte morphology and meiotic status at 200× magnification.

References 1. Dekel N, Aberdam E, Goren S, Feldman B, Shalgi R. Mechanism of action of GnRH-induced oocyte maturation. J Reprod Fertil Suppl 1989; 37: 319–27. 2. Lindner HR, Tsafriri A, Lieberman ME, et al. Gonadotropin action on cultured Graafian follicles: induction of maturation division of the mammalian oocyte and differentiation of the luteal cell. Recent Prog Horm Res 1974; 30: 79–138. 3. Dekel N. Hormonal control of ovulation. In: Litwack G, ed. Biochemical Action of Hormones, Vol. 13. Orlando, Florida: Academic Press, 1986; 57–90. 4. Buccione R, Vanderhyden BC, Caron PJ, Eppig JJ. FSH-induced expansion of the mouse cumulus oophorus in vitro is dependent upon a specific factor(s) secreted by the oocyte. Dev Biol 1990; 138: 16–25. 5. Salustri A, Yanagishita M, Hascall VC. Mouse oocytes regulate hyaluronic acid synthesis and mucification by FSH-stimulated cumulus cells. Dev Biol 1990; 138: 26–32. 6. Vanderhyden BC, Caron PJ, Buccione R, Eppig JJ. Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation. Dev Biol 1990; 140: 307–17. 7. Vanderhyden BC. Species differences in the regulation of cumulus expansion by an oocyte secreted factor(s). J Reprod Fertil 1993; 98: 219–27. 8. Lin Y, Mahan K, Lathorp W, Myles D, Primakoff P. A hyaluronidase activity of the sperm plasma membrane protein PH-20 enables sperm to penetrate the cumulus cell layer surrounding the egg. J Cell Biol 1994; 125: 1157–63. 9. Cherr G, Meyers S, Yudin A, et al. The PH-20 protein in cynomologus macaque spermatozoa: identification of two different forms exhibiting hyaluronidase activity. Dev Biol 1996; 175: 142–53. 10. Oversreet J, Lin Y, Yudin A, et al. Location of the PH20 protein on acrosome-intact and acrosome-reacted spermatozoa of cynomologus macaques. Biol Reprod 1995; 52: 105–14.

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Preparation and evaluation of oocytes for ICSI 11. Sabeur K, Cherr G, Yudin A, et al. The PH-20 protein in human spermatozoa. J Androl 1997; 18: 151–8. 12. Meyers SA, Rosenberger AE. A plasma membraneassociated hyaluronidase is localized to the posterior acrosomal region of stallion sperm and is associated with spermatozoal function. Biol Reprod 1999; 61: 444–51. 13 Bleil JD, Wasserman PM. Autoradiographic visualization of the mouse egg’s sperm receptor bound to sperm. J Cell Biol 1986; 102: 1363–71. 14. Beaver EL, Friend DS. Morphology of mammalian sperm membranes during differentiation, maturation, and capacitation. J Electron Microsc Tech 1990; 16: 281–97. 15. Mortillo S, Wasserman PM. Differential binding of gold-labeled zona pellucida glycoproteins mZP2 and mZP3 to mouse sperm membrane compartments. Development 1991; 113: 141–9. 16. Jones R. Interaction of zona pellucida glycoproteins, sulphated carbohydrates and synthetic polymers with proacrosin, the putative egg-binding protein from mammalian spermatozoa. Development 1991; 111: 1155–63. 17. Urch UA, Patel H. The interaction of boar sperm proacrosin with its natural substrate, the zona pellucida, and with polysulphated polysaccharides. Development 1991; 111: 1165–72. 18. Yanagimachi R. Mechanisms of fertilization in mammals. In: Mastroianni L, Biggers JD, eds. Fertilization and Embryonic Development in Vitro. New York: Plenum Press, 1981: 133–4. 19. Dunbar BS, Budkiewicz AB, Bundman DS. Proteolysis of specific porcine zona pellucida glycoproteins by boar acrosin. Biol Reprod 1985; 32: 619– 30. 20. Brown CR, Cheng WTK. Limited proteolysis of the porcine zona pellucida by homologous sperm acrosin. J Reprod Fertil 1985; 74: 257–60. 21. Dunbar BS, Prasad SV, Timmons TM. Comparative Overview of Mammalian Fertilization. New York: Plenum Press, 1991. 22. Yanagimachi R. Time and process of sperm penetration into hamster ova in vivo and in vitro. J Reprod Fertil 1966; 11: 359–70. 23. Phillips DM, Shalgi RM. Sperm penetration into rat ova fertilized in vivo. J Exp Zool 1982; 221: 373–8. 24. Shalgi R, Phillips D. Mechanics of sperm entry in cycling hamsters. J Ultrastruct Res 1980; 71: 154–61. 25. Shalgi R, Phillips DM, Jones R. Status of the rat acrosome during sperm–zona pellucida interactions. Gamete Res 1989; 22: 1–13. 26. Cohen J, Malter H, Fehilly C, et al. Implantation of embryos after partial opening of oocyte zonal pellucida to facilitate sperm penetration. Lancet 1988; 2: 162. 27. Cohen J, Malter H, Wright G, et al. Partial zona dissection of human oocytes when failure of zona pellucida is anticipated. Hum Reprod 1989; 4: 435–42. 28. Cohen J, Talanski BE, Malter HM, et al. Microsurgical fertilization and teratozoospermia. Hum Reprod 1991; 6: 118–23. 29. Tucker MJ, Bishop FM, Cohen J, et al. Routine application of partial zona dissection for male factor infertility. Hum Reprod 1991; 6: 676–81.

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30. Laws-King A, Trounson A, Sathananthan H, et al. Fertilization of human oocytes by microinjection of single spermatozoon under zona pellucida. Fertil Steril 1987; 48: 637–42. 31. Ng SC, Bongso A, Ratnam SS, et al. Pregnancy after transfer sperm under zona. Lancet 1988; 2: 790. 32. Bongso TA, Sathananthan AH, Wong C, et al. Human fertilization by microinjection of immotile spermatozoa. Hum Reprod 1989; 4: 175–9. 33. Palermo G, Joris H, Devoroey P, et al. Induction of acrosome reaction in human spermatozoa used subzonal insemination. Hum Reprod 1992; 7: 248–54. 34. Palermo G, Joris H, Devoroey P, et al. Pregnancies after intracytoplasmic injection of a single spermatozoon into an oocyte. Lancet 1992; 340: 17–18. 35. Palermo G, Joris H, Devoroey P, et al. Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertil Steril 1993; 59: 826–35. 36. Van Steirteghem AC, Liu J, Nagy Z, et al. Use of assisted fertilization. Hum Reprod 1993; 8: 1784–5. 37. Van Steirteghem AC, Liu J, Joris H, et al. Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. Report of a second series of 300 consecutive treatment cycles. Hum Reprod 1993; 8: 1055–60. 38. Van Steirteghem AC, Nagy Z, Joris H, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1993; 8: 1061–6. 39. Pickering SJ, Braude PR, Johnson MH, Cant A, Currie J. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990; 54: 102–8. 40. Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. Limited recovery of meiotic spindle in living human oocytes after cooling–rewarming observed using polarized microscopy. Hum Reprod 2001; 16: 2374–8. 41. Magistrini M, Szollosi D. Effects of cold and isopropyl-N-phenylcarbamate on the second meiotic spindle of mouse oocytes. Eur J Cell Biol 1980; 22: 699–707. 42. Rienzi L, Ubaldi F, Anniballo R, Cerulo G, Greco E. Preincubation of human oocytes may improve fertilization and embryo quality after intracytoplasmic sperm injection. Hum Reprod 1998; 13: 1014–19. 43. Yanagida K, Yazawa H, Katayose H, et al. Influence of preincubation time on fertilization after intracytoplasmic sperm injection. Hum Reprod 1998; 13: 2223–6. 44. Van de Velde H, De Vos A, Joris H, Nagy ZP, Van Steirteghem AC. Effect of timing of oocyte denudation and micro-injection on survival, fertilization and embryo quality after intracytoplasmic sperm injection. Hum Reprod 1998; 13: 3160–4. 45. Joris H, Nagy Z, Van de Velde H, De Vos A, Van Steirteghem A. Intracytoplasmic sperm injection: laboratory set-up and injection procedure. Hum Reprod 1998; 13(Suppl 1): 76–86. 46. Van de Velde H, Nagy ZP, Joris H, De Vos A, Van Steirteghem AC. Effects of different hyaluronidase concentrations and mechanical procedures for cumulus cell removal on the outcome of intracytoplasmic sperm injection. Hum Reprod 1997; 12: 2246–50.

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47. Iritani A. Micromanipulation of gametes for in vitro assisted fertilization. Mol Reprod Dev 1991; 28: 199–207. 48. Flaherty SP, Payne D, Swann NG, et al. Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection. Hum Reprod 1995; 10: 2629–32. 49. Junca AM, Mandelbaum J, Belaisch-Allert J, et al. Oocyte maturity and quality: value of intracytoplasmic sperm injection. Fertility of microinjected oocytes after in vitro maturation. Contracept Fertil Sex 1995; 23: 463–645. 50. Mandelbaum J, Junca AM, Balaisch-Allert J, et al. Oocyte maturation and intracytoplasmic sperm injection. Contracept Fertil Sex 1996; 24(7–8): 534–8. 51. Hassan-Ali H, Hisham-Saleh A, El-Gezeiry D, et al. Perivitelline space granularity: a sign of human menopausal gonadotropin overdose in intracytoplasmic sperm injection. Hum Reprod 1998; 13: 4325–30. 52. De Vos A, Van de Velde H, Joris H, Van Steirteghem A. In-vitro matured metaphase-I oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-II oocytes after intracytoplasmic sperm injection. Hum Reprod 1999; 14: 1859–63. 53. Coetzee K, Windt ML. Fertilization and pregnancy using metaphase I oocytes in an intracytoplasmic sperm injection program. J Assist Reprod Genet 1996; 13: 768–71. 54. Nagy ZP, Cecile J, Liu J, et al. Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal-vesicle stage oocytes: case report. Fertil Steril 1996; 65: 1047–50. 55. Jaroudi KA, Hollanders JMG, Sieck UV et al. Pregnancy after transfer of embryos which were generated from in-vitro matured oocytes. Hum Reprod 1997; 12: 857–9. 56. Liu J, Katz E, Garcia JE, et al. Successful in vitro maturation of human oocytes not exposed to human chorionic gonadotropin during ovulation induction, resulting in pregnancy. Fertil Steril 1997; 67: 566–8. 57. Menezo YJ, Nicollet B, Rollet J, Hazout A. Pregnancy and delivery after in vitro maturation of naked ICSIGV oocytes with GH and transfer of a frozen thawed blastocyst: case report. J Assist Reprod Genet 2006; 23: 47–9. 58. Edrishinghe WR, Junk SM, Matson PL, Yovich JL. Birth from cryopreserved embryos following in-vitro maturation of oocytes and intracytoplasmic sperm injection. Hum Reprod 1997; 12: 1056–8. 59. Trounson A, Anderiesz C, Jones GM, et al. Oocyte maturation. Hum Reprod 1998; 13(Suppl 3): 52–62; discussion 71–5. 60. Russell JB. Immature oocyte retrieval with in-vitro oocyte maturation. Curr Opin Obstet Gynecol 1999; 11: 289–96. 61. Goud PT, Goud AP, Qian C, et al. In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium. Hum Reprod 1998; 13: 1638–44. 62. Mikkelsen AL. Strategies in human in-vitro maturation and their clinical outcome. Reprod Biomed Online 2005; 10: 593–9.

63. Al-Sunaidi M, Tulandi T, Holzer H, et al. Repeated pregnancies and live births after in vitro maturation treatment. Fertil Steril 2007; 87: 1212. e9–12. 64. Sousa M, Tesarik J. Ultrastructural analysis of fertilization failure after intracytoplasmic sperm injection. Hum Reprod 1994; 9: 2374–80. 65. Xia P. Intracytoplasmic sperm injection: correlation of oocyte grade based on polar body, perivitelline space and cytoplasmic inclusions with fertilization rate and embryo quality. Hum Reprod 1997; 12: 1750–5. 66. Loutradis D, Drakakis P, Kallianidis K, et al. Oocyte morphology correlates with embryo quality and pregnancy rate after intracytoplasmic sperm injection. Fertil Steril 1999; 72: 240–4. 67. De Sutter P, Dozortsev D, Qian C, Dhont M. Oocyte morphology does not correlate with fertilization rate and embryo quality after intracytoplasmic sperm injection. Hum Reprod 1996; 11: 595–7. 68. Alikani M, Palermo G, Adler A, et al. Intracytoplasmic sperm injection in dismorphic human oocytes. Zygote 1995; 3: 283–8. 69. Serhal PF, Ranieri DM, Kinis A, et al. Oocyte morphology predicts outcome of intracytoplasmic sperm injection. Hum Reprod 1997; 12: 1267–70. 70. Balaban B, Urman B, Sertac A, et al. Oocyte morphology does not affect fertilization rate, embryo quality and implantation rate after intracytoplasmic sperm injection. Hum Reprod 1998; 13: 3431–3. 71. Oldenbourg R, Mei G. New polarized light microscope with precision universal compensator. J Microsc 1995; 180: 140–7. 72. Silva CP, Kommineni K, Oldenbourg R, Keefe DL. The first polar body does not predict accurately the location of the metaphase II meiotic spindle in mammalian oocytes. Fertil Steril 1999; 71: 719–21. 73. Wang WH. Spindle observation and its relationship with fertilization after ICSI in living human oocytes. Fertil Steril 2001; 75: 348–53. 74. Moon JH, Hyun CS, Lee SW, et al. Visualization of the metaphase II meiotic spindle in living human oocytes using the Polscope enables the prediction of embryonic developmental competence after ICSI. Hum Reprod 2003; 18: 817–20. 75. Dekel N, Ayalon D, Lewysohn O, et al. Experimental extension of the time interval between oocyte maturation and ovulation: effect on fertilization and first cleavage. Fertil Steril 1995; 64: 1023–8. 76. Eichenlaub-Ritter U, Schmiady H, Kentenich H, et al. Recurrent failure in polar body formation and premature chromosome condensation in oocytes from a human patient: indicators of asynchrony in nuclear and cytoplasmic maturation. Hum Reprod 1995; 10: 2343–9. 77. Aboulghar MA, Mansour RT, Serour GI, Ramzy AM, Amin YM. Oocyte quality in patients with severe ovarian hyperstimulation syndrome. Fertil Steril 1997; 68: 1017–21. 78. Shoham Z, Jacobs HS, Insler V. Luteinizing hormone: its role, mechanism of action, and detrimental effects when hypersecreted during the follicular phase. Fertil Steril 1993; 59: 1153–61.

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9 Oocyte in vitro maturation Daniela Nogueira, Sergio Romero, Leen Vanhoutte, Daniel Gustavo de Matos, Johan Smitz

Theoretical overview The relation between oogenesis, meiotic maturation, and developmental competence Oocyte growth and quality are dependent on the normal growth and differentiation of the oocyte’s harboring follicle. However, the oocyte itself also plays a direct part in the follicular environment, for example by preventing premature luteinization by regulating the secretion of cumulus mucification-enabling factors, luteinizing hormone (LH) receptor expression on cumulus cells, and kit ligand expression in granulosa cells.1–4 Human oocytes obtained for in vitro maturation (IVM) are aspirated from 6–14-mm follicles, and have not completed their growth and final maturation Previous work has shown that, during the period just preceding the final meiotic maturation stage, the synthesis and packaging of RNA and translational products are essential for determining further developmental events.5,6 When retrieving oocyte–cumulus complexes from small antral follicles for IVM, it is our aim to substitute for those intrafollicular maturation conditions which seem to be fundamental for further embryonic development. The small antral follicles which are aspirated for the purpose of IVM have already undergone a growth period of several months, and have moved into a state of gonadotropin dependence (Fig 9.1).7

The regulation mechanisms governing meiotic arrest The exact molecular nature by which the oocyte is held in nuclear arrest is still incompletely understood. Inhibitory signals originating in theca and granulosa cells are positively influenced by follicle-stimulating hormone (FSH), and are conducted via the gap junctions and follicular fluid into the oocyte (Fig 9.2). The meiosis-arrester, named oocyte meiotic inhibitor (OMI), present within the somatic compartment of the follicle has not yet been fully characterized.8 The arrest has to be seen as the result of the action of diverse molecules. Several candidate molecules, peptides, have shown a meiosis-arresting activity, such as transforming growth factor-β (TGF-β), anti-Müllerian

hormone (AMH), activin, inhibin, or follistatin. This meiosis-arresting activity is rather the result of the contribution by many factors originating in the theca interna, granulosa, and follicular fluid. Purine bases such as hypoxanthine and adenosine present in follicular fluid inhibit phosphodiesterase activity, and retain by this means a sufficiently high intra-oocyte cyclic adenosine monophosphate (cAMP) concentration to maintain oocyte meiotic arrest.9,10 Cyclic AMP is produced in granulosa cells by gonadotropins binding to their G-protein-coupled receptors (GPCR), causing activation of stimulatory guanine nucleotide-binding proteins (Gs proteins), which sequentially activate the adenyl cyclase (AC) expressed in granulosa cells, generating cAMP from adenosine triphosphate (ATP).11 The cAMP generated is transferred to the oocyte via gap junctions between the granulosa cells and the oocyte. cAMP activates the cAMP-dependent protein kinase A (PKA), which, in the absence of cAMP, exists as an inactive tetramer, comprising a dimer of regulatory (R) subunits bound to two catalytic subunits (C).12 PKAs identified in mammals include four R subunits (RIα, RIβ, RIIα, and RIIβ). The RI of PKA was initially described as the predominant form, found in mouse oocytes, and RII in granulosa cells.13 The presence of RII subunits in rat oocytes has been documented.14 The R subunits can bind four cAMP molecules, resulting in the release of C monomers, and the dissociated C subunits may phosphorylate substrates, which are inhibitory to oocyte maturation (Fig 9.3).15 There is recent evidence that the mammalian oocyte also has the capacity to produce cAMP due to the presence of G proteins,16,17 AC,18 and GPCR.19,20 Different forms of AC (ACDY) are expressed within the rodent oocyte species. While mRNA ACDY3 is the predominant gene expressed in rat oocytes, the ACDY1 and ACDY9 are the most predominant forms present in mice oocytes.18 In rat and mice, ACDY is differentially expressed in somatic and germ cells, with mRNAs amplification predominating for types ACDY6 and ACDY9 in granulosa and cumulus cells. Several mRNA GPCR have been detected in rat and mouse oocytes, including GPR12 and GPR56 mRNA,19,30 and some of the GPR receptors expressed

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FSH-dependent

FSH-independent rescue

selection

dominance

LH action

primary

secondary early terciary 0.2–2 mm

(antral) 2–5 mm

small antral 5–8 mm

middle-large antral 10–20 mm

preovulatory > 20 mm meiosis reinitiation

prophase I (GV) meiotically incompetent

GVBD-competent MII-competent early embryonic developmental-competent full embryonic developmental-competent -

ovulation of a mature & developmentally competent oocyte

Fig 9.1 Inter-relation of in vivo follicular and oocyte developmental stages. The earliest stages of follicular growth up to early antral stage occur independently of FSH stimulus. Antral follicles are rescued from atresia by the FSH action, which stimulates few antral follicles to growth, followed by a selection and a further growth of a single dominant follicle destined to ovulate. During this period, oocytes are kept arrested at prophase I, germinal vesicle (GV) stage. Oocyte developmental competence in humans is gradually acquired throughout folliculogenesis. At the earliest stages of folliculogenesis, oocytes are meiotically incompetent due to intrinsic oocyte factors. The meiotically competent oocytes enclosed in antral follicles are arrested at the GV stage because of inhibitory factors present in follicle fluid and derived from somatic cells. Upon further development, the oocyte acquires the capability to complete nuclear maturation. This is followed by a further period of cytoplasmic development in which oocytes acquire the capability to sustain early embryonic development post-fertilization. At the end of follicle growth, oocytes have attained the potential to sustain full embryonic development. Therefore, although oocytes have acquired the potential to complete nuclear maturation at an earlier stage of folliculogenesis, oocytes, cytoplasmic maturation is completed only by the end of follicle development.

Fig 9.2 (a) The oocyte is surrounded by a compacted mass of granulosa cells which holds the oocyte in the germinal vesicle (GV) stage. (b) Staining of actin with fluorochromes and confocal microscopy analysis can demonstrate the intricate transzonal connections between the granulosa cells and the oocyte. (c) A semithin section through a cultured cumulus–oocyte complex (COC) demonstrates the intact transzonal projections. (d) An electron microscopy (EM) view of a GV oocyte with apposition of the corona cells. (e) EM view at large magnification demonstrates cytoplasmic projections of corona and cumulus cells towards the oocyte. Note the short microvilli of the oolemma. (f) Part of the oolemma at large EM magnification illustrates a tight junction between the transzonal projection of a granulosa cell and the oolemma.

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OOCYTE Gs AC

ATP cAMP

GRANULOSA

MP

GPCR Gs

ATP

AMP

CREB

P

cAMP

+ P

P

AMPK

PKA I active

R

PKA I inactive

cAMP + P

A cAMP K A R cAMP P

AMP

C R

AC

PDE 4

C

cA

PDE3A

C

RR A K R A P cAMP

cAMP R

P

cAMP

cAMP

C

cAMP

GVBD

C

R

P ?

cAMP C

PKA II active

cAMP

nucleus

cA

MP

cA

P

MP

cAM

Fig 9.3 Paradoxical effect of cyclic adenosine monophosphate (cAMP) during oocyte maturation. Cyclic AMP generated by adenylyl cyclase (AC) in granulosa cell (GC) and transferred to oocyte via gap junctions activates the regulatory (R) subunits of protein kinase A (PKA) anchored to A-kinase anchor proteins (AKAPs) for their localization. The oocyte also has the capacity to produce cAMP due to the presence of AC. RI subunits of PKA can bind four cAMP molecules, resulting in the release of catalytic (C) monomers phosphorylating unknown substrates in the oocyte which are inhibitory to maturation. PKA RII is also present in oocytes but its effect on maturation is unknown. Activation of phosphodiesterase 3 (PDE3) in oocytes leads to a decrease in cAMP by hydrolysis, forming the AMP product. The decrease in cAMP leads to a decrease in PKA activity and the arresting influence on oocyte maturation is no longer in existence. In rodent oocytes, the cAMP product, AMP, activates the regulatory enzyme AMP-activated protein kinase (AMPK) and an increase in AMPK activity cooperates with oocyte maturation. In somatic cells, activation of PKA results in the phosphorylation (P) of certain transcription factors (cAMP response element binding protein, CREB), regulating transcription of several genes. Activation of PKAII in cumulus cells is stimulatory to oocyte meiosis reinitiation. Gs, stimulating subunit of G-protein complex; ATP, adenosine triphosphate; GVBD, germinal vesicle breakdown.

in oocytes are receptors with unknown ligands (orphan receptors). GPR3 is the predominant form present in mice oocytes, playing an essential role in regulating meiotic arrest: follicle-enclosed mice oocytes lacking GPR3 gene undergo spontaneous oocyte maturation independently of an increase in LH.21 Oocytes from Gpr3-/- females resume meiosis prematurely in vivo in about one-third of antral follicles.22 While juvenile Gpr3-/- females are fertile but have a reduction in litter size, aging Gpr3-/- females present a reduction of fertility because of increasing number of abnormal oocytes and arrested embryos upon ovulation.22 Studies on rat oocytes suggest a role for the protooncogene c-kit in the participation of meiotic arrest. Kit ligand (in granulosa cells) could be implicated as an oocyte meiosis-inhibiting substance.23 Lately, it has been suggested that the oocyte itself would be able to produce enough cAMP and thus maintain the meiotic arrest. A G-protein-coupled receptor family, GPR3, was shown to be present in the oocyte and induce activation of Gs protein, which in turn stimulates the adenyl cyclase, leading to intra-oocyte cAMP

production. This is supported by studies in knockout mice for GPR3, which cannot sustain meiotic arrest once follicles reach the antral stage.16,17,24

Competence to resume meiosis and mechanisms driving reinitiation of meiosis Meiotic competence is sequentially acquired during the final phase of oocyte growth. The oocyte initially becomes able to undergo germinal vesicle breakdown (GVBD), but arrests at metaphase I (MI). With further development it acquires the ability to reach metaphase II (MII), and becomes meiotically competent. The relation between oocyte volume and the competence to reinitiate meiosis has been well established for different mammalian species. Once the oocyte has acquired a critical amount of its final volume it can reinitiate meiosis when it is retrieved from the follicle.25 This oocyte volume has been related to a certain follicular diameter in the different species: 1–2 mm in mice;26 2–4 mm in cattle;27–29 and 7–10 mm in humans.30–33

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Textbook of Assisted Reproductive Technologies P2A cdc25

cdc25-P

inactive

active

ASSEMBLY cdc2-kinase

INACTIVE MPF

P34

ACTIVE MPF

cdc2-kinase cyclin-B

cdc2-kinase

DESTRUCTION

Tyr cyclin-B

Thr

cyclin-B

cdc2-kinase

Tyr

P

Thr

P

P34

P2A wee1

wee1-P

active

inactive

mRNA CYCLIN-B PROTEOLYSIS

DNA

Fig 9.4 M-phase-promoting factor (MPF) activation: activation of MPF by complex formation of p34cdc2 and cyclin B and dephosphorylation. P, phosphorylation; Tyr, tyrosine; Thr, threonine.

It is clear that cAMP molecules are key meiotic regulators in mammalian oocytes. The debate centers on whether the major source of intra-oocyte cAMP is produced intrinsically or extrinsically via diffusion from somatic cells. Most studies show that maintenance of basal or high steady-state levels of cAMP in oocyte blocks maturation. Upon release of oocytes from their follicles, it is supposed that a drop of intra-oocyte cAMP is enough to cause meiotic arrest release in vitro, i.e. spontaneous oocyte maturation. Cyclic AMP can be provided by the somatic cells diffusing via gap junctions. However, evidence supports the hypothesis that cAMP can be also produced within the oocyte in sufficient amounts to maintain the meiotic arrest state, and external factors stimulate the intra-oocyte synthesis of cAMP.9 Consistent with this idea, microinjection of a dominant negative form of Gs protein into mouse and Xenopus follicleenclosed oocytes causes meiosis resumption34 and the meiotic arrest can be released in mice by microinjecting the follicle-enclosed oocytes with an antibody that inhibits the Gs protein.34 Intra-oocyte cAMP level is also regulated by a paracrine system mediated by gonadotropin actions involving transcripts from theca cells which stimulate oocyte G-inhibitory (Gi) proteins and releasing oocytes from meiotic arrest.19 These data support the concept that Gs-regulated generation of cAMP by the oocyte is a common mechanism for maintaining meiotic prophase arrest. However, mechanisms regulating G-protein expression in oocytes remain to be elucidated in mammals. Although there is considerable evidence to support cAMP-dependent meiosis arrest in meiotically

competent oocytes, high levels of cAMP in granulosa cells leads to meiosis resumption. Several proposed models have been researched to clarify these paradoxical effects of cAMP preceding oocyte maturation: •

•

•

•

Stimulation of cAMP production in granulosa cells followed by the decrease in the cAMP in the oocyte, as a result of gap junctions uncoupling, which leads to oocyte maturation.35–37 On gonadotropin-induced cAMP production in granulosa cells, signals are activated to degrade intra-oocyte cAMP at increased rates.38 The somatic cells, via paracrine means, activate mechanisms in oocyte for inhibition of cAMP production.19 Positive cAMP-mediated signal(s) within cumulus cells activate mechanisms in the oocyte, overriding the inhibitory cAMP levels,39,40 because meiotic resumption can occur in response to gonadotropin – also epidermal growth factor (EGF), EGF-like factors, GH, TNF-α, and other substances (FSH and FF-MAS) – in the presence of cAMP analogs and hypoxanthine.9,41,42 Thus, cAMP does not necessarily have to fall below basal levels for meiosis to occur; a decrease in cAMP following an increase may therefore be necessary to induce meiosis resumption.

Resumption of meiosis involves several signal transduction pathways. Downstream of PKA deactivation, M-phase-promoting factor (MPF), mitogen-activating protein kinase (MAPK), and other cyclin-dependent kinases (cdks) function simultaneously and interact as crucial regulators in the oocyte meiotic cell cycle.

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GRANULOSA AND THECA

115

In vitro maturation

OMI

OMI

oocyte

oocyte

GAP JUNCTIONS

OBSERVATIONS FIRST

RUPTURE CONTACTS CUMULUS EXPANSION

NUCLEAR MEMBRANE CHANGES

SECOND

NUCLEAR MEMBRANE CHANGES

RUPTURE CONTACTS

The rate of synthesis and control of activation of these factors are species-dependent. Meiotic competence is a reflection of the activation of MPF, a heterodimer composed of p34cdc2, a serine–threonine kinase, and the regulatory subunit, cyclin B (Fig 9.4). MPF is stored in immature oocytes in its inactive form (pre-MPF), and its amount differs depending on the animal species. MPF activation is a result of dephosphorylation of threonine residue 14 and tyrosine 15 of p34cdc2 at the entry into MI. This last step of dephosphorylation is under the control of the gene products wee1 and cdc25 phosphatase. There exists furthermore an autocatalytic amplification of MPF.43 By aspirating the oocyte–granulosa cell (cumulus) complexes (COCs) from the follicle, connections to surrounding cells are broken. The factors from theca and mural granulosa cells responsible for keeping meiosis arrested are no longer transferred to the oocyte via the junctional contacts, and the nuclear maturation program is started.8 Experience has shown that, in contrast to the (natural) hormonal induction of final maturation, the first sign of meiosis reinitiation after mechanical disruption is folding of the oocyte’s nuclear membrane instead of rupture of the gap junctional processes, testifying that there might be factors other than those provided via gap junctions that trigger germinal vesicle breakdown (Fig 9.5).44 Activation of MPF in the human requires protein synthesis, as in other domestic mammalian species. Work from Crozet et al45 in the goat emphasized that a deficiency in the expression of p34cdc2, the catalytic subunit of MPF, may be a limiting factor for acquisition of GVBD competence, the oocytes being already equipped with the regulatory subunit cyclin B in meiotically incompetent oocytes.45 Experiments in bovine oocytes showed that cyclin B plays a major part in the initiation of p34 activation, and that this protein represents the limiting factor for meiotic resumption.46

Fig 9.5 Differences in sequential morphological events leading to reinitiation of meiosis I. Left panel: intrafollicular induction of meiosis by the luteinizing hormone (LH) signal. Right panel: reinitiation of meiosis by disruption of the contacts between oocyte and follicular wall. OMI, oocyte meiotic inhibitor.

The need for neosynthesis of cyclin B explains the lag time between the signal for meiosis and GVBD and the transition from metaphase I to metaphase II. The transition between MI and MII is associated with cyclin B degradation, where in anaphase I there is a decrease of MPF (owing to cyclin degradation) to a lower level of activity, which is sufficient for the maintenance of chromosome condensation. After a secondary rise of MPF (owing to cyclin resynthesis), chromosomes align on the metaphase II plate up to the moment of eventual fertilization. The arrest in metaphase II is governed by a cytostatic factor (CSF), which is activated by the gene product of the proto-oncogene c-mos.47 The reinitiation of meiosis can be provoked by a transient fall in cAMP concentrations within the oocyte.48,49 The delicate balance in cAMP concentrations within the granulosa cells and oocyte is maintained by an inflow of cAMP, driven by the gonadotropin environment, by production of cAMP in the oocyte and degradation of cAMP by cell-specific phosphodiesterases (PDEs) (Fig 9.6 and Fig 9.7). The cAMP stabilizes interphase microtubules,52 and the relevant mediators for MPF activation are retained in a cortical cytoskeletal scaffold.53 The PDEs exist in several isoforms that are differently expressed in somatic cells and the oocyte. The effectors of cAMP are the protein kinases, among which there is also a compartmentalization of different isoforms. The signaling pathway controlling meiotic resumption is dependent on switching off PKA activity that is required for maintaining meiotic arrest. Inactivation of PKA is the result of a decrease of cAMP that might be caused by PDEmediated degradation13,54 (see also Fig 9.3). PDEs are large groups of proteins consisting of several gene families identified in mammals. PDEs inactivate cyclic nucleotides by hydrolytically cleaving the 3′-phosphoester bond to form the corresponding inactive 5′-nucleotide monophosphate products. PDEs are distinct by their regulatory subunits. Two PDEs are differentially expressed in the

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Textbook of Assisted Reproductive Technologies Activation of IP3 /DAG

Peak LH signal

Ca2+ Mobilization Dispersion of cumulus cells Ca2+ Influx Disruption of connections Ca2+-Calmodulin No inflow of inhibitory signal PKC Transitory decrease of cAMP PDE Inactivation of PKA (cAMP dependent) cAMP Reinitiation of meisois PKA

PKC MPF

GVBD Chromosomal condensation

MAPK

Spindle formation

from: Homa et al., 199350 Homa, 199551 Downs and Hunzicker-Dunn, 19953 Tsafriri et al., 199648

Fig 9.6 Hypothetical molecular mechanisms of reinitiation of meiosis. Hypothetical links between signal transduction factors and M-phase promoting factor (MPF) activation. LH, luteinizing hormone; cAMP, cyclic adenosine monophosphate; PKA, protein kinase A; IP3, inositol triphosphate; DAG, diacyl glycerol; PKC, protein kinase C; PDE, phosphodiesterase; MAPK, mitogen-activated protein kinase; GVBD, germinal vesicle breakdown.

MAPK activity

MPF activity

GV

GVBD

Pro-MI

MI

anaphase / telophase

MII meiotic stage

extrusion of 1st PB

disappearance of nuclear membrane

n 2C

2n 4C

2n 4C

2n 4C

n 2C

n 2C

Ploidy, Chromatin

Fig 9.7 Evolution of M-phase promoting factor (MPF) activity during oocyte meiosis. During anaphase/telophase MPF drops, but remains elevated above baseline; this causes the extension of the condensed state of the chromatin. Mitogen-activated protein kinase (MAPK) activity increases in oocytes during resumption of meiosis and remains high throughout progression to MII. After GVBD, this component of the serine–threonine protein kinase family is involved in microtubule organization and spindle formation. GV, germinal vesicle; GVBD, germinal vesicle breakdown; MI, metaphase I; MII, metaphase II; PB, polar body; MAPK, mitogen-activated protein kinase.

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PDE3 specific inhibitors (Cilostamide, Org9935, Milrinone)

Mural GC

IBMX/ Hypoxanthine

PDE3A PDE4 Cumulus GC

PDE4 specific inhibitor (Rolipram)

PDE4D

Fig 9.8 Intrafollicular localization of phosphodiesterases (PDEs) and respective selective (right from figure) and nonselective (left from figure) inhibitors of PDE isoforms represented in a cultured antral mouse follicle. The PDE4 gene type expressed in cumulus cells has not been identified.49 IBMX, isobutylmethylxanthine; GC, granulosa cell.

ovarian follicle and associated with cAMP diffusion control between the cellular compartments. 55 The PDE4 types D and B are present in mural granulosa and theca cells, and PDE3 type A is expressed in oocytes of several species.48,54,56 The PDE3 family is known as cyclic guanosine monophosphate (cGMP)inhibited cAMP PDE, and the PDE4 is highly selective for cAMP. While PDE3 inhibitors effectively inhibit oocyte maturation, PDE4 inhibitors cause oocyte maturation in follicle culture in the absence of gonadotropin stimulation48 (Fig 9.8). In vivo, the primary stimulus for meiosis resumption is the rise of LH, which binds to the LH receptor on granulosa cells and induces a positive yet unknown

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meiosis-inducing substance originating in the somatic compartment that triggers GVBD in the continuous presence of meiotic inhibitor. The meiosis inducer from the somatic cells either is vehiculated via the connections between granulosa and oocyte, or diffuses to the oocyte extracellularly in a paracrine mode. Marco Conti’s laboratory has recently characterized a whole network of members of the epidermal growth factor family as paracrine mediators that propagate the LH signal throughout the follicle.40 Within granulosa cells, LH generates first, an initial cAMP increase and second, the liberation of intracellular calcium stores (via phospholipase C and inositol triphosphate (a calciumreleasing ligand) and diacyl glycerol) as stimulator of protein kinase C (PKC).51 The effects of the protein kinases PKA and PKC on oocyte maturation parallel each other, and determination of the meiotic outcome is entirely dependent on the site of stimulation. PKA activation leads to phosphorylations that maintain meiotic arrest.58 PKA type 1 residing in the oocyte is inhibitory, and PKA type 2 in the cumulus leads to meiosis reinitiation.13 Therefore, cAMP can have paradoxical effects on oocyte maturation; it is the level of cAMP reached and the duration of the cAMP flux that will determine the meiotic status. PKC activation in the somatic compartment overrides the direct (inhibitory) effect of the same kinase into the oocyte and has a positive effect on meiosis (for review, see reference 59). PKA and PKC exert their action via a cascade of phosphorylations and dephosphorylations, which finally lead to MPF activation in the oocyte. The LH trigger propagates a microtubule-labilizing factor (calcium perhaps), which provokes intermixing of cell cycle molecules and, by such, activation of MPF.60–62 PKA

Cumulus/granulosa cells

Oocyte

Glucose ? Hormone

ATP + Positive GTP Stimulus cAMP (MAPK,?)

(MAS)

cAMP Type 3 PDE PKA* 5′-AMP Positive MPF + Stimulus + AMPK AMPK* MAS

GV MPF* GVB

? ATP

Fig 9.9 Proposed model for meiotic induction in mice. Hormone binding to the granulosa cells produces, through a cyclic adenosine monophosphate (cAMP)-dependent pathway, a positive stimulus that traverses gap junctions to activate phosphodiesterase 3 (PDE3) within the oocyte. This results in the degradation of cAMP, which inactivates protein kinase A (PKA) but simultaneously activates AMP-activated protein kinase (AMPK) via generation of AMP. The combined loss of PKA activity but gain in AMPK activity leads to stimulation of M-phase promoting factor (MPF) and germinal vesicle (GV) breakdown (GVB). Note that since AMPK is activated by an increase in the AMP/adenosine triphosphate (ATP) ratio, such activation can be antagonized by increases in ATP. In this model, meiosis-activating sterols (MAS) can be produced in the somatic compartment in response to gonadotropin stimulation, and may enter the oocyte either through gap junctions or by paracrine means, but it is not the principal stimulus driving meiotic resumption. The asterisk denotes the active state of the respective kinase. GTP, guanosine triphosphate. Reproduced from reference 63 with permission from Downs SM, The biochemistry of oocyte maturation. In: Eppig J, Hegele-Hartung C, Lessl M, eds. The Future of the Oocyte: Basic and Clinical Aspects. Ernst Schering Research Foundation, Worksop 41. Berlin: SpringerVerlag, 2002: 81–99. Copyright Springer-Verlag Berlin Heidelberg 2002; Figure p. 93 Chapter 6.

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The as yet unknown ‘positive factor’ will build up in the oocyte and stimulate PDE3. This will provoke a degradation in the local cAMP concentration and an increase in AMP. Work from Steve Downs’ laboratory proposes that AMP activates an important regulatory enzyme, AMP-activated protein kinase (AMPK). This enzyme is activated by increases in the AMP/ATP ratio, and there is evidence from experiments with mouse oocytes that AMPK acts downstream of PDE but upstream of MPF (Fig 9.9). Downs proposes that meiotic resumption is caused by a two-tiered process involving loss of PKA activity and increase of AMPK activity.48

Oocyte maturation after superovulation The relation between oocyte maturity and follicle diameter in superovulated cycles for IVF/ICSI in the human The relation between the follicle diameter and the competence of its enclosed oocyte for normal embryonic development has been studied in humans with the aim of optimizing ovarian superovulation treatments for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).64 The small antral follicles (2–8 mm in diameter) present at the onset of the natural menstrual cycle are dependent for their further growth on both gonadotropins FSH and LH, and develop generally into an asynchronous cohort of follicles from which a dominant follicle emerges after day 8 of the cycle.65 In a superovulated cycle using exogenous gonadotropins, the size of the cohort of follicles that is aspirated is largely dependent on the moment of decision to inject the ovulatory dose of human chorionic gonadotropin (hCG). Very commonly, in superovulation protocols without suppression by gonadotropinreleasing hormone analog (GnRHa), the clinician decides to administer hCG when at least three large follicles reach a mean follicular diameter of 17 mm, to avoid as much as possible the triggering of an endogenous mid-cycle LH rise. After the introduction of GnRHa, some teams using assisted reproductive technologies (ART) prefer to use, as criterion for hCG injection, the presence of a majority of follicles with diameters between 17 and 22 mm. On the day that is decided for hCG injection, punctured follicles have generally been showing a progressive growth profile for 6–8 days. It seems from a study by Tan et al that a window of decision for hCG injection of 3 days can be tolerated without influencing pregnancy outcome.66 Some researchers evaluated the outcome of IVF or ICSI in relation to the different classes of follicular size from which the oocytes were aspirated. Decreased oocyte recovery, increased polyspermy, abnormal fertilization, and cleavage were observed when oocytes originated from follicles larger than 6.5 ml or >23 mm in diameter.67,68 Studies from Nayudu et al64 and others69

found that most normal pregnancies after IVF came from follicles in the 2–5 ml volume or 15–23 mm diameter ranges. Studies were conducted by us to analyze the relation between fertilizability, embryo cleavage, and clinical pregnancy rates in GnRHa- and gonadotropinstimulation cycles, showing superior developmental capacity of oocytes aspirated from fairly large follicles with a diameter between 20 and 22 mm (Smitz, unpublished personal observations). All IVF studies consistently show that follicles ≤2 ml (volume) or ≤14 mm (diameter) generate oocytes leading to a very low proportion of clinical pregnancies. Nogueira et al70 showed that PB-extruded oocytes retrieved from small follicles (7–12 mm) generate embryos of lower developmental potential than oocytes derived from larger follicles (>12 mm) following ICSI. Most commonly, matured oocytes from small follicles do not develop after fertilization, and even if they succeed to implant, often early abortion occurs.64,69 An analysis from Plachot revealed that approximately 15% of the oocytes collected after super ovulation for ICSI were still in prophase I (GV) or metaphase I,71 with a higher proportion of GV oocytes recovered from these small follicles.72 When GV oocytes from ICSI cycles are injected, fertilization fails, and when denuded oocytes are injected 24–30 hours after an IVM period, most preimplantation embryos are of poor quality, have a high aneuploidy rate, and yield karyotype anomalies.73 It has been recently proven that these in vitro matured oocytes have a high aneuploid rate; a further increase on the length of the IVM period to 36 hours results in an increased rate of multiple aneuploidies.74 A similar tendency had been observed by Smitz et al:33 GV oocytes from follicles measuring 6–12 mm and analyzed after an IVM period of 36 hours vs 30 hours showed an increased aneuploidy rate. As for the metaphase I oocytes retrieved, although matured within a short incubation period of 4 hours, embryos obtained were of a lower postimplantation developmental potential75–77 and had increased cytogenetic abnormalities.78 It should be made clear that immature oocytes obtained in regular ART cycles are disappointingly low because they represent an already compromised group of follicles that did not respond to the hCG stimulus. These follicles might suffer from intrinsic anatomic (vascularization) or local metabolic (paracrine) defects. The cycles that yielded a higher proportion of immature oocytes had experienced poor stimulation management, with aspiration of smaller follicle diameters (<14 mm). Follow-up of these patients with a poor oocyte maturity rate revealed that only 0.7% of all patients had repeatedly poor oocyte maturity rates after controlled ovarian hyperstimulation (COH) by using a combined GnRHa/ gonadotropin-stimulation regimen.72 This suggests that intrinsic meiotic maturation defects in infertile couples are very rare events. Figures on the failure of completion of meiosis I in an increased proportion of oocytes after hCG injection in infertile women programmed for ART treatment have not been systematically reported. Only a

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handful of cases suffering from repeated maturation failure in successive ovarian stimulations have been described in the recent literature.79 Occasionally some women show abnormal meiosis progression, but it is difficult to propose exact figures on the prevalence of this defect.

Rationale to develop a clinical program for IVM Immature oocytes from superovulated cycles for IVF or ICSI are not comparable to immature oocytes resulting from unstimulated or slightly stimulated cycles. Moreover, the maneuver of enzymatic decoronization of immature oocytes in view of ICSI also compromises further maturation progression. Granulosa cells are the production site of steroids, growth factors (insulin-like growth factor-I [IGF-I], epidermal growth factor [EGF]), peptides, and proteins that have as yet not been characterized, and other compounds that contribute to cytoplasmic maturation of oocytes.80–82 Immature oocytes – considered a side product in ART cycles – among mature MII oocytes were most often not cocultured in a controlled or appropriate way to enable valuable conclusions for the future management of this material. Current oocyte IVM technology involves the practice of intentionally retrieving immature oocytes from small antral ovarian follicles and culturing these oocytes in a cumulus-enclosed fashion. Although there is a tendency today to apply superovulation drugs more cautiously and to monitor the IVF cycles closely, the serious complication, severe ovarian hyperstimulation syndrome (OHSS), cannot be completely prevented.83–85 Considering the long treatment time (several days/weeks),86 the development of an efficient IVF system without stimulation may reduce the patient’s burden by cutting short both the duration and the costs of the treatment.

Results from clinical experience with in vitro maturation in the human Pioneering groups in the 1990s have shown that COCs can be retrieved in a reproducible way from small antral follicles. In unstimulated or stimulated normoovulatory women or polycystic ovary (PCO) patients, 10–15 COCs were obtained.87–89 Two-thirds of the aspirated COCs showed spontaneous nuclear maturation within 36–38 hours and, although fertilization and cleavage were apparently normal, clinical pregnancy rates remained low.87–90 Nowadays, IVM has been applied in ART clinics, resulting in full-term pregnancies; however, further research is still recommended to refine IVM protocols before introducing IVM as a treatment that is as effective as conventional IVF/ICSI. Data based on human studies are fairly recent for final conclusions to be drawn on the safety of the technique. Owing to the lack of standardized procedures, very limited scientific information could be drawn from these studies on immature human oocyte culture. Several aspects influence the

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success of an IVM program, including patient selection, follicular priming, endometrium preparation, and culture conditions for oocyte maturation and derived embryo. The patients scheduled for IVM were pretreated with several stimulation-drug regimens (Table 9.1). Various reports are anecdotal, and the culture material (COC) was always poorly characterized as to its follicular origin. The COCs were retrieved at different moments in the menstrual cycle and probably from follicles of different sizes. Various media were used for culture. These basal media contained different hormones and/or growth factors, and the concentrations and natures of the protein source differed. Some researchers used co-culture with a variety of primary feeder cells. The diversity of all these protocols results in a wide range of pregnancy and implantation rates. Summarizing these clinical reports, results showed that in PCO patients, hCG priming can give rise to pregnancy rates per embryo transfer of 20–38% and implantation rates of about 15%;93,97,100,101 FSH priming give rises to about 33% pregnancy rate and 10–21% implantation rates;96,100 and unstimulated cycles in PCO patients can result in 0–33% pregnancy rates and 0–34% implantation rates.92,96,103,104 In women with regular cycles, few studies have been conducted applying hCG priming, pregnancy rates attained were 4%97 and 25%;103 in FSH priming these rates varied from 0 to 20%91 and in unstimulated cycles, pregnancy rates attained were 17–33%, with implantation rates varying from 6.5 to 19%.91,95,98,103 The concept of performing IVM in unstimulated cycles has been exposed successfully as an alternative to conventional IVF protocol for poor ovarian response105 and for oocyte donation (Fig 9.10).106 Currently, about a 1000 babies have been born from IVM. Only 400 of these were included in a registry. There are no reports of adverse pregnancy events or baby malformations.107 An efficient IVM registry that reaches IVF centers worldwide would be appropriate. Children born from IVM have been deemed as healthy, with the oldest child being 9 years old. Nevertheless, we should welcome more scientific and clinical studies to assure that the technique is completely innocuous for the future child’s health. Recently, epigenetic studies have warned us how environmental conditions can alter the imprinting balance (see Ref 108 for a review). To date, it is not possible to predict the extent to which IVM can cause imprinting disturbances. Safety studies such as cytogenetic evaluation of embryos is a powerful predictor of the risks caused by IVM technology, but also in this field few studies have so far been conducted.

Oocyte retrieval from immature follicles The endocrine milieu at oocyte retrieval Early in the follicular phase, small antral follicles of between 2 and 8 mm in diameter can be observed by vaginal ultrasound scan. These antral follicles are recruited by the rising FSH concentrations that follow

PCO

hCG-primed

Child et al Normo-ovulatory hCG-primed et al 200197 idem

Grouped in 1 ml of TCM-199

Singly in microdroplets of TCM-199

Unstimulated vs FSH-primed

Mikkelsen PCO et al 200196

FSH-primed

Under oil in TCM-199 idem Singly in microdroplets of TCM-199

PCO

Normo-ovulatory FSH-primed

Unstimulated vs hCG-primed

Mikkelsen Normo-ovulatory Unstimulated et al 200095

Suikkari et al 200094

Chian PCO et al 200093

Grouped, in 1 ml of TCM-199

0.29 mM pyruvate

0.29 mM pyruvate idem

0.075 IU/ml FSH 3 mM pyruvate 0.5 IU/ml hCG 1 µg/ml estradiol

0.075 IU/ml FSH 3 mM pyruvate 0.5 IU/ml hCG 1 µg/ml estradiol

0.075 IU/ml FSH +0.5 IU/ml hCG idem

75 mIU/ml hMG 25 mM pyruvate

10 IU/ml PMSG* +10 IU/ml hCG

20% heat75 mIU/ml hMG 25 mM pyruvate inactivated maternal serum idem idem idem

10% heatinactivated patient serum

10% heatinactivated patient serum

idem

10% FBS

20% FBS

COCs grouped, in 20% FBS 1 ml of TCM-199

Unstimulated

Other additives

0.075 IU/ml FSH 3 mM pyruvate 0.5 IU/ml hCG 1 µg/ml estradiol

Gonadotropin supplementation

24h/48h

24h/48h

28–36h

28–36h

44h

44h

24h/48h

48h

28–36h

Hours of IVM culture

60%/76%

59%* 70%/79%

44%

60%

78%

64%

5%/69% 78%∗/84%∗

85% 62%

76%

Percent maturation

idem

Medicult IVF medium

Medicult IVF medium

Medicult IVF medium

–

–

Medicult IVF medium

TCM199+20% FBS

Medicult IVF medium

Embryo culture medium

idem

5% CO2 and air

5% CO2 and air

5% CO2 and air

5% CO2 and air idem

5% CO2 and air

5% CO2 and air

5% CO2 and air

Conditions during culture

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PCO

10% heatinactivated patient serum

Protein supplementation

Cha et al 200092

Patient treatment Singly in microdroplets of TCM-199

Patient profile

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Mikkelsen Normo-ovulatory Unstimulated vs et al 199991 FSH-primed

Author (Ref)

IVM culture condition, basal media, and methodology

Culture conditions for oocytes and derived embryos

Reported conditions mostly used in clinical IVM program for culture of immature oocytes and for the derived embryos postinsemination

120

Table 9.1

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PCO

PCO

Normal + PCO

Lin et al 2003100

LeDu et al 2005101

Son et al 2005102

Unstimulated

idem

TCM-199 or Medicult IVM medium idem

10% patient serum

30% HFF

20% heatinactivated maternal serum

20% heatinactivated patient serum

idem

0.075 IU/ml rFSH 0.1 IU/ml hCG

1 IU/ml FSH 10 IU/ml hCG

0.75 IU FSH + 0.75 IU LH

idem

Pyruvate

10 ng/ml EGF

–

75 mIU/ml hMG 0.2 mM pyruvate

PMSG, pregnant mare serum gonadotropins; FBS, fetal bovine serum; hff, human follicle fluid. ∗ Significant difference between treatment groups.

PCO

Unstimulated

YS medium

Grouped in 1 ml of TCM-199

—

24–36h/48h

24–36h/48h

24–30/48–52h

24h/48h

24h/48h

24h/48h

49%/61%

55%/67%

52%/65%

54%/63%

43%/76% 39%/72%

–/76%

5% CO2 and air

5% CO2 and air

5% CO2 and air

5% CO2 and air

idem

idem

Co-culture with 5% O2 patients cumulus 5% CO2 cells + 10% HFF and 90% N2 IVF universal 5% CO2 media Medicult and air

ISM1 or Medicult

P1 medium

Medicult IVF medium

24h/48h/56h 41%/71%/74% YS + 10% human follicular fluid

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hCG-primed

hCG-primed -

FSH+hCG-primed Grouped in vs hCG-primed TCM-199

—

20% heat75 mIU/ml hMG 25 mM pyruvate inactivated maternal serum

70% HFF

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SoderstromNormal Anttila et al 2005103

Grouped in 1 ml of TCM-199

PCO

Child et al 200299

hCG-primed

YS medium

Yoon Normo-ovulatory Unstimulated et al 200198

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Fig 9.10 Diagram representing the diversified IVM clinical protocols applied in PCO, PCOS, or normo-ovulatory women. These protocols reflect on morphology and maturation timing of oocytes retrieved. (a) Immature oocytes are retrieved prior to the formation of a dominant follicle during a natural cycle: oocytes retrieved are enclosed in a compact mass of cumulus cells and the majority of the oocytes reach maturation between 28 h and 36 hours of culture. (b) Immature oocytes are collected following a mild stimulation: from patients primed only with FSH, the oocytes retrieved have a full compacted cell morphology and the majority of oocytes mature within 28–36 hours of culture; with the administration of hCG with or without FSH priming, maturation is more rapid, within 24–30 hours of culture, and the oocytes retrieved from the most advanced follicles have a more expanded cell morphology. (c) Immature oocytes collected from patients undergoing controlled ovarian hyperstimulation for IVF/ICSI treatment are often in vitro matured in the absence of adjacent surrounding granulosa cells and the majority of the oocytes mature within a time frame of 24–30 hours.

regression of the corpus luteum of the preceding cycle. The follicles differ in size and in capacity for further growth. In the natural cycle, paracrine interaction and decreasing FSH levels with the progressive growth of a leading follicle are at the basis of the inequality of growth progression of recruited antral follicles. During an immature oocyte aspiration procedure, some follicles from this cohort might be on the verge of undergoing atretic changes. Part of the oocyte–granulosa complexes from these small follicles might lack influx of inhibitory signals which finally result in reactivation. A study by Yuan and Guidice analyzed human ovaries from normally cycling women and quantified

the atretic changes in the different classes of follicles.109 Their data suggest that when small (2.1–9.9 mm) follicles are aspirated, about half of these might have initiated a cell death program. These findings in humans are similar to those in domestic animals, in which 85% of antral follicles found in an ovary at any time of the cycle are atretic.110,111 Others have studied the availability of small antral follicles that are healthy and suitable for culture (Table 9.2). However, early stages of follicular atresia may not be an impediment to full meiotic maturation and further embryonic development, as was shown in sheep81 and cattle.116 The first signs of atresia in antral follicles are

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Table 9.2 Historical data on number of cumulus–oocyte complexes (COCs) obtained and data on apoptosis in human follicles of 4–10 mm in unstimulated cycles in normal women Author(s)

Year

Nonatretic follicles (4–10 mm)

Expected number of viable oocytes

McNatty et al112 Gougeon113 Chikazawa et al114 Yuan and Giudice109 Mikkelsen and Lindenberg115

1979 1986 1986 1997 2001

<10% Low 50% Low 50%

1–2 Not mentioned 3–4 Not mentioned Not mentioned

Table 9.3

Follicles recruited by follicle-stimulating hormone (FSH) pretreatment in normo-ovulatory women Follicles of 6–12 mm at day after last FSH dose

Author(s)

Year

Regimen

Mannaerts et al118 Salha et al119 Schipper et al65 Smitz et al33

1996 1998 1998 2007

150 U r-hFSH/day, 5 days 100 U r-hFSH/day on days 2,4,6 375 U as single dose on day LH + 14 600 IU r-hFSH total dose on days 5 (300 IU), 7 (150 IU), 9 (150 IU) after contraceptive use

Table 9.4

Up to 22 8.9 (2–20) 6.0 (1–22) 10.6 (1–29)

Oocyte recovery rate in relation to basal endocrine profile and follicle-stimulating hormone (FSH) priming

Normal endocrinology Unstimulated FSH primed

Polycystic ovary syndrome Unstimulated

FSH primed

Authors(s)

Year

Mean number of COCs at retrieval (range)

*Wood Mikkelsen et al95 Mikkelsen and Lindenberg96 Mikkelsen et al95 Salha et al119 Smitz et al33

1995 2000 2001 2000 2001 2007

3.9 5.2 (2–9) 4.7 4.7 (0–17) 5.0 (0–14) 6.5

Trounson et al88 Mikkelsen and Lindenberg96 Child et al124 Mikkelsen and Lindenberg96

1994 2001 2001 2001

13.8 (0–33) 7.0 (2–12) 10.3±7.6 7.0 (2–16)

*Personal communication. COC, cumulus–oocyte complex.

manifested by pyknosis in the granulosa cell compartment, whereas the oocyte is affected last by atresia.117 In cattle, Blondin and Sirard surprisingly found that slightly atretic or nonatretic follicle status had no impact on the further developmental competence of the oocyte.116 The induction of the naturally occurring atretic process can be prevented by maintaining increased FSH concentrations, ensuring a climate of intrafollicular growth progression at the moment of follicular puncture. Several reports from the recent literature on follicle recruitment after a short course of FSH treatment are summarized in Table 9.3. In humans, there are cumulative evidences that the developmental pattern of the immature oocytes retrieved from small antral follicles is greatly influenced by the endocrine environment in which the follicles are exposed. In consequence, many conflicting

data exist in relation to IVM from natural or from gonadotropin-stimulated cycles. Cha and Chian illustrated a more rapid progression of meiosis I and first polar body extrusion rate in stimulated cycles compared with unstimulated cycles.120 Data from Gomez et al121 and Toth et al122 showed a higher MII maturation yield after gonadotropin priming. Prospective work from Wynn et al123 showed improved meiotic maturation yields after priming with a short course of FSH, whereas Mikkelsen et al91 could not find any beneficial effect on oocyte maturation, fertilization, and preimplantation embryo development in normo-ovulatory patients. However, the same author showed that FSH-priming significantly increased maturation, pregnancy, and implantation rates in PCO patients.96 An overview of the numbers of oocytes retrieved

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per procedure in relation to indication and FSH pretreatment is shown in Table 9.4. A recent completed study by the authors’ laboratory evaluated the oocyte retrieval rate in young volunteers who were pretreated with oral contraceptives followed by a short course of maximally 4–5 days of recombinant hFSH (r-hFSH). When three or more follicles of size >6 mm were seen on ultrasound, oocyte retrieval was scheduled the next day. With this policy, an average of 6.5 oocytes were retrieved per patient, and 68% of the oocytes originated from follicle sizes of between 6 and 12 mm.33 Follicles of diameter >7 mm had a maturation capacity superior to that of the smaller ones. The MII rate was between 70 and 80%. In a search for the optimal moment during the unstimulated cycle for immature oocyte aspiration, Cobo et al125 showed that follicular diameter at the time of oocyte pick-up plays an important part in further development. Oocytes with the best developmental competence originated when they were aspirated before the leading follicle reached 10 mm in diameter (earlier than day 6 of the cycle). Similar observations were reported by Smith et al,126 who found that 62% of all aspirated COCs were suitable for maturation, and 28 hours after collection 73% of oocytes were metaphase II. Clinical implantation rates were not different whether oocytes matured in vitro for 28 hours (10% implantation) or 36 hours (12% implantation). An interesting observation was made first by Chian et al93 that injection of hCG even with a cohort of small follicles was beneficial for oocyte retrieval and IVM outcome. Similar data were recently published by Nogueira et al56 describing a visible effect of LH action on cumulus expansion in GV oocytes from small follicles from normo-ovulatory patients, and an unexpectedly favorable nuclear maturation rate in these oocytes. In PCO patients, cumulus cells presenting an expanded pattern have a higher expression of mRNA LH receptor than compacted cumulus cell pattern.127 The distinct COC patterns resulted in differences in the capability of oocytes to mature in vitro: about 70% of expanded COC had already matured by 24 hours of culture compared with 30% of oocytes enclosed in fully compacted cell mass and, by 48 hours of culture, this difference dropped to ~20%. Furthermore, there is indication of a positive relationship between expanded COC and embryo developmental capacity in these cycles, i.e. 40% of fertilized oocytes derived from expanded COC formed blastocyst vs 23% derived from compacted cumulus cells. FSH priming in combination with hCG priming in these patients is indifferent to the IVM capability of the oocytes.100 Important is the fact that hCG priming may induce IVM of the oocytes within small antral follicles in PCO or regular cyclers.93,128,129,102 It is likely that this rate of maturation might be a reflection of the size of follicles on day of hCG-priming. Some authors administer hCG

when follicles reach 10 mm on day 8, with subsequent retrieval performed between days 9 and 14 of the cycle and oocytes are deemed immature at time of collection.93,92,97,100 It seems that further growth of follicles ensures that few oocytes mature at the time of retrieval. This was observed in an approach used by Son et al,102 in which aspirates from follicles measuring between 11 and 13 mm 36 hours post-hCG resulted in 12% of oocytes with polar body extruded at retrieval. In their study, in vivo matured oocytes derived blastocysts of higher developmental quality than the in vitro matured oocytes. Yang et al127 reported 8% of matured oocytes retrieved from PCOS patients when hCG was administered on the day that follicles reached a 10 mm diameter. Recently, it has been revealed that upon hCG priming, about 50% of cycles in PCO patients have already at least one MII oocyte retrieved and, interestingly, 62% of these matured oocytes were derived from follicles sized ≤10 mm.130 These data emphasize the importance of assessing the nuclear maturity of the oocytes on the day of egg collection to improve the chances of the patient achieving pregnancy.

Methods The technique of immature oocyte retrieval Aspiration of small follicles is nowadays performed via the transvaginal ultrasound-guided route. The ultrasound machine should have very good resolution to permit clear detection of small antral follicles. A double-lumen needle offers the possibility for flushing in case COCs are not easily detached from the follicular wall. Pre-warmed 4-(2-hydroxyethyl)-1piperazine ethanesulfonic acid (HEPES)-buffered solution containing 2–10 U/ml heparin is used for flushing the punctured follicles, because the aspiration fluid is generally very bloody as successive small follicular structures are punctured subsequently, causing damage to the well-vascularized theca cell compartment. Reduction of the aspiration pressure from the usual 100 mmHg for recovery of mature oocytes to 60 mmHg is necessary to avoid the detachment of cumulus from the oocyte and for a better oocyte recovery.

Identification and typing of the cumulus–corona–oocyte complexes The morphology of the retrieved immature COCs often differs from the mature COC retrieved from superovulated cyles for conventional IVF treatment. In aspirates from smaller follicles (≤12 mm in diameter), immature oocytes can be found enclosed within fully compacted layers of cumulus mass, which brings up difficulties in recognizing the oocytes. Often the clumps of COCs are floating between more compact masses of mural granulosa cells (Fig 9.11) and the prolonged searching time for COCs necessitates strict control of temperature and pH

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conditions. COCs from large follicles are graded on the basis of expansion of surrounding cells from the corona and cumulus. As the mucification process has often not yet taken place within ovarian follicles of this size, detachment of COCs from the follicle wall is more difficult, and multiple flushing cycles have to be applied. In routine IVF practice, grading of the nuclear maturation stage is approximated by the degree of expansion of the surrounding cumulus and corona cells.131 Early work of Testart et al132 established that in natural cycles there is good synchrony between the development of the cumulus cells and the nuclear maturation stage. This synchrony in maturation is observed less often in superovulation cycles for ART.133 The degree of cumulus expansion is influenced by the size of follicle and it will depend on whether there has been an effect of LH on the follicles, which progressively acquire LH receptors on the mural granulosa cell layers. A preliminary FSH effect and the subsequent LH signal lead to mucification of the differentiated granulosa cells neighboring the oocyte. Fully compacted CEO can be also retrieved from ovaries exposed to exogenous hCG56,127 but are probably derived from less developed follicles. A classification of aspirated COCs from small follicles is proposed in Table 9.5 and illustrated in Fig 9.12. For easy search and accurate manipulation of the COCs we use a stereomicroscope at magnifications from 5× to 60×. At first the entire Petri dish is overviewed at a magnification of 5×, going field by field. Alternatively the isolation of immature COCs from the red blood cells can be done by filtering through a commercialized cell-strainer device of 70 µm pores. Tissue clumps suspected of containing an oocyte are sucked up in the pipette and transferred to a new plate for rinsing. In this way, follicular fluid and blood cells coming with the aspirated fluid are separated from the oocyte. The oocytes are washed in three steps in HEPES-buffered media before being placed in IVM media for incubation. The selected complexes are placed in culture dishes, evaluated, and classified following the cumulus and corona expansion criteria (Table 9.5). A more thorough evaluation can be performed on the inverted microscope with Hoffman modulation contrast system (Modulation Optics, NY, USA). A magnification of 200× is used, and an attempt is made to visualize the germinal vesicle to grade nuclear maturation. In oocytes enclosed within a compacted cell mass, a GV can be easily visualized under the inverted microscope. However, in expanded COCs it is difficult to assess oocytes’ nuclear maturity, due to the mucified-like cells that cover the oocyte. By briefly ‘spreading’ the oocyte on a dish plate containing a small amount of media, one is able to recognize a GV (Fig 9.13). During all IVM manipulation steps, extreme attention is given to maintaining the physiologic temperature (37°C) by adapting heating stages on the stereomicroscope and on the inverted microscope.

125

Table 9.5 Maturity grading of cumulus–oocyte complexes (COCs) and oocyte (after cumulus–corona denudation) during prematuration days in culture 1. Grading of granulosa cell mass: cumulus expansion and oocyte coverage Cumulus mass (CM): 3 or fewer layers of (CM0) cumulus cells more than 3 but fewer than (CM1) 10 layers of cumulus cells 10 or more layers of (CM2) cumulus cells Cumulus expansion (CE): tight, dense cells (CE0) moderate expansion of cells (CE1) fully expanded cells (CE2) Contact (CO) between cumulus cells and oocyte: naked (CO0) partially naked (CO1) fully enclosed (CO2) fully enclosed and part of (CO3) follicle wall 2. Assessment of oocyte nuclear maturation stage GV GVBD PB 3. Assessment of oocyte morphology Oocyte diameter (µm) Oocyte cytoplasm presence of inclusions: vacuoles/refractile bodies darkness: clear/dark granularity: homogeneous/granular Zona: normal/abnormal Perivitelline space: normal/enlarged Oocyte shape: regular/irregular Polar body: intact/fragmented GV, germinal vesicle; GVBD, germinal vesicle breakdown; PB, polar body

Within the laminar flow, a gassed table mini-incubator can preserve optimal temperature and pH of the culture medium outside the incubator. For further maturation, COCs are placed one by one in microdroplets of culture medium or in groups of maximum 5 into 4-well nunc dishes which have been preincubated for a minimum of 3 hours. The culture dishes are kept in a 5% CO2 and a 100% humidified incubator maintained at 37°C. The COCs are cultured for a minimum of 24–36 or even 48 hours, depending on the type of clinical protocol applied, and oocyte maturation is scored for cumulus expansion, nuclear maturation, and oocyte morphology (Table 9.5).

Noninvasive techniques to evaluate COC culture procedures An optimal culture medium has still to be defined, and a suitable strategy for culturing immature oocytes has

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Fig 9.11 After follicle fluid aspiration, a cumulus–corona–oocyte complex (COC) has to be found floating between other granulosa cell debris (arrow). Arrow in (a), (b), and (c) shows the COC from low to large magnification as visualized under the stereomicroscope. Bar is 500 µm.

a

b

c

d

Fig 9.12 Classification of cumulus–corona–oocyte complex (COC) retrieved from small antral follicles. (a) CM1, CEO, CO2: the oocyte is surrounded by a compact mass of four to five layers of granulosa cells. The germinal vesicle is clearly visible within the oocyte at a peripheral position. (b) CM1, CE2, CO2: there is expansion of the distal layer of granulosa cells (cumulus), the proximal granulosa cells surrounding the oocyte are still compacted. (c) CM1, CE1, CO2: there is expansion of both distal (cumulus) and proximal (corona cells) layers of granulosa cells. (d) Distal granulosa cells are dispersed. The oocyte is partially naked and connections between cumulus cells and oocytes are lost.

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Fig 9.13 Photomicrograph of a cumulus–oocyte complex with an expanded cell morphology pattern (CM2, CE2, CO2). (a) Nuclear maturity of oocyte is not visible under an inverted microscope. (b) After cell ‘spreading,’ the GV is visualized within the ooplasm (arrow).

Fig 9.14 Spindle images of human in vitro matured oocytes using the PolScope software showing different locations of spindle in the ooplasm in relation to polar body position. (Courtesy of Lin Liu and David Keefe, University of Florida, Florida.)

yet to be developed. Depending on the grade of oocyte immaturity a differential approach to culturing technique could lead to improved oocyte quality. The grade of maturity of an individual COC can be defined using morphologic criteria. Making use of the inverted microscope equipped with Hoffman modulation can allow the grading of nuclear maturity and degree of mucification of the cumulus cells as explained previously. The induction of follicle growth by supraphysiologic levels of FSH dissociates nuclear and cumulus maturity.133 When puncturing COCs from unstimulated, FSH-stimulated, or hCG-induced patients, the morphology of the COC is already very different at the start of the IVM procedure. The PolScope, a microscope equipped with a polarization optics system coupled with image processing software, is a useful tool to examine microtubule structures such as the meiotic spindle in living oocytes.134 It enables one to verify the location and to evaluate the dynamic architecture of the spindles in human oocytes (Fig 9.14). In the near future, with technologic modifications, it might also become a useful tool to investigate chromosome segregation during the meiotic and mitotic divisions in oocytes and embryos.135

Correlates for oocyte nuclear maturity Chromosome spreading and analysis techniques The technique of IVM is still in an experimental phase. Before any clinical application, it is necessary to determine whether the process of IVM of oocytes

derived from small antral follicles bears an increased risk of aneuploidy. There is very limited information on the cytogenetic constitution of human in vitro matured oocytes. The majority of reports are from in vitro matured oocytes derived from superovulation for IVF which failed to mature despite exposure to hCG. For example, Boiso et al136 and Plachot137 reported an aneuploidy rate of 24% and 23%, respectively. Clyde et al138 reported aneuploidy at maternal meiosis I in 39% of noninjected in vitro matured oocytes, while Pujol et al139 identified a 47.5% aneuploidy rate. In these studies fluorescence in situ hybridization (FISH) or M (multiple)-FISH techniques were used to probe a limited number of chromosomes. It is hypothesized that oocytes that remain immature in spite of ovarian stimulation may be of inherently reduced quality. Therefore, the conclusions in the current literature may not be representative for oocytes from small follicles. A phase I clinical study was carried out to determine the frequency of aneuploidy in a large number of in vitro matured oocytes.33 These GV-stage oocytes were derived from small antral follicles (6–12 mm) using a mild stimulation protocol with solely FSH administration for 4–5 days. Oocytes were donated by a well-defined population of 134 healthy volunteers, between 18 and 37 years of age, with unknown infertility history. They received limited ovarian stimulation with r-hFSH for 4–5 days. Follicles of ≥6 mm and ≤12 mm in diameter were punctured. A total of 680 COCs were put in defined IVM medium (tissue culture medium TCM-199) without

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Giemsa

18 −23 chromosomes

< 18 chromosomes

No analysis

SKY

Success

Failure

Multivision FISH Chromosomes: 13, 16, 18, 21, and 22

serum for 30 or 36 hours. After culture, COCs were denuded mechanically and 363 (53%) oocytes extruded the first polar body. Finally, 354 oocytes were spread for chromosome analysis. A total of 301 healthy endocrinologically normal volunteers aged 18–37 years old with regular menses were randomized 1 to 6 to conventional IVF (n = 43) and IVM (n = 258). For IVM, there were 1075 COC randomized over the 6 IVM media and 2 maturation times (30 hours and 36 hours). IVM medium (TCM199 based, supplemented with FSH, insulin-like growth factor, energy substrates) was supplemented or not with four concentrations of FF-MAS (Novo Nordisk A/S, DK). The % PB rates in IVM were about 70%. Polar body extruded oocytes (MII) were spread for chromosome analysis by spectral karyotyping (SKY) or FISH. Control metaphase II donated oocytes had an aneuploidy rate of 8%. In the IVM groups (30 hours and 36 hours IVM), the controls (without FF-MAS) had at least a two-fold increase in chromosomal abnormalities compared to the IVF group.33 This indicates that more oocytes punctured from small follicles and in vitro matured showed lower competence to resume correct chromosome segregation compared to oocytes from larger follicles and matured in vivo.

Technique of fixation for chromosome spreading of in vitro matured oocytes Different procedures for chromosome spreading are described in the literature. Most of them are variants from the original air-dry method described by Tarkowski.140 The fixation method used is often a limitation for accurate chromosome analysis. The authors have obtained the best chromosome preparations using the modification of a method initially described by Racowsky et al.141 Fixation is performed in a closed small room. A waterbath at a temperature of 40°C is placed next to the microscope:

Fig 9.15 Procedure for chromosome analysis of in vitro matured oocytes. SKY, spectral karyotyping; FISH, fluorescence in situ hybridization.

this creates a relative humidity of around 50%. After hypotonic treatment in 1.2% sodium citrate (ambient temperature, 1–5 min) oocytes are exposed to a protease solution (0.7 IU/ml; Pronase 16592, Roche) for 20 seconds, followed by a second hypotonic treatment in 0.6% sodium citrate (ambient temperature, 3–6 min). The whole process is performed under a stereomicroscope (Olympus SZX12, Hamburg, Germany). Adjustment of hypotonic treatment is necessary and is very patient-related. Each oocyte is then transferred onto a microscope slide placed beforehand on an inverted microscope with phase contrast (Olympus CK40). Ice-cold fixative (ethanol:acetic acid 3:1 v/v) is added dropwise on top of the oocyte. Often, the oocyte moves after the first drop of fixative; watching the phase contrast image of the microscope, it can be found again quite quickly. A single drop of fixative is usually sufficient for adequate spreading. If not, an additional drop can be added. Compared with freshly recovered in vivo matured MII oocytes, spreading of IVM oocytes demands increased incubation time in hypotonic solution to break the oocyte membrane and to obtain adequate spreading of the chromosomes. (Perhaps the culture procedure used for these oocytes induces zona hardening.) Using this spreading technique, more than 90% (n = 339) of the above attempted preparations were successfully completed.

Analysis of oocyte chromosomes by fluorescent hybridization techniques To be analyzable, preparations must be selected for adequate chromosome spreading with little overlapping, overspreading, and presence of excess cytoplasm. A schematic overview of the procedure for chromosome analysis performed in the above study is presented in Fig 9.15. All preparations are first stained with Giemsa in order to perform a chromosome

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Polar body chromosomes: d

a

b d

c

Oocyte chromosomes: e

h f h

g

Fig 9.16 Representative spectral karyotyping of oocyte and polar body metaphases. (a–d) Normal 23,X polar body: (a) red–green–blue (RGB) image; (b) Giemsa image; (c) classified 24-color image; (d) composite karyotype showing, from left to right, RGB, Giemsa, and classified 24-color image of each chromosome. (e–h) Normal metaphase II (MII) 23,X oocyte: (e) RGB image; (f) Giemsa image; (g) classified 24-color image; (h) composite karyotype showing, from left to right, RGB, Giemsa, and classified 24-color image of each chromosome.

count. Preparations with <18 chromosomes are considered to be technical artifacts and are not further analyzed. When there are 18–23 chromosomes visible, oocytes are karyotyped by spectral karyotyping (SKY), which can identify each one of the 23 chromosome types present in the oocyte142 (see Fig 9.16). However, if the hybridization by SKY appears to be unsuccessful, oocytes are analyzed for chromosomes 13, 16, 18, 21, and 22 by FISH. Of 339 preparations, 15 (4%) were excluded from the above study, because chromosomes were lost during hybridization or fewer than 18 chromosomes were present. Fifty-eight (17%) were classified as nonevaluable when hybridization with SKY or FISH was unsuccessful or when the result was noninformative (e.g. overlapping chromosomes, presence of excess cytoplasm, etc.). Of 266 evaluable preparations, 101 (38%) were analyzable with SKY. These data are in accordance with a previous publication on the use of SKY in human oocytes.143 Better fixation methods may help to increase the yield of analyzable oocytes with this technique. FISH could provide reliable data in 165 chromosome preparations (62%). In total, results were interpretable for 78% of the oocyte preparations. Whenever possible, chromosomes of the corresponding polar bodies were analyzed as well. The

chromosomes in the oocyte and in the first polar body complement each other and provide an internal control to differentiate between aneuploidy and technical errors. In 80% (n = 272) of the preparations, polar body chromosomes were present. However, degeneration of the first PB occurs rapidly, and accurate analysis is seldom possible. A total of 130 (48%) polar body metaphases were analyzable with FISH and only four (1.5%) with SKY.

Quick chromosome assessment by DNA staining with Hoechst Extrusion of the first polar body is not a sufficient criterion to evaluate completion of nuclear maturation. It must be ascertained that, after a specific time in culture, the oocyte has not only extruded its PB but also formed a well-aligned second metaphase plate (2 h after PB extrusion). A convenient way to assess nuclear maturation and abnormalities is to identify the oocyte’s meiotic stage by staining the DNA with Hoechst 33342 (Molecular Probes, Leiden, The Netherlands) and visualizing the nuclear stage with ultraviolet (UV) light (Fig 9.17). After first polar body extrusion it is possible to observe a physically unarranged chromosome plate (pro-metaphase II), or a disarrangement of the chromosomes or a normal MII

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a

b

Fig 9.17 (a) and (b) DNA content of the oocyte stained with Hoechst after 48 hours’ culture: both oocytes had polar body extrusion with a well-aligned chromosome metaphase II (MII) plate. (a) The arrow shows the DNA content within the polar body. In (b) the arrow shows the well aligned MII chromosome plate.

a

b

c

Fig 9.18 Electron micrographs taken from cultured oocytes. (a) Immature oocyte at germinal vesicle (GV) stage with few cortical granules (CGs) aligned (arrow). Clumps of mitochondria indicate the immature stage of the oocyte (arrowhead). (b) Oocyte after 48 hours’ culture: although the mitochondria have spread over the cytoplasm (arrowhead) as is typical for a meiotically competent oocyte, complete cytoplasmic maturation has not occurred and double layers of CGs are found at the periphery (arrow). (c) Oocyte with polar body extruded after 48 hours’ culture. CGs are well-aligned under the oolemma forming a single layer (arrow). Stars in figures indicate the zona pellucida.

plate. If abnormal, some chromosomes are not well aligned on the metaphase plate, or there might even be a dislocation of chromosomes in the cytoplasm of the egg. With Hoechst staining, it is also possible to identify whether the oocyte underwent activation: in this instance, a clump of chromosomes can be visualized in the cytoplasm. The presence of chromosomes in the polar body will be clearly detected by DNA staining, testifying whether segregation of the chromosomes had occurred on the occasion of polar body extrusion.

Correlates for oocyte cytoplasmic maturity Cortical granules During oocyte maturation, fully grown oocytes undergo ultrastructural and functional modifications that allow them to continue monospermic fertilization and development. Some of the changes throughout oogenesis are the redistribution of cortical granules (CGs) originating in the Golgi apparatus during the late preantral and antral stages. The cortical granules in mammalian eggs are electron-dense small spherical vesicles (300–500

nm in diameter) surrounded by a single membrane. CGs contain mucopolysaccharides, proteases, tissue-type plasminogen activator with serine-protease activity, acid phosphatase, and peroxidase enzyme activity. Cortical granule exocytosis normally occurs after sperm penetration in response to intracellular calcium mobilization. This leads to modification of the zona pellucida by the hydrolytic enzymes released from the CG. The zona pellucida hardens, and polyspermy is prohibited by this.144,145 During the transition of the GV to MII, migration and dispersal of CGs take place in such way that in a mature egg they are lined up just below the oolemma. Incomplete dispersion of CGs is a reliable marker for a disturbance in cytoplasmic maturation. Localization of CGs can be done by electron microscopy analysis (Fig 9.18) and/or by staining CGs with lectins labeled with a fluorescent marker and analysis on a confocal laser microscope (Fig 9.19).

Cytoskeleton: actin and tubulin microfilaments Mammalian oocytes possess cortical filaments of actin that play an important part in CG migration during

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b

Fig 9.19 Confocal images representing the distribution of cortical granules (CGs) in bovine immature (a) and in vitro maturing (b) oocyte. CGs display a green fluorescence owing to fluorescein isothiocyanate–peanut agglutinin (FITC–PNA) staining. The chromatin displays a red fluorescence owing to ethidium homodimer-1 (EthD-1) staining. (a) CGs as large aggregates located over the entire cytoplasm. In red, nuclei of some granulosa cells surrounding the oocyte. (b) CGs are localized at periphery surrounding the entire cortex of the oocyte.

oocyte maturation, polar body extrusion, and cell division.146 Cortical microfilaments are also involved in the peripheral migration of the first meiotic spindle. Analysis using staining for actin and microfilament inhibitors has shown that microfilaments predominate in the cortical region of the cytoplasm, forming a polymerized actin layer which can participate in cytokinetic functions. Therefore, observation of the formation and localization of microfilaments might also be an important marker for oocyte maturity. In rodents, the second meiotic spindle is localized paratangentially to the cell surface, and the microfilaments participate in rotation of the second meiotic spindle after fertilization. In primates the second meiotic spindle is localized radially to the cell surface, thus avoiding this specific function of the microfilaments. The exact role of microfilaments in primate maturation is still uncertain. Microtubules are essential for chromosome movements during first and second meiosis, for the movement of the sperm after fertilization, and also for syngamy. Microtubules are dynamic structures of tubulin protein constituting the meiotic spindle during maturation. In mammals, the second meiotic spindle is stable until the moment of fertilization. During primate oocyte maturation, the microtubule configuration, which has been investigated by immunocytochemical techniques, is anastral, barrel-shaped, and oriented radially to the cell surface.147,148 Microtubules in human meiotic spindles are highly sensitive to temperature variation149 and environmental perturbances.150 Perturbances of the spindle microtubules can be irreversible and affect the genetic balance, since the segregation and alignment of the chromosomes during meiosis involves a complex interaction between chromosomes and cytoskeleton.151 Therefore, evaluation of abnormalities of the spindle structure after oocyte maturation might be important

for control of manipulation during culture, and a checkpoint for assessment of the microenvironment surrounding the developing oocyte (Fig 9.20). At the transition of interphase to M-phase, microtubule reorganization and stability are influenced by factors such as protein kinase activity, centrosome-based microtubule nucleation, and post-translational modifications of tubulin.152 These factors have been recently investigated in human oocytes that failed to mature after normal superovulation treatment. The baseline M-phase markers such as mitotic phosphoprotein monoclonal-2 reactive protein (MPM-2), phosphorylation of histone-3 (PH3), and microtubules (α-tubulin) were studied by Combelles et al153 in immature human oocytes during IVM in defined culture medium. By using immunofluorescence and confocal microscopy, Combelles et al153 documented the cell cycle-dependent modifications in chromatin and microtubules in human oocytes during IVMs, and demonstrated specific cell cycle deficiencies in IVM oocytes.

Mitochondria Work in hamsters documented the structural changes by mitochondrial redistribution in the oocytes after fertilization. Alterations in the normal pattern of mitochondrial distribution correlated with abnormal development of the embryo.154 Mitochondrial redistribution was also noted in ova of primates and cattle pre-fertilization during IVM. In cattle GV oocytes, mitochondria are arranged in a cortical distribution but relocate during IVM.155 In metaphase II oocytes matured in a medium that supports further development, the mitochondrial distribution might be an interesting indicator of oocyte developmental competence. Active mitochondria can be stained by fluorescent dyes, such as Rhodamine 123 or MitoTracker® (Molecular Probes, Leiden, The Netherlands), and

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Fig 9.20 Three distinct progression stages of meiosis are illustrated. Mouse oocytes are fixed and extracted in a microtubule-stabilizing buffer and stained with monoclonal antibodies against α- and β-tubulin (label green). Chromosomes are stained with ethidium homodimer-2 (label red). The left panel shows a prophase I oocyte, with the chromosomal material packed into the germinal vesicle and the tubulin dispersed throughout the cytoplasmic compartment. The upper right panel shows a metaphase I spindle. The lower right panel demonstrates a normal barrelshaped metaphase II spindle and the extruded and degrading first polar body in the perivitelline space. The metaphase plate shows well-aligned chromosomes.

scanning of the entire oocyte can be done by confocal laser microscopy. By image processing the distribution patterns can be analyzed.156

Proteins in IVM oocytes The mature human oocyte has a diameter of 110–120 µm and contains some 150 ng of total protein. During mammalian oocyte growth and maturation there are transcriptional processes that lead to the synthesis and accumulation of proteins necessary for regulation of nuclear and cytoplasmic maturation.157 Sensitive twodimensional (2D) gel electrophoresis techniques allow us to distinguish nuclear and cytoplasmic maturation by analyzing the difference in protein neosynthesis over an arbitrarily chosen time interval (such as the IVM period), and informing us how the changes in protein depend on the culture conditions and the maturation stages of the oocyte. Significant differences were found between protein profiles of MII oocytes, namely oocytes in vitro matured for 48 h (collected in unstimulated cycles) that were compared with in vivo collected MII oocytes after stimulation. However, there was no difference between profiles of matured (MII) oocytes obtained from GV oocytes from superovulated cycles.158 The missing protein spots in IVM oocytes were probably a result of the shortened oocyte growth phase of oocytes retrieved from small follicles, and may explain the poor developmental capacity of embryos obtained from small follicles. Gonadotropins are known to promote the synthetic capacity of the

oocyte.158 This may explain the differences in protein spots between oocytes obtained from unstimulated cycles and those obtained after superovulation.159 Collaborative work between Centre de Recherche en Reproduction Animale (CRRA) (A. Goff, St Hyacynthe, Quebec, Canada), and our laboratory (Follicle Biology Laboratory, Vrije Universiteit Brussel (VUB), Brussels) analyzed protein profiles of mouse oocytes in different stages of nuclear development obtained from follicle culture. It was shown that the competence to resume meiosis was reflected in the protein synthesized at the GV and MII stages in the oocyte.119 Analysis of protein neosynthesis in the cumulus cells from corresponding oocytes did not show major changes in relation to duration of follicle culture. 2D sodium dodecyl sulfate–polyacrylamide gel (SDS–PAGE) analysis was used to evaluate protein profiles of aspirated immature human oocytes from small follicles (2–8 mm). Goff et al reported that in a serum-free culture system, addition of 10 ng/ml EGF induced the synthesis of at least 12 proteins.160 These data further showed that EGF supplementation (but not FSH/LH supplements) yielded similar results to those with serum-based culture medium. This approach, using 2D gel electrophoresis, could allow the exploration of factors involved in cytoplasmic maturation. As at least some aspects of cytoplasmic maturation are under cytoplasmic control, it might be interesting to investigate the pattern of proteins synthesized after maturation under a specific culture condition.

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Timing of oocyte maturation and insemination The time of maturation influences the fertilization and developmental capacity of the oocyte and depends on the hormonal treatment conditions of the patient93 and on the conditions in which the oocyte has been cultured (see Table 9.1).164 Studies of the kinetics of oocyte maturation have found that by 20 hours of IVM the first oocytes extrude their first PB, and that this timing is influenced by gonadotropin priming of patients.95,120 In gonadotropin-primed patients (normo-ovulatory women), at least 24% of oocytes extruded the first PB after 23 hours.126 In a first group of patients, COCs were denuded at 28 hours; 73% of oocytes showed the presence of a PB. In a second group, denudation was performed at 36 hours and showed that 77% of the ova were MII. Son et al102 reported that cumulus-enclosed GVstage oocytes from hCG-primed patients maturing within 30 hours of culture are developmentally more competent than oocytes necessitating longer time to mature. In their evaluation, there was a 20% higher cleavage and 40% higher blastocyst formation when oocytes were matured for 30 hours compared to oocytes maturing after 48 hours. Others have shown that the PB extrusion rate of cumulus-enclosed oocytes cultured for at least 36 hours was improved compared with denuded ones, and fertilizability and cleavage rates were comparable to those of fresh MII oocytes.88,165,166 There are data available on spindle stability only from ‘left-over’ immature cumulus-denuded oocytes obtained after hCG injection in superovulated patients for ICSI. In a recent study, it was determined that optimal IVM of cumulus-enclosed human oocytes obtained after a short FSH treatment course of 4–5 days was obtained 30 hours after aspiration. From 36 hours, oocytes showed a doubling in aneuploidy rate.33 These oocytes have been matured in defined culture conditions in a TCM-199 medium supplemented with FSH; insulin tranferring selenium (ITS); IGFI long; and 0.8% human serum albumin. The nuclear maturation potential (MII) of IVM oocytes in this medium was comparable to that of in vivo matured oocytes from an age-matched IVF control group (71% vs 73%). The use of a PolScope to examine the spindles in in vitro matured denuded oocytes revealed that after 24 hours, 77% of oocytes reached the metaphase II stage, with 52% of oocytes showing birefringent spindles.134 These data need to be confirmed using in vitro matured cumulus-enclosed MII oocytes obtained from small follicles. Indications from Combelles et al153 stressed that the immaturely derived GV oocytes from ICSI cycles have rather quickly degenerating metaphase II spindles after IVM. Interestingly, it was observed using a Polscope that overall retardance of light was also lower in the in

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vitro as compared with the in vivo matured human oocytes163 (Shen et al, unpublished results). Since there is a link between good pronucleus (PN) score and high retardance, these observations confirm that the immature oocytes from ICSI cycles have a low developmental potential even when they are capable of emitting a first polar body and developing in vitro to metaphase II and possessing a spindle. In conventional IVF, it was shown that a prematuration time before insemination is beneficial for the outcome.88,131 Insemination by conventional IVF of in vitro matured oocytes has been performed with lower success compared with in vivo matured oocytes (45% vs 73%, respectively).87 Hardening of the zona pellucida of the oocyte caused by a prolonged culture period could be responsible for the decreased fertilization,161,162 and theoretically, ICSI is used to overcome the problem of zona hardening. In a comparative study Hwang et al167 showed that in vitro matured oocytes had higher fertilization rates by ICSI but similar embryonic developmental quality than IVF. In a more recent work, a higher fertilization rate was obtained with ICSI but higher pregnancy and implantation rates with IVF.103 In this work the insemination method was chosen based on sperm quality. These studies point out that the functional mechanisms needed for the fertilizability process have been accomplished in in vitro matured human oocytes approaching those of oocytes matured in vivo. After polar body extrusion, the oocyte needs a short period to form its second meiotic spindle in preparation for sperm entrance. The most appropriate time for microinjection of IVM oocytes is probably between 2 and 6 hours after polar body extrusion. Fertilization rates have shown to be highest when oocytes are injected between 1 and 2 hours following PB extrusion, but embryonic developmental quality was similar if oocytes were injected earlier or later (6 hours post-PB extrusion).168,169

In vitro oocyte aging In postmature oocytes, changes in localization of some cytoplasmic organelles may occur. Numerous CGs may conglomerate beneath the oolemma, or they may migrate centripetally.170 After fertilization, postmature oocytes may have inhibition of cortical granule release or poor zona reaction.171 Clumping of mitochondria, which are normally distributed, an increase in their electron density accompanied by changes in shape, and their association with large vacuoles become more pronounced in aged oocytes. Metaphase I human oocytes that fail to complete maturation or MII oocytes that age in culture often have deep-seated spindles with fewer microtubules or spindles with attenuated poles. Chromosomes may clump together or scatter in the cytoplasm.172 Fragmentation of the polar body may be a sign of aging in human oocytes.

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Fukuda et al compared short and long culture of mouse oocytes after subzonal sperm injection with reference to spontaneous zona hardening.173 It was observed that zona digestion required a significantly longer time for long culture compared with short culture, and significantly higher blastocyst formation and hatching were observed in short than in long oocyte culture, which seems to indicate that hardening of the zona is an indicator of oocyte aging. Calcium signaling is involved in important events in oocytes, such as meiotic competence acquisition174 and oocyte activation.50 Modification in the regulation of intracellular Ca2+ is one of the major changes taking place during oocyte maturation.175 Normally, Ca2+ is released from the inositol 1,4,5-triphosphate (InsP3) channels of the endoplasmic reticulum (ER) membrane, which affects Ca2+ oscillations in fertilized oocytes. In vitro aging-related changes in Ca2+ release after fertilization have recently been shown. Microinjection of InsP3 into the cytoplasm of eggs demonstrated a lowering in the maximum rate of increase in Ca2+ in aged compared with fresh mouse oocytes. This is due to a depletion of the ER Ca2+ stores with aging.176

Fertilization and embryonic development as a function of oocyte competence Assessing oocyte quality after IVM can be done by studying its fertilizability and embryo development capacity. Inadequate cytoplasmic maturation will impair the function of cytoplasmic organelles which control polar body and pronuclear formation within the oocyte. Microtubules and microfilaments are the major cytoskeleton components in the mammalian ovum, and provide the framework for chromosomal movement and cell division.177,178 In porcine oocyte IVM, the involvement of microtubules and microfilaments in chromosomal dynamics was proved during transition from GV to MII.179 Thus, disturbances in cytoskeleton organization may result in abnormal development patterns and a lower incidence of embryonic development. Damiani et al compared IVM of cow and calf oocytes. They demonstrated some indications of intrinsic ooplasmic deficiencies causing abnormal fertilization characterized by lack of sperm aster formation, asynchronous development of pronuclei, or extrusion of maternal chromatin.180 All these features can be investigated using the Hoechst and immunofluorescence techniques already described in this chapter. Even simple observation under a light microscope can reveal eventual defects induced by culture. The morphology of pronuclear pre-embryos can be used as a first step in the evaluation of fertilization after IVM. Scott and Smith, in a retrospective study, described characteristics of the pronuclear morphology of pre-embryos in relation to

their development to term.181 After fertilization (16–17 hours), a good pre-embryo possesses close proximity of pronuclei, aligned nucleoli, and heterogeneous cytoplasm with a clear halo present. It might be possible to apply the same criteria to analyze the competence of IVM oocytes. During subsequent days of development, it is important to observe the cleavage rate, synchrony of blastomere division, and incidence of multinucleation of the blastomeres.182 Several studies showed a high incidence of cleavage arrest after IVM and increased multinucleation owing to cell division arrest.73 Possible causes of cleavage arrest include inadequate culture conditions,183,184 inherent or induced abnormalities,150,185 and failure of embryonic gene expression.186 Moor and Trounson obtained a low cleavage rate after IVM of oocytes retrieved from PCO patients (54% cleavage).81 Consequently, the incidence of polyploidy and mosaicism is also increased, owing to cell division arrest.187,188 It has been shown that multinuclear blastomeres are related to some extent to chromosomal abnormalities.189,190 Again, it should be stressed that this poor embryo quality is observed mainly after the use of immature oocytes (GVs) from superovulated ICSI-treated patients. These ‘left-over’ oocytes from ICSI cycles cannot be compared to immature oocytes obtained from small follicles (6–12 mm) from unstimulated or slightly gonadotropin-primed patients. Goodquality embryos can be obtained from IVM oocytes,115 and reasonable pregnancy rates were obtained with a maximum of 2–3 embryos transferred.191 Another interesting way of evaluating embryo quality is the spreading technique for the FISH method. Analysis of the chromosomes by FISH is straightforward, to obtain information on the nuclear status of IVM embryos.73 Finally, evaluation of blastocyst formation rate might be an important alternative. In large mammals it is known that in vivo matured oocytes fertilized and cultured in vitro give almost twice as many blastocysts as in vitro matured oocytes fertilized and cultured under the same conditions.192–194

The basal medium for in vitro maturation The types of media and laboratory conditions used can affect nuclear and cytoplasmic maturation of oocytes during IVM. The pH of the culture medium is an important factor because it affects the internal pH of the oocyte. Resumption of meiosis was shown as a result of increasing pH,195 and changes in pH during isolation of mammalian oocytes can alter their developmental capacity.196 Changes in pH from 6.8 to 7.4 during IVM of mouse oocytes affected their response to meiotic inhibitors and inducers.197 During IVM of mouse oocytes, the use of different culture media, or even minor changes in culture conditions, can lead to

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a significant variation in spontaneous oocyte maturation, in the ability of meiotic inhibitors to suppress GVBD, or in the efficacy of meiosis-inducing ligands.197 Numerous media have been formulated for the purpose of somatic cell culture that support the spontaneous IVM of oocytes. For human IVM, the following basal media have been used by different authors: TCM-199, α-modified Earle’s medium (MEM), HTF, synthetic oviduct fluid (SOF), Ham’s F10, and more recently commercialized IVM media. As far as these culture media are appropriate to maintain essential metabolism, growth, and molecular expression patterns, preimplantation embryo development can be obtained from a wide range of these media. The real challenge is to provide the culture conditions which are responsible for the generation of factors critical for further embryo development.

Additives shown to improve in vitro maturation GVBD can be induced by treatment with FSH and epidermal growth factor.198 Many different media are available for oocyte maturation, and whether or not they induce or arrest oocyte maturation does not necessarily depend on their composition or complexity. Addition or extraction of certain compounds from a particular medium can sometimes have an effect that could be interfering with the experimental design. Some additives or compounds that have a proven effect on IVM are described, and knowledge of these requirements is essential to determine optimal IVM media.

Energy substrates Considerable attention has been given to the energy substrates for oocyte maturation in rodent species. In mice, the presence of pyruvate is important, and the oocyte uses it as a direct source of energy for IVM,199,200 while cumulus cells mediate glucose utilization. Cumulus cells metabolize glucose to pyruvate that will be used by the oocyte.201,202 In denuded oocytes (DOS) cultured with pyruvate, meiotic arrest is maintained in the presence of dibutyryl cAMP (dbcAMP). However, the arrest is not maintained in cumulus-surrounded oocytes (COCs),203 suggesting that metabolism of pyruvate by cumulus cells overcomes the meiosis-arresting action of dbcAMP. Downs and Mastropolo showed the importance of pyruvate in promoting the completion of nuclear maturation.197 The absence of pyruvate in different IVM media decreases the maturation potential. Cumulus cells also produce lactate from glucose, which is probably passed directly by gap junctions to the oocyte (see reference 204) but may also be transported by monocarboxylate transporters and their chaperones, such as basigin, into the oocyte. Expression of basigin mRNA has thus been considered as a marker of oocyte

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cytoplasmic maturation in human and mouse species.205 Lactate becomes accumulated in follicular fluid, and may be contributed by granulosa cells similar to paracrine factors for maintaning oocyte health. During folliculogenesis, glycolysis increases and lactate is produced, in order to meet the enhanced energy requirements within the probably increasingly hypoxic internal portions of the follicle, due to the decrease in local oxygen concentration by diffusion into the considerably grown follicular compartment. While lactate production was constant throughout maturation of bovine COCs, glucose consumption increased, suggesting that glucose supports formation of the extracellular matrix,80 in addition to providing high-energy substrates. Recently, there has been evidence that culture of naked oocytes of the mouse in the absence of lactate may adversely affect timing of chromatid segregation at maturation. There was precocious chromatid segregation prior to anaphase II in the absence of lactate in the medium, although pyruvate, glucose, and glutamine were available.206 Addition of lactate to the medium, or, alternatively, culture of oocytes within their cumulus in the absence of lactate, restored chromosome cohesion.207 Precocious chromatid segregation predisposes oocytes to errors in chromosome segregation.206 In the mouse model, the absence of lactate also affected the association of mitochondria with the spindle in the oocytes, suggesting that there are tentative links between metabolic activities and health of the somatic cells in the follicle, and mitochondrial function, timing, and fidelity of chromosome segregation in the oocytes, and, possibly, also nondisjunction in mitosis of the preimplantation embryo. In agreement with this assumption, Wilding and co-workers208 observed that human oocytes with mitochondria with a low redox potential may generate aneuploid, mosaic embryos after fertilization. In fact, it was shown in humans that lactate dehydrogenase levels in follicular fluid are related to follicle size and also to patient age,209 indicating that the metabolic activity of the follicle cells critically influences the microenvironment of the maturing oocyte, and possibly in this way also the fate of the embryo. The importance of the energy source has been studied more recently in larger animals such as cattle and primates. In an attempt to reduce confounding factors in the study of nutrient requirements, serum and bovine serum albumin (BSA) were not added to IVM media as they contain undefined substances,210 but a rather simple salt solution with 11 amino acids formed the basal medium. These studies emphasized the importance of glutamine in combination with glucose or lactate, or glucose plus lactate, for acquisition of developmental competence. Optimal metabolism of glutamine requires intact COCs, and was enhanced by LH.211 Glutamine plays a primary part in oocyte maturation. Glucose is essential for acquisition of developmental competence in cattle, and is metabolized by COCs but not by denuded oocytes or cumulus cells alone.212

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There is a need for more fundamental studies to assess possible energy sources during IVM of human COCs.

The protein source in IVM Although protein supplements in IVM media are used successfully to produce viable embryos, they introduce many unknown factors into the system and are generally at the basis of the variability in experimental outcomes between groups.213,214 It appears as though the addition of human follicular fluid or inactivated autologous patient serum sustains the efficacy of the media for human IVM96,100,103 instead of the previously used human serum albumin or synthetic serum substitute.89,123,125,215,216 Some authors use heterologous human serum instead or apply protein supplementation from animal sources;92–94,104 however, a fair comparison has never been conducted. Moreover, it becomes impossible to study the impact of specific factors on ooycte maturation and embryo development. Besides the large variation in components in serum or albumin preparations, there is also an essential aspect of safety: yet undefined infectious agents might contaminate the cultured embryos. Furthermore, one of the factors most often mentioned in relation to the ‘large offspring syndrome’ is serum, which provides a rich but undefined environment to the gamete and embryo. Serum contributes to ammonia formation in culture, which can damage the mitochondria.217 These facts will preclude the future use of proteins from human or animal origin in maturation media. On the other hand, the serum source functions as a scavenger for possible toxicities, and by this means can protect the embryo. When serum is replaced by albumin or another macromolecule (polyvinylpyrrolidone [PVP] or polyvinylalcohol [PVA]), the maturation or culture system becomes more sensitive to possible negative influences. When the serum component is omitted, the medium has to be balanced by using supplements. This strategy is currently being investigated with the recently developed semidefined sequential media for blastocyst culture. Possible safer alternatives for serum or purified albumin preparations are recombinant albumin or inert matrices such as hyaluronan (HA), PVA, or PVP. Making use of inert matrices might also promote contact between the oocyte and surrounding cumulus cells during prolonged in vitro culture.218

Glutathione metabolism The oxidative modification of cell components via reactive oxygen species (oxidative stress) is one of the most potentially damaging processes for proper cell function. In most cells, efficient antioxidant systems can attenuate the effect of oxidative stress by scavenging reactive oxygen species.219 Glutathione (GSH) is the major nonprotein sulfhydryl compound in mammalian cells, and is known to play an

important role in protecting the cell from oxidative damage. Synthesis of GSH during oocyte maturation has been reported in mice,220 hamsters,221 pigs,222 and cattle.223,224 GSH content increases during development and oocyte maturation in the ovary, as the oocyte approaches the time of ovulation. After fertilization, GSH participates in sperm decondensation in parallel with oocyte activation, and in the transformation of the fertilizing sperm head into the male pronucleus.220–222,225,226 Glutathione is synthesized by the γ-glutamyl cycle,227,228 and its synthesis is dependent on the availability of cysteine in the medium (Fig 9.21). In a review, Eppig suggested that GSH production is a critical part of cytoplasmic maturation. Cytoplasmic maturation entails numerous molecular events, including synthesis, protein phosphorylation, and activation of particular metabolic pathways.229,230 These changes are essential for normal fertilization and embryo development. Funahashi et al suggested that the intracellular GSH content of porcine oocytes at the end of IVM appears to reflect the degree of cytoplasmic maturation.231 Moreover, results obtained with bovine oocytes are in agreement with the hypothesis that measurement of GSH after IVM may be a valuable indicator of cytoplasmic maturation.232–234 It has been shown that β-mercaptoethanol and cysteamine reduce cystine to cysteine and promote the uptake of cysteine, enhancing GSH synthesis.235–238 It was shown that cysteamine, β-mercaptoethanol, cysteine, or cystine supplementation of IVM medium increased the intracellular GSH content of oocytes after IVM and improved embryo development and quality.239 When β-mercaptoethanol was added during ovine oocyte IVM, GSH synthesis was also stimulated, but this increase in intracellular oocyte GSH levels did not improve subsequent embryo development.240 The availability of cysteine in the IVM medium seems to be the limiting factor for glutathione synthesis in mammalian oocytes.241 The concentration of cysteine in TCM-199 used for routine IVM of bovine oocytes is very low (0.6 mmol/l) compared with that of cystine (83.2 mmol/l), and because of auto-oxidation, essentially no cysteine is present. Consequently, GSH synthesis may be impaired owing to the lack of substrate, generating suboptimal culture conditions for IVM. It is possible that the cystine generated by auto-oxidation is converted into cysteine by cumulus cells and then incorporated into GSH synthesis.233 The pool of GSH generated during IVM, by stimulating its synthesis, is very important for the fertilization process and first stages of embryo development in view of the fact that increased levels are maintained over the fertilization process and are still present at the beginning of embryo culture.239

Meiosis-activating sterols Several factors, such as high concentrations of cAMP and hypoxanthine (Hx) in the compartment surrounding

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Glutathione (GSH) Synthesis and its Inhibition Reduction Glutamic Acid + Cysteine Cystine (Cys-Cys) (Cys) Oxidation Buthionine y-Glutamylcysteine Sulfoximine Synthetase - BSO - GCS γ-glutamyl~cysteine

Reduction NADPH

Glycine

Glutathione Peroxidase - GPX -

Glutathione Synthetase

R-O-O-H + 2

GSH +H2O2

GSH: Reduced glutathione (reduced) GSSG: Oxidized glutathione BSO: Specific inhibitor of GSH synthesis R-O-O-H: Organic peroxides

GSSG +H2O

Oxidation

the oocyte, prevent meiotically competent oocytes from resuming meiosis spontaneously.242–244 Keeping the concentration of cAMP in the oocyte high inhibits resumption of meiosis.244,245 Byskov et al discovered a group of endogenous meiosis-activating sterols (MAS) in follicular fluid occurring naturally in the biosynthetic pathway between lanosterol and cholesterol.246 One of these sterols was isolated from human follicular fluid and was named FF-MAS (4,4-dimethyl-5α-cholesta-8,14, 24trien-3β-ol). This sterol induces meiosis resumption in cumulus-enclosed mouse oocytes in a dose-dependent manner when the oocytes are kept arrested in meiosis artificially with hypoxanthine (Hx), isobutylmethylxantine (IBMX), dbcAMP, and inhibitor of PDE3 (although with slower kinetics)247 (personal unpublished observations). Notably, MAS sterols could be LH downstream messenger candidates, since FF-MAS has been observed to be under the influence of gonadotropin regulation in vivo, especially LH,248,249 and, furthermore, FF-MAS has been observed to induce mitogen-activated protein (MAP) kinase activity in the mouse oocyte.250 Reversal of the Hx-induced meiotic block by FF-MAS is dependent on protein synthesis.247 The dynamics of microtubule- and actin-mediated organelle movement is well established for processes such as extension of endoplasmic reticulum,251 changes in mitochondrial distribution,252 and movement of the cortical granules to the cell surface.253 The studies with FF-MAS clearly show that it is possible to delay the kinetics of nuclear maturation in the presence of Hx without disturbing the general cortical polarization in the plasma membrane.254 In addition, FF-MAS treatment of mouse oocytes not only delays the migration of cortical granules to the oolemma but also appears to increase the reuptake of cortical granular material.254 These studies suggest that FF-MAS may have beneficial effects on IVM, although it may not be an indispensable component at the initiation of meiotic resumption in vivo.255

Fig 9.21 The metabolic pathways involved in the synthesis of glutathione and in its action as an antioxidant. Glutathione is synthesized by the γ-glutamyl cycle. The activity of γ-glutamyl cysteine synthetase (GCS) can be inhibited by buthionine sulfoximine (BSO). Reduced glutathione (GSH) detoxifies reactive oxygen species by action of glutathione peroxidase (GPX) which is stage-dependently expressed in oocyte and early embryo. NADPH, reduced nicotinamide adenine dinucleotide phosphate.

Even though FF-MAS has been shown in several models to induce nuclear maturation, it is still debated whether FF-MAS constitutes a pivotal meiosis-mediating signal downstream from gonadotropins.248,249,256,257 FF-MAS has also been reported to signal in rat oocytes in vitro.258 Importantly, entire ovaries have been cultured as ex vivo perfused organ culture ad modum Brännström,259 and FF-MAS has been observed to overcome this purely physiologic meiosis inhibition exerted by the full functional follicle in rats in a dose-dependent manner.258 Furthermore, FF-MAS has been reported to signal in bovine oocytes.260 In recent years, FF-MAS has been applied to human oocyte IVM in a small number of oocytes. Encouraging data show improvements in maturation rate.261,262 In a large prospective study in healthy volunteers and IVF patients, the safety of FF-MAS was studied when incubated for 30 hours or during IVM. Results of karyotyping oocytes with the SKY technique showed no increase in aneuploidy rate by FF-MAS. Consequently, this compound could be applied to improve oocyte quality after IVM.33 Further study is needed to unravel the cellular targets for FF-MAS and to understand its signaling cascade in the COC.

Hormonal and growth factor requirements Gonadotropins Follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) are generally included as components of IVM media. IVM of bovine oocytes in the presence of FSH retards nuclear maturation via a cAMP-mediated pathway, while it enhances fertilizability and developmental ability of bovine oocytes. A delay in resumption of meiosis in the presence of FSH has also been reported in rodent oocytes.198,263,264 In the presence of FSH, a transient rise of cAMP was

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observed in COCs,36 and this can stimulate oocyte maturation.198,265 There is experimental evidence that FSH induces cumulus expansion in bovine oocytes.266 Addition of FSH in the culture of human oocytes retrieved from unstimulated ovaries increased steroid secretion by cumulus cells without interfering with capability for maturation to metaphase II.267 A 1:10 ratio of FSH:LH following 24 hour culture with FSH seemed to benefit the maturation of oocytes collected from unstimulated ovaries, and lead to a higher embryonic developmental capacity than when only FSH is added to the culture of oocytes.268 In the same report, using bovine oocytes, the effect of FSH:LH ratio during IVM culture significantly improved embryonic development. The ovulatory gonadotropin surge, mediated by LH, is a key event of the estrous cycle, and it is responsible for various ovarian processes, including the resumption of meiotic division of the oocyte. It was suggested that the progressive reduction of gap junctions between granulosa cells and oocyte, induced by the gonadotropin surge, stops the diffusion of inhibitory molecules to the oocytes. As a consequence, resumption of meiosis ensues.35 On the other hand, resumption of meiosis has been seen in many species before reduction of functional communication.269–272 Earlier studies showed that high concentrations of LH during IVM of bovine oocytes improves embryo development up to the 4–8-cell stages.273 LH induction of maturation via progesterone synthesis has only been proven in mouse oocytes.274 Studies on intra-preovulatory follicular factors demonstrated that LH/hCG causes cumulus expansion and oocyte maturation via paracrine means, partly by inducing expression of EGF-like growth factors in mural granulosa cells which are then diffused to the cells neighboring the oocyte.40,275 For IVM of human oocytes, gonadotropins are often added to the medium in concentrations ranging from 0.075 IU/ml of FSH to 10 times higher and 0.1–10 IU/ml hCG (see Table 9.1). There are few reports available elucidating the effects of the use of selective gonadotropins during IVM on the developmental competence of human oocytes. Bovine antral follicles <8 mm possess only FSH receptor mRNA and no LH receptor mRNA in mural granulosa cells. Van Tol et al, using small- to mediumsized follicles, showed the presence of FSH receptors in granulosa cells and cumulus cells, but not in oocytes.276 LH receptors were present only in thecal cells. There is evidence that the effect of FSH on oocyte maturation is mediated via a cAMP signal transduction pathway, and as the oocyte does not contain FSH receptors,276 it is possible that the effect of FSH on oocyte maturation is exerted through the cumulus cells. Izadyar et al277 and Alberio and Palma278 found that FSH added during bovine oocyte IVM improved fertilization and had beneficial effects on embryo development. It is debatable whether LH or hCG addition to culture medium could have any

positive effect on IVM outcome. Hreinsson et al279 showed no differences on laboratory outcomes and clinical pregnancies when 0.5 IU/ml of recombinant hCG or recombinant LH was added in culture of immature oocytes retrieved after a mild stimulation with FSH. Recently, it was shown that hCG is unnecessary for the maturation of oocytes from PCO patients in medium containing 20% fetal bovine serum and FSH.280 The optimal dosage of gonadotropins for oocyte culture and its effect on oocyte competence for subsequent development are still uncertain. Animal studies have alerted us that exposure of oocytes to high levels of FSH during IVM speeds up the process of nuclear maturation, inducing chromosomal abnormalities.281 Besides, depending on the developmental status of the immature oocyte, a differential approach could be more appropriate to the developmental competence of the oocyte.

Growth factors: insulin and insulin-like growth factor I Various paracrine actions for insulin-like growth factor I (IGF-I) have been demonstrated in the ovary. The insulin signaling cascade is active in oocytes; gonadotropins influence oocyte insulin receptor expression during meiosis.281a It was found that exposure to high doses of insulin of oocytes in their growth phase was detrimental for meiotic chromatin remodeling and induces condensation errors.281a IGF-I stimulates rat granulosa-cell mitogenesis, and its effect is made more potent by FSH.282 FSH and IGF-I act synergistically, as evidenced by the maximal estradiol secretion in cultured human granulosa cells.283 IGF-I is a potent mitogen for granulosa cells,284 and enhances nuclear maturation in oocytes surrounded by compact cumulus cells in cattle,285 humans,121 rats,41 and rabbits.286,287 It was reported that FSH stimulates intraovarian IGF-I production.288 In the buffalo it was suggested that IGF-I was effective in stimulating nuclear maturation but not cumulus expansion, without impairment of fertilization and embryo development. IGF and FSH stimulate progesterone secretion in granulosa cells,289,290 and their synergistic coupling induces a steroidogenic activity in both bovine cumulus and granulosa cells.291 IGF-I plays a multifunctional role in follicle development and oocyte maturation. Using human COCs from 6–12-mm follicles, it was found that the presence of physiologic amounts of insulin and gonadotropins in serum-free medium were essential to obtain a good nuclear maturation rate made during pilot studies of the FM-MAS trial33 (personal observations 2004).

Epidermal growth factor Peptide growth factors have been implicated as autocrine–paracrine regulators of ovarian function. Among many growth factors, epidermal growth factor (EGF) has been shown to stimulate IVM in rats,37

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mice,198,292 cattle,285 pigs,293 and humans.294 EGF can stimulate cumulus expansion of bovine oocytes in serum-free conditions.284,295 In pigs, in the presence of gonadotropins and serum, EGF did not stimulate cumulus expansion.296 In cattle, the presence of EGF during IVM influences the protein neosynthesis pattern in the oocyte.213 It has been proposed that some of the proteins synthesized during oocyte maturation may be essential for the normal oocyte maturation process, while others may be required for fertilization and embryo development.297 Recently, the discovery of the LH messengers within the ovarian follicle increased the understanding of the ovulatory events. EGF-like factors, epiregulin, amphiregulin, and betacellulin were shown to be upregulated in granulosa cells after the LH source. Furthermore, once they are shed from the cells membranes, these factors exert their action on cumulus cells, inducing upregulation of genes related to mucification and expansion (Ptgs2, Has2, Tnfaip6, and Ptx3). Since the LH receptor is very low expressed (or absent) in cumulus cells, the potential use of these proteins in IVM protocols relies on its direct effects on these cells (Fig 9.22).

Growth hormone The addition of growth hormone (GH) during IVM of bovine oocytes accelerates nuclear maturation, induces cumulus expansion, and promotes subsequent cleavage and embryonic development.298 Moreover, it also improves cytoplasmic maturation, as testified by improved migration of cortical granules and sperm aster formation, leading to improved fertilization of bovine oocytes.272 Culture of rat and porcine oocytes in the presence of GH also showed an acceleration of the process of nuclear maturation.42,299 In human IVM there has been no systematic in vitro study to evaluate whether GH could improve cytoplasmic maturation.

Activins and inhibins In different animal species and in the human COCs obtained from small- and medium-sized follicles, expression of activin A, inhibin, follistatin, and activin receptor type II proteins has been shown in both the cumulus cells and oocyte during IVM.300 The data suggest a possible role of these developmentally regulated proteins during IVM. Follistatin, which is cycle-regulated, has been measured in serum and follicular fluid of rats,301 humans,302 pigs,303 and cattle.304 It functions as a high-affinity binding protein of inhibins and activins.305 Within the ovary, these peptides have both autocrine and paracrine functions during folliculogenesis,306 but the actions of activin during IVM maturation are conflicting. Receptors for activin A, a homodimer composed of two disulfide linked β-subunits of inhibin A, are present on rat and mouse granulosa cells,307–309 as well as in

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cumulus cells of the rat and mouse.310,311 Hulshof demonstrated the presence of activin receptors in granulosa cells and oocytes of bovine antral follicles.312 Experimental work from Van Tol et al313 and Izadyar et al300 showed that activin A has no effect on nuclear and cytoplasmic maturation of bovine oocytes, but more recent work indicates that activin increases the developmental capacity of both cumulus-enclosed and cumulus-free bovine oocytes.314,315 A study from Alak et al in nine women found a stimulating effect of activin (100 ng/ml) on oocyte maturation.316 These data still need confirmation by others before they can be applied routinely.

Oxygen tension and oocyte in vitro maturation When cumulus-denuded oocytes were used for IVM, adverse effects of oxygen on nuclear maturation were observed, and a 5% oxygen concentration appeared to be optimal.317,318 However, Eppig and Wigglesworth failed to see deleterious effects on fertilization and embryo development, regardless of the concentration of oxygen used during the oocyte maturation period alone.319 In our laboratory, as a standard procedure, IVM of mouse,320 bovine,233 and human73 oocytes is done in 20% oxygen. In a recent collaborative study by Hu et al, the maturation rate, spindles, and chromosome alignment in oocytes were analyzed after exposure of in vitro cultured mouse follicles to low (5%) or normal oxygen tension in the atmosphere during the final IVM period (after hCG/EGF administration).321 The presence of only 5% O2 in the atmosphere during the final maturation stages decreased the percentage of oocytes with ordered chromosomes. This in vitro study underlined the importance of providing a correct oxygen environment to prohibit errors in chromosome segregation at meiosis I. The culture medium composition is also important in relation to the oxygen concentration which is provided to the cultured tissues, and might cause oxidative damage.322 It is possible that the presence of several layers of cumulus cells around the maturing oocyte buffers the deleterious effects of oxygen tension. It was suggested that most of the oxygen entering follicles is consumed by the outer layers of granulosa cells. The available oxygen diminishes with progression toward the oocyte,323 so that the oocyte is located in a relatively anoxic environment. No studies have addressed the oxygen tension in human IVM. This point might become important if prolonged culture times are considered as an option to improve cytoplasmic maturation.

Future directions Strategies to improve in vitro maturation of oocytes Studies in large mammals showed that the competence to undergo meiotic maturation and to sustain embryonic development is gradually acquired during oocyte

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LH-R

EGF-like

ErbB

FW FF

MAPK3/1 Activation CC

- Ptgs2 -Has2 -Tnfaip6 -Ptx3

Cumulus expansion

Meiotic resumption

FW: Follicular wall Oo FF: Follicular fluid CC: Cumulus cells Oo: Oocyte : Matrix metaloproteinases

Fig 9.22 Ovulatory stimulus. Right before ovulation the ovarian follicle becomes responsive to the Luteinizing Hormone (LH) surge. During the ovulatory process LH binds to its receptors on the follicular wall inducing expression of EGF-like proteins which are displayed on the cell surface as pre-proteins. Shedding by matrix metalloproteinases (MMP) ensures that mature EGF-like proteins reach their receptors or cumulus cells. As a result, and depending on MAPK31 activation, cumulus cells will produce factors required for mucification and expansion such as Ptgs2, Has 2, Tnfaip6, and Ptx3. Simultaneously, meiotic resumption occurs, most likely triggered by a decrease in the oocyte cAMP content; an event which has been related to both the activation of oocyte phosphodiesterases and the limited flow of cAMP from surrounding cumulus cells to the oocyte, after closure of gap junctions.

growth.6,29,324 The importance of terminal differentiation and maturation of the oocyte has been emphasized by studies showing a relation between follicle diameter and competence for normal embryogenesis. The molecular substrate of this oocyte capacitation is as yet not characterized, but refers to transcripts and proteins that are stabilized in the oocyte and mobilized during early development when the embryonic genome is still quiescent. These reserves for early development are acquired very late in oogenesis, in the period just before ovulation. During the last phase of its growth, the oocyte produces different RNA species of prolonged stability, with an average half-life of 28 days.325 These RNA species are functional in different levels at different times.326 This is made possible by the different degrees of polyadenylation (poly-A). Long poly-A tails (~150Å residues) are for immediate use (during maturation), whereas shorter poly-A tails (<90Å) will have to be polyadenylated later to become functional.327 These mRNAs are stockpiled

within specialized microribonucleoparticles in the cytoplasm for later use after fertilization, during early embryonic development. The reduced potential to produce clinical pregnancies after IVM of oocytes can be explained by shortcomings of the above-described mechanisms at different levels. First of all, there is a very heterogeneous population of oocytes collected during ultrasound-guided transvaginal retrieval. The oocytes from the smallest follicles might still not contain all required reserves for normal preimplantation development. Experimental evidence in large mammals showed that nuclear and cytoplasmic maturation have to go hand in hand to enable normal further development. Delays in cytoplasmic maturity lead to disturbances in nuclear meiotic maturation rate, besides causing inadequate redistribution of organelles (CG migration and dispersal) and an altered ability to decondense the sperm chromatin after sperm aster formation.328–330

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Future studies on in vitro maturation The causes of oocyte deficiency (e.g. aneuploidy, embryonic developmental failure) remain enigmatic. Some of the defects in in vitro matured oocytes have been identified as the inability of the cytoplasm to maintain M-phase characteristics during meiosis progression, a propensity to undergo spontaneous activation after metaphase arrest, and a deficient coordination between nuclear and cytoplasmic maturation. The origins of deficient quality of the oocyte might be diverse and multifactorial: •

Defects can be inherent to the GV oocyte caused by a suboptimal intrafollicular environment prior to oocyte retrieval. For example, in vivo maturation of oocytes is based on a more natural selection by the exogenous hCG stimulation. However, collection of immature oocytes from small follicles is

va

l

Several DAYS depending on maturity stage 30 hours

re tri e C C C

Furthermore, the culture principles used until today might have been inadequate to support normal maturation. The basal culture media for IVM used in the literature have been chosen on an empirical basis, and might lack essential constituents. By the act of retrieval of meiotically competent oocytes, the spontaneous reinitiation of meiosis is triggered, leading to an almost immediate arrest of transcription. When immature oocytes are retrieved from small follicles, optimal maturation (cytoplasmic maturation) is truncated by the arrest of all transcriptions once nuclear maturation is reinitiated. In order to obtain optimal cytoplasmic maturation, the reinitiation of meiosis should be inhibited and the correct culture environment provided for a critical period. The achievement of these principles is possible only if technical developments can provide the essential noninvasive tools objectively to distinguish some grading in the concept of ‘cytoplasmic maturation.’ To this purpose, more basic research needs to sort out methods that are rapid and adapted to routine practice and that could allow oocyte maturity grading, before deciding in which culture medium and for how long they should be matured. Perhaps a prematuration phase of immature oocytes could be beneficial to enable the biochemical processes accompanying the cytoplasmic rearrangements to develop in a more physiologic way (Fig 9.23). Several possible strategies could be adopted to reach this goal, either by co-culture of follicle shells or by using pharmacologic agents interfering with protein synthesis, cAMP metabolism, or the generation of pre-MPF, or inhibiting MPF activity. Efforts to design a better in vitro environment for these GV oocytes by developing a three-dimensional co-culture condition have resulted in improved spindle and chromatin organization after IVM with MPF and MAPK activity patterns approaching those of in vivo-matured oocytes.331

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PRE-Maturation

MATURATION

Time

GC-enclosed oocyte PDE3INH or MPF Blocker

FERTILIZE

Fig 9.23 The scheme illustrates the principle of including a prematuration phase involving a pharmacological compound to maintain meiotic arrest for a number of days before letting the oocytes become metaphase II (MII) oocytes. CCC, cumulus–corona complex; PDE3INH, phosphodiesterase 3 inhibitor; MPF, M-phase promoting factor; GC, granulosa cell.

• •

nonselective, and even those oocytes that would not have the potential to mature in vivo at a later stage in the development process under the hCG influence, when harvested, are able to undergo in vitro maturation. Defects caused by the in vitro environment in which the oocyte is cultured. The retrieved GV oocytes might be cytoplasmic immature to support IVM and further development.

Improvement of the quality of in vitro matured oocytes might involve a combination of cultures that sustain a prolonged period of development in vitro prior to meiosis resumption (e.g. prematuration culture) in combination with a favorable in vitro environment prior to and during maturation. An extension of the developmental period of the immature oocyte in culture might be necessary to achieve a better nuclear/cytoplasmic synchrony of maturation in oocytes. Arrest of nuclear maturation can be achieved by using several pharmacologic compounds (Fig 9.24). Research has already been done along this line of thinking, involving cattle and human oocytes. In the bovine IVM model, arrest of nuclear maturation could be obtained by co-culturing COCs with granulosa or theca cells. This system, although quite effective, would not be suitable for human assisted reproductive technologies (ART) routine practice owing to its heaviness.332 The in vitro methods to control meiosis progression in rodent COCs such as hypoxanthine, dbcAMP, and IBMX are easier to use, but not completely effective in COCs from large species.333 Some researchers have suggested prolonged inhibition of meiosis (>24 hours) by applying substances that involve the inhibition of protein synthesis or phosphorylation processes.334 After the use of cycloheximide (1 mg/ml), a protein synthesis inhibitor, or 6-dimethylaminopurine (2 mmol/l), an inhibitor of phosphorylation, approximately 80% of temporarily (for a minimum of 24 hours) blocked oocytes developed to MII (after wash-out of inhibitors). Some 10–20% of these oocytes developed to blastocysts, showing that

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LH/hCG or mechanical disruption of follicle integrity

INTERVENTION

Closure of gap junctions

Decrease of cAMP

− coculture with follicular shells − isobutylmethylxanthine (IBMX) (adenylyl cyclase activator) − dibutyryl (cAMP analogs) − phosphodiesterase inhibitors (PDEs) (inhibit cAMP degradation)

Inactivation of PKA − cycloheximide (inhibits protein synthesis) Synthesis of pre-MPF

Formation of MPF

− 6-dimethylaminopurine (6-DMAP) (inhibits phospho- and dephosphorylation) − butyrolactone (Roscovitine) (inhibits MPF)

MAPK activation

this option is feasible but should be further improved.335 Inhibition of MPF kinase activity by roscovitine (25 µmol/l) could also reversibly keep H1 kinase activity very low, after which a comparable blastocyst formation rate (36% vs 40% in controls) was observed, as without roscovitine but matured in TCM199 plus 10 ng/ml EGF. This experiment proved the feasibility of arresting nuclear maturation for 24 hours to allow for cytoplasmic maturation without compromising the resulting developmental potential.336 Inhibition of cyclin-dependent kinases (p34cdc2 and MAP kinase) by butyrolactone I for 24 hours was reversible, 90% of the oocytes reached MII, and a 70% 2-PN formation rate was observed.337 Similar work was done in humans by Anderiesz et al158 involving 6-dimethylaminopurine (DMAP), a serine–threonine protein kinase inhibitor of histone H1 kinase, but not interfering with protein synthesis.338 DMAP could temporarily (for at least 24 hours) inhibit human meiotic maturation without affecting subsequent maturation to MII. However, as these kinase inhibitors are not cellspecific, they interfere with the somatic cell compartment. Specific inhibitors of cAMP hydrolytic enzymes, phosphodiesterases (PDEs), have recently attracted more attention in the field of reproduction. Proof of principle has been addressed for selective PDE inhibitors during follicle and oocyte development.48,49,57 By using the selective PDE3 inhibitor, interference with cAMP pathway mechanisms in the somatic cells can be avoided. PDE3 acts directly in the oocyte, whereas PDE4 is involved mainly in the metabolization of cAMP in granulosa cells.47 Selective PDE3 inhibitors (Org9935, cilostamide, milrinone) have emphasized the importance of the PDE3A enzyme in the regulation of oocyte meiosis.54 The in vitro use of PDE3 inhibitor on human COCs retrieved from small follicles (<12 mm in diameter) before or after

Fig 9.24 Possible interventions to maintain nuclear maturation arrest. LH, luteinizing hormone; hCG, human chorionic gonadotropin; cAMP, cyclic adenosine monophosphate; PKA, protein kinase A; MPF, M-phase promoting factor; MAPK, mitogen-activated protein kinase.

exposure to an LH trigger resulted in 88.9% and 83.3% GV arrest without signs of GVBD, respectively, and the inhibitory effect on meiosis progression was reversible.56 In further experiments with human oocytes, early embryonic development could be achieved after 48-hour arrest by PDE3 inhibitor.339 Postimplantation development has been achieved in mice.340 Using this animal model, following reversal from 24 hours of meiotic arrest by PDE3 inhibitor, fertilization was significantly improved and embryonic preimplantation development and live offspring rates were successfully achieved. This culture system with PDE3-I proved more effective for obtaining normal spindle and chromosome configurations in in vitro matured oocytes than in controls counterpart.341,342 The ability to regulate the human oocyte’s nuclear maturation might provide a useful starting point towards creating favorable medium conditions that could enhance cytoplasmic maturity during a prematuration phase.

Conclusions Development of a culture system for COCs from small antral follicles There is good rationale to schedule immature oocyte retrieval before the intraovarian selection processes have taken place or after a short treatment course of FSH. When targeting 6–12-mm diameter follicles for IVM, these follicles would in vivo still undergo a growth phase of 4–5 days before ovulation. Hence, a prolonged ‘prematuration system’ could be beneficial for IVM. Although oocyte nuclear and cytoplasmic maturation can proceed independently from each other, both processes need to be coordinated to ensure developmental competence. Therefore, the intimate transzonal connections between granulosa cells and

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oocyte must be kept patent for transfer of regulatory substances between the two cellular compartments. Keeping the oocyte meiotically arrested (by using either a co-culture approach or pharmacologic agents that arrest the meiosis process) and providing growth factors and hormonal supplements to sustain completion of the oocyte’s cytoplasmic maturation could theoretically lead to an improvement of development after fertilization. This strategy is actually pioneered in bovine and human oocytes.56,158,336,337 Safety restricts the use of protein sources from other species in human IVM media. Serum substitutes might be found necessary to cover the ‘serum functions.’ The presence of steroids in the culture medium might be important for the maintenance of intercellular contacts (estrogens and progesterone) and for the oocyte’s cytoplasmic maturation. It remains to be proved whether r-hCG or r-hFSH in IVM media are essential components during the in vitro prematuration phase. Additions of a meiosis-activating sterol,246 growth hormone,298 activin/inhibin ratios,260,339 or enhancers of glutathione synthesis in the cytoplasm231,239 are possible approaches towards increased cytoplasmic maturation. Owing to the large interspecies differences in regulation of meiosis, the authors would advocate a stepwise approach to the development of ‘robust IVM’ in the human. In a first step, safety is tested on the reinitiation of meiosis by performing karyotyping of MII oocytes. If safe, matured oocytes are injected with sperm and the aneuploidy rate tested in day 3 embryos with state-of-the-art technology. Further emphasis should be placed on blastocyst quality obtained after IVM, and, finally, transferred embryos should be monitored closely during implantation. As with all new techniques in ART, a registry for all children born has to be set in place.

Acknowledgments Professor Raphael Ron-El and his clinical IVF team at Assaf Harofeh (Israel) made a major contribution to our work on human IVM back in 2004 by mobilizing the whole lab for the project. Peter Platteau and Carola Albano provided most of the human oocytes from small follicles for IVM research at UZ Brussels. Co-workers from the Follicle Biology Laboratory are acknowledged for their participation in the initial NM studies at UZ Brussels: Mrs Rita Cortvsindt, Mrs Kelly Van Wemmal, Dr. Christian Grönolahl The authors acknowledge the Fund for Medical Research Flanders (FWO Grant no G.0166.98), The Belgian Technical Cooperation (BTC), and the Belgian Embassy in Lima (Peru), for the study grant of Sergio Romero, Ares Serono International (GF9405), and Novo Nordisk Fertility Team (NN 5492-1312) for financially supporting these research projects. Organon is acknowledged for providing the PDE3 inhibitor Org9935.

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279. Hreinsson J, Rosenlund B, Friden B, et al. Recombinant LH is equally effective as recombinant hCG in promoting oocyte maturation in a clinical in-vitro maturation programme: a randomized study. Hum Reprod 2003; 18: 2131–6. 280. Ge HS, Huang XF, Zhang W, et al. Exposure to human chorionic gonadotropin during in vitro maturation does not improve the maturation rate and developmental potential of immature oocytes from patients with polycystic ovary syndrome. Fertil Steril 2008; 89: 98–103. 281. Roberts R, Iatropoulou A, Ciantar D, et al. Folliclestimulating hormone affects metaphase I chromosome alignment and increases aneuploidy in mouse oocytes matured in vitro. Biol Reprod 2005; 72: 107–18. 281a Acevedo N, Ding J, Smith GD. Insulin signaling in mouse oocytes. Biol Reprod 2007; 77: 872–9. 282. Bley MA, Simon JC, Estevezo AG, DeAsua LJ, Baranao JL. Effect of follicle stimulating hormone on insulin-like growth factor-I stimulated rat granulosa cell deoxyribonucleic acid synthesis. Endocrinology 1992; 131: 1223–9. 283. Erickson GF, Danforth DR. Ovarian control of follicle development. Am J Obstet Gynecol 1995; 172: 736–47. 284. Hernandez ER, Resnick CE, Svoboda ME, et al. Somatomedin-C/insulin-like growth factor-I as an enhancer of androgen biosynthesis by cultured rat ovarian cell. Endocrinology 1998; 122: 1603–12. 285. Lorenzo PL, Illera MJ, Illera JC, Illera M. Enhancement of cumulus expansion and nuclear maturation during bovine oocyte maturation in vitro by the addition of epidermal growth factor and insulin-like growth factor I. J Reprod Fertil 1994; 101: 697–701. 286. Lorenzo PL, Rebollar PG, Illera MJ, et al. Stimulatory effect of insulin-like growth factor-I and epidermal growth factor on the maturation of rabbit oocyte in vitro. J Reprod Fertil 1996; 107: 109–17. 287. Yoshimura Y, Ando M, Nagamatsu S, et al. Effect of insulin-like growth factor I on follicle growth, oocyte maturation and ovarian steroidogenesis and plasma activator activity in the rabbit. Biol Reprod 1996; 55: 152–60. 288. Adashi EY, Resnick CE, D'Ercole J, Svoboda ME, Van Wyk JJ. Insulin-like growth factor as intraovarian regulator of granulosa cells. Endocrinol Rev 1985; 6: 400–20. 289. Amsterdam A, May JV, Schomberg DW. Synergistic effect of insulin and follicle stimulating hormone on biochemical and morphological differentiation of porcine granulosa cells in vitro. Biol Reprod 1988; 39: 379–90. 290. May JV, Schomberg DW. Granulosa cell differentiation in vitro: effect of insulin on growth and functional integrity. Biol Reprod 1981; 25: 421–31. 291. Armstrong DT, Xia P, Gannes G, Teckpetey FR, Khamsi F. Differential effect of insulin-like growth factor I and follicle-stimulating hormone on proliferation and differentiation of bovine cumulus cells and granulosa cells. Biol Reprod 1996; 54: 331–8. 292. Smitz J, Cortvrindt R, Hu Y. Epidermal growth factor combined with r-HCG improves meiotic progression in mouse follicle-enclosed oocyte culture. Hum Reprod 1998; 13: 664–9.

293. Ding J, Foxcroft GF. Epidermal growth factor enhances oocyte maturation in pigs. Mol Reprod Dev 1994; 39: 30–40. 294. Das K, Stout LE, Hensleigh HC, et al. Direct positive effect of epidermal growth factor on the cytoplasmic maturation of mouse and human oocytes. Fertil Steril 1991; 55: 1000–4. 295. Kobayashi K, Yamashita S, Hoshi H. Influence of epidermal growth factor and transforming growth factor-α on in vitro maturation of cumulus cellenclosed bovine oocytes in a defined medium. J Reprod Fertil 1994; 100: 439–46. 296. Wang W, Niwa K. Synergetic effects of epidermal growth factor and gonadotropins on the cytoplasmic maturation of pig oocytes in a serum-free medium. Zygote 1995; 3: 345. 297. McGaughey RW, Van Blerkom J. Patterns of polypeptide synthesis of porcine oocytes during maturation in vitro. Dev Biol 1977; 56: 241–54. 298. Izadyar F, Colenbrander B, Bevers MM. In vitro maturation of bovine oocytes in the presence of growth hormone accelerates nuclear maturation and promotes subsequent embryonic development. Mol Reprod Dev 1996; 45: 372–7. 299. Hagen DR, Graboski RA. Effects of porcine pituitary growth hormone (pGH) on cytoplasmic maturation of porcine oocytes in vitro. J Anim Sci 1990; 68: 446. 300. Izadyar F, Zeinstra E, Colenbrander B, Vanderstichele HMJ. In vitro maturation of bovine oocytes in the presence of activin A does not affect the number of embryos. Anim Reprod Sci 1996; 45: 37–45. 301. Saito S, Nakamura T, Titani K, Sugino H. Production of activin-binding protein by rat granulosa cells in vitro. Biochem Biophys Res Commun 1991; 176: 413–22. 302. Krummen LA, Woodruff TK, de Guzman G, et al. Identification and characterization of binding proteins for inhibin and activin in human serum and follicular fluids. Endocrinology 1993; 132: 431–43. 303. Ueno N, Ling N, Ying SY, et al. Isolation and partial characterization of follistatin: a single chain Mr 35000 monomeric protein that inhibits the release of follicle stimulating hormone. Proc Natl Acad Sci USA 1987; 84: 8282–6. 304. Robertson DM, Klein R, de Vos FL, et al. The isolation of polypeptides with FSH suppressing activity from bovine follicular fluid which are structurally different to inhibin. Biochem Biophys Res Commun 1987; 196: 388–95. 305. Shimonaka M, Inouye S, Shimasaki S, Ling N. Follistatin binds to both activin and inhibin through the common β-subunit. Endocrinology 1991; 128: 3313–15. 306. Ronghao LI, Philips DM, Mather JP. Activin promotes ovarian follicle development in vitro. Endocrinology 1995; 136: 849–56. 307. Sugino H, Nakamura T, Hasegawa Y, et al. Identification of a specific receptor for erythroid differentiation factor on follicular granulosa cell. J Biol Chem 1988; 263: 15249–52. 308. Feng ZM, Madigan MB, Chen CLC. Expression of type II activin receptor genes in the male and female reproductive tissues of the rat. Endocrinology 1993; 132: 2593–600.

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Oocyte in vitro maturation 309. Vanderstichele H, Delaey B, Winter JD, et al. Secretion of steroids, growth factors and cytokines by immortalized mouse granulosa cell lines. Biol Reprod 1994; 50: 1190–203. 310. Sadatsuki M, Tsutsumi O, Yamada R, Muramatsu M, Taketani Y. Local regulatory effects of activin A and follistatin on meiotic maturation of rat oocytes. Biochem Biophys Res Commun 1993; 196: 388–95. 311. Tsung-chieh JWU, Ming HJIH, Lai W, Yu-Jui YW. Expression of activin receptor II and IIB mRNA isoforms in mouse reproductive organs and oocytes. Mol Reprod Dev 1994; 38: 9–15. 312. Hulshof SCJ. Bovine preantral follicles and their development in vitro. Thesis, Utrecht University, The Netherlands, 1995. 313. Van Tol HTA, de Loos FAM, Vanderstichele HMJ, Bevers MM. Bovine activin A does not affect the in vitro maturation of bovine oocytes. Theriogenology 1994; 41: 673–9. 314. Stock AE, Woodruff TK, Smith LC. Effects of inhibin A and activin A during in vitro maturation of bovine oocytes in hormone- and serum-free medium. Biol Reprod 1997; 56: 1559–64. 315. Silva CC, Knight PG. Modulatory actions of activinA and follistatin on the developmental competence of in vitro matured bovine oocyte. Biol Reprod 1998; 58: 558–65. 316. Alak BM, Coskun S, Friedman CI, et al. Activin A stimulates meiotic maturation of human oocytes and modulates granulosa cell steroidogenesis in vitro. Fertil Steril 1998; 70: 1126–30. 317. Haidri AA, Miller IM, Gwatkin RBL. Culture of mouse oocytes in vitro, using a system without oil or protein. J Reprod Fertil 1971; 26: 409–11. 318. Gwatkin RBL, Haidri AA. Oxygen requirements for the maturation of hamster oocytes. J Reprod Fertil 1974; 37: 127–9. 319. Eppig JJ, Wigglesworth K. Factors affecting the developmental competence of mouse oocytes grown in vitro: oxygen concentration. Mol Reprod Dev 1995; 42: 447–56. 320. Cortvrindt RG, Smitz JE. Follicle culture in reproductive toxicology: a tool for in vitro testing of ovarian function? Hum Reprod Update 2002; 8: 243–54. 321. Hu Y, Betzendahl I, Cortvrindt R, Smitz J, Eichenlaub-Ritter U. Spindles and chromosomes in mouse oocytes from preantral follicle culture: effects of low O2 and ageing. Hum Reprod 2001; 16: 737–48. 322. Eppig JJ, O’Brien MJ, Pendola FL, Watanabe S. Factors affecting the developmental competence of mouse oocytes grown in vitro: follicle-stimulating hormone and insulin. Biol Reprod 1998; 59: 1445–53. 323. Gosden RG, Byatt-Smith JG. Oxygen concentration gradient across the ovarian follicular epithelium: model, predictions and implications. Hum Reprod 1986; 1: 65–8. 324. Hyttel P, Fair T, Callesen H, Greve T. Oocyte growth, capacitation and final maturation in cattle. Theriogenology 1997; 47: 23–32. 325. Wassarman PM. Oogenesis. In: Adashi EY, Rock JA, Rosenwacks Z, eds. Reproductive Endocrinology, Surgery and Technology. Philadelphia: LippincottRaven, 1996: 341–57.

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326. Decker C, Parker R. Mechanisms of mRNA degradation in eukaryotes. Trends Biochem Sci 1994; 19: 336–40. 327. Bachvarova R. A maternal tail of poly(A): the long and short of it. Cell 1992; 69: 895–7. 328. Van der Westerlaken LAJ, Van der Schans A, Eyestone WH, de Boer HA. Kinetics of first polar body extrusion and the effect of time of stripping of the cumulus and time of insemination on developmental competence of bovine oocytes. Theriogenology 1994; 42: 361–70. 329. Nimura S, Hosoe M. Changes in cortical granule distribution within bovine oocytes during maturation and fertilization in vitro. J Reprod Dev 1995; 41: 103–8. 330. Moor RM, Gandolfi F. Molecular and cellular changes associated with maturation and early development of sheep eggs. J Reprod Fertil 1987; 34: 55–69. 331. Combelles CM, Fissore RA, Albertini DF, Racowsky C. In vitro maturation of human oocytes and cumulus cells using a co-culture three-dimensional collagen gel system. Hum Reprod 2005; 20: 1349–58. 332. De Loos FAM, Zeinstra E, Bevers MM. Follicular wall maintains meiotic arrest in bovine oocytes cultured in vitro. Mol Reprod Dev 1994; 39: 162–5. 333. Bilodeau S, Fortier MA, Sirard MA. Effect of adenylate cyclase on meiotic resumption and cyclic AMP content of zona-free and cumulus-enclosed bovine oocytes in vitro. J Reprod Fertil 1993; 97: 5–11. 334. Lonergan P, Khatir H, Carolan C, Mermillod P. Bovine blastocyst production in vitro following inhibition of oocyte meiotic resumption for 24h. J Reprod Fertil 1997; 109: 355–65. 335. Mermillod P, Lonergan P, Carolan C, et al. Maturation ovocytaire in vitro chez les ruminants domestiques. Contracept Fertil Sex 1996; 24: 552–8. 336. Mermillod P, Tomanek M, Marchal R, Meijer L. High developmental competence of cattle oocytes maintained at the germinal vesicle stage for 24 hours in culture by specific inhibition of MPF kinase activity. Mol Reprod Dev 2000; 55: 89–95. 337. Kubelka M, Motlik J, Schultz RM, Pavlok A. Butyrolactone I reversibly inhibits meiotic maturation of bovine oocytes, without influencing chromosome condensation activity. Biol Reprod 2000; 62: 292–302. 338. Fulka A, Leibfried-Rutledge ML, First NL. Effect of 6dimethylaminopurine on germinal vesicle breakdown of bovine oocytes. Mol Reprod Dev 1991; 29: 379–84. 339. Nogueira D, Ron-El R, Friedler S, et al. Meiotic arrest in vitro by phosphodiesterase 3-inhibitor enhances maturation capacity of human oocytes and allows subsequent embryonic development. Biol Reprod 2006; 74: 177–84. 340. Nogueira D, Cortvrindt R, De Matos DG, Vanhoutte L, Smitz J. Effect of phosphodiesterase type 3 inhibitor on developmental competence of immature mouse oocytes in vitro. Biol Reprod 2003; 69: 2045–52. 341. Vanhoutte L, De Sutter P, Nogueira D, et al. Nuclear and cytoplasmic maturation of in vitro matured human oocytes after temporary nuclear arrest by phosphodiesterase 3-inhibitor. Hum Reprod 2007; 22: 1239–46. 342. Vanhoutte L, Nogueira D, Gerris J, Dhont M, De Sutter P. Effect of temporary nuclear arrest by phosphodiesterase 3-inhibitor on morphological and functional aspects of in vitro matured mouse oocytes. Mol Reprod Dev 2008; 75: 1021–130.

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10 Use of in vitro maturation in a clinical setting Anne-Maria Suikkari

What is in vitro maturation? The basic concept of in vitro maturation (IVM) of oocytes for IVF and embryo transfer (ET) in a clinical setting is a simple one. Immature oocytes are collected from small antral follicles before spontaneous ovulation, and the oocytes are then allowed to mature in vitro, after which routine in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and ET can be performed. IVM is based on the observations of Pincus and Enzmann in 1935 and Edwards in 1965 that oocytes undergo spontaneous nuclear maturation when removed from the follicular milieu.1,2 This alone does not guarantee full developmental competence of the oocytes, but these findings were the first cornerstones of the research of oocyte maturation. Further understanding of the complicated orchestration of nuclear and cytoplasmic maturation, resulting in a developmentally competent oocyte, is still limited in humans and there is a clear need for more basic research in animal models.3 Nevertheless, IVM has been practiced in a clinical setting for over a decade, but it is only recently when a larger body of studies have accumulated on the clinical outcomes.4– 7 The benefits of a clinical IVM program have been widely acknowledged. First and foremost, IVM is simple and safe for the woman, because of lack of hormone stimulation and the risk of ovarian hyperstimulation syndrome (OHSS). IVM may also be very cost-effective, depending on the health care and reimbursement systems of the country. As a result, clinical applications of IVM have evolved ahead of basic research, thereby raising many questions and perhaps also limitations to further development and success rates. In the course of developing a clinically useful IVM program, several protocols have been applied and not all have resulted in embryo transfer, making it difficult to compare the results in terms of healthy children born.3,7,8 In this chapter, IVM is defined as the primary intention to collect immature oocytes from hormonally unstimulated or minimally primed follicles to achieve a live birth; this is distinct from the so-called rescue IVM, in which immature oocytes are retrieved from fully stimulated follicles and matured in vitro.9 Today, no universal IVM protocol exists, and each

group has developed its own modifications to achieve the best results. There are differences in patient selection, hormonal priming, cycle monitoring, oocyte collection, and in vitro culture techniques between the groups.7,10 Even after evaluating the clinical outcomes of the various protocols, one cannot reach a singular recommendation for one good protocol. In this chapter, the advantages and possible disadvantages and the clinical outcomes of the different protocols are discussed.

Patient selection Choosing the right patients for IVM is an important part of a successful clinical program. IVM of human oocytes was primarily developed to make IVF safer and simpler for women with polycystic ovaries (PCOs) and high risk of OHSS using conventional stimulation protocols.5 Recently, the indications for IVM have been extended to other causes of infertility such as male factor and unexplained infertility.11–14 However, polycystic ovary syndrome (PCOS) is the most widely used indication for IVM. Several studies show that the number of oocytes obtained for IVM is higher in women with PCOS compared with non-PCO women.13,15,16 This is thought to result in better pregnancy rates. It is the antral follicle count rather than the diagnosis of PCOS itself which is an important predictive factor of the number of immature oocytes obtained from an unstimulated cycle and should be used as a main criterion to select patients for IVM.17 The use of IVM in women with regular cycles and normal ovaries is more controversial. One obvious indication is male factor infertility, when the woman does not require fertility drugs. Although IVM has been applied to IVF poor responders, it has mostly been used in younger patients with none or a few previous IVF attempts.11,13,18,19 The most important prognostic criterion for IVM seems to be the number of antral follicles in a baseline ultrasound scan.16 If the antral follicle count is less than five per ovary, IVM is not recommended.20 Thus, low ovarian reserve can be considered as a contraindication for IVM. The clinical experience thus far suggests that IVM is a first-line treatment rather than the last resort. Patients with

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unexplained infertility and repeatedly failed controlled ovarian hyperstimulation–intrauterine insemination (COH–IUI) cycles fulfilling the inclusion criteria should be offered IVM before COH–IVF.

Hormonal priming The initial purpose of IVM was to obtain immature oocytes from antral follicles without any prior hormone stimulation.5 However, as the pregnancy rates initially were low, there was a need to try to increase the number of good-quality oocytes available for IVM and subsequent IVF–ET. One approach was to use mild ovarian stimulation with gonadotropins.8 Theoretically, early follicular phase FSH/hMG (follicle-stimulating hormone/human menopausal gonadotropin) priming of the follicles could enhance oocyte maturation, increase the oocyte yield, and make the collection of the oocytes easier. As a result, various priming protocols have been studied, both in women with irregular cycles and PCOS and in women with regular cycles and normal ovaries.8,11,12,21–3 The results from these studies are conflicting. Although FSH priming may increase the number of oocytes collected, it does not seem to improve the pregnancy rate in regularly cycling non-PCO women.11,22 In a small study of PCOS women, no pregnancies were obtained in women with no FSH priming whereas seven clinical pregnancies (29%) were achieved in the FSHprimed group.20 On the other hand, Lin et al showed no difference in clinical pregnancies between PCOS women with or without FSH priming.23 It is, however, difficult to draw conclusions from these studies, because of differences in the priming protocols and culture conditions. In their studies, Mikkelsen et al used FSH 150 IU on cycle days 3–5, whereas Lin et al used both FSH (75 IU for 6 days) and hCG (human chorionic gonadotropin) priming.20,23 In a recent retrospective study by Son et al, the effect of follicle priming with either low-dose hMG or hCG was compared with nonprimed cycles in 56 PCOS women. They found more metaphase II oocytes at the time of immature oocyte retrieval and faster maturation time and blastocyst formation in patients primed with hCG.24 The main reason to use hCG priming in women with PCOS has been the finding that in vivo administered hCG enhanced the nuclear maturation of the oocytes.25 This observation was based on a prospective randomized study of 24 cycles in 17 patients, and the improved maturation rate was not reflected in the pregnancy outcome. In non-PCO women with regular cycles, hCG priming has not been shown to have any beneficial effect.13,16 However, these studies, like the majority of clinical studies on IVM, lacked sufficient power to draw statistically significant conclusions on the clinical outcomes. The mechanism by which the developmental potential of the oocyte originating from a small antral follicle would be enhanced by in vivo hCG priming is still unclear. At least in the

bovine the granulosa cells of <8 mm diameter seem to lack LH receptor expression.26 As shown by Son et al, some of the oocytes from hCG-primed ovaries are already mature at immature oocyte collection and require no further in vitro maturation.24 Whether these oocytes originate from small or large follicles is not known. Contrary to most groups, we at Väestöliitto Fertility Clinic in Helsinki have not used any FSH or hCG priming in PCOS or non-PCO patients in our IVM program over the last 6 years13,27 (Tables 10.1 and 10.2).

Cycle monitoring and timing of oocyte collection The management of an IVM cycle depends on whether it is an ovulatory or an anovulatory cycle. In women with PCOS the cycles are usually anovulatory without dominant follicle development, in which case the monitoring of the cycle and the timing of the oocyte pick-up are more flexible than in an ovulatory cycle.13,16 A baseline ultrasound examination is always necessary to exclude ovarian cysts and to count the antral follicles. Another ultrasonography a few days later is used to schedule for oocyte retrieval. The criteria for oocyte pick-up in anovulatory patients is the thickness of the endometrium, which should be >6 mm, although successful pregnancies have been reported with an endometrium as thin as 3 mm.28,29 Thus, the day of the cycle does not play a role in the timing of the oocyte pick-up in anovulatory cycles of PCOS women, which allows more flexibility in scheduling for the IVF laboratory and the clinic. In women with regular cycles and selection of the dominant follicle, the timing of the oocyte retrieval is more critical than in women with no dominant follicle selection. Based on the published literature, there is no clear consensus as to when is the best time to collect the immature oocytes. The prevalent understanding is that once the dominance of a follicle is established, the cohort follicles undergo atresia.11,30,31 However, based on animal studies, there is an undetermined period during which some of the small antral follicles contain early atretic but still developmentally competent oocytes.32 The aim is to retrieve the oocytes at a time when the cohort follicles are undergoing early atresia, just after the selection of the dominant follicle: i.e. not too early and not too late. As very little is known about the cellular events involved in recruitment and selection of follicles, no specific marker has yet been developed to determine the optimal time for immature oocyte collection. The clinical usefulness of the measurements of serum estradiol and inhibin A concentrations has still to be proven.20,33 Ultrasonography remains the best method to monitor the cycle for the timing of immature oocyte collection. How wide then is the window for oocyte retrieval? Based on the published literature, the size

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Use of IVM in a clinical setting Table 10.1

Cha50 Cha52 Chian

25

Child16

Child

28

Mikkelsen

20

Lin23

Le Du

35

SöderströmAnttila13

Son

51

Clinical outcome of IVM cycles in women with PCOS or PCO

No. of cycles

Reference

157

Priming

Mean no. of oocytes retrieved

Percent maturation (time)

Percent fertilization Percent Mean no. Percent (type of cleaved of embryos PR Percent Percent No. of insemination) embryos transferred per ET IR Sab live births

94

None

13.6

62 (48)

68 (ICSI)

88

4.9

27.1

6.9

20

20

203

None

15.5

NA

NA

NA

5.0

21.9

5.5

37

24

13

hCG

7.8

78/85 (24/48)

91 (ICSI)

95

2.8

38.5

16.6

40

3

11

None

7.4

5/68 (24/48)

84 (ICSI)

96

2.5

27.3

14.8

0

3

53 (PCO) hCG

10.0

76 (48)

76 (ICSI)

95

3.3

23.1

8.9

40

9

68 (PCOS) hCG

11.3

77 (48)

79 (ICSI)

91

3.2

29.9

9.6

52

10

9.5

26

17

107

hCG

10.3

76 (48)

78 (ICSI)

74

2.5

21.5

12

None

6.8

44 (24)

69 (ICSI)

64

1.7

0

0

0

0

24

FSH

6.5

59 (24)

70 (ICSI)

56

1.8

33.0

21.6

63

3

35

FSH + hCG

21.9

43/77 (24/48)

76 (ICSI)

89

3.8

31.4

9.7

13

21

33

hCG

23.1

39/72 (24/48)

70 (ICSI)

88

3.8

36.4

11.3

45

hCG

11.4

54/63 (24/48)

70 (ICSI)

96

2.5

22.5

10.9

40

6

20 (PCO) None

9.3

55 (30)

35 (IVF) 72 (ICSI)

86 62

1.7 2.0

22.2 0

13.3 0

0 0

2 0

28 (PCOS) None

14.3

58 (30)

44 (IVF)

83

1.7

52.9

34.5

33

6

78 (ICSI)

71

1.8

22.2

12.5

50

1

80 (ICSI)

NA

4.0

33.2

12.2

NA

NA

521

hCG

16.2

75

PR, pregnancy rate; ET, embryo transfer; IR, implantation rate; Sab, spontaneous abortion; NA, not available; hCG, human chorionic gonadotropin; FSH, follicle-stimulating hormone; PCO, polycystic ovary; PCOS, polycystic ovary syndrome; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization. Data rearranged and updated from Jurema and Nogueira.7

of the leading follicle with successful outcome varies between less than 10 mm to 14 mm.6,11,13,34,35 When the leading follicle has reached 13 mm in diameter, significantly less oocytes were collected, matured, and fertilized, and fewer embryos transferred, than in cycles with a leading follicle <13 mm, whereas others have recommended cancellation of the cycle if the leading follicle is >10 mm.34–36 All these studies are observational and no prospective data are available. Along with the size of the leading follicle, one needs to consider the thickness of the endometrium, which has to be sufficient to sustain the pregnancy, although there are no prospective studies to evaluate the impact of endometrial thickness on the clinical outcome of IVM. The concept of developmentally challenged cohort follicles in the presence of a dominant follicle has been debated by Chian et al, who have retrieved several competent oocytes along with an in vivo matured oocyte.37 Recent publications have also suggested that the combination of natural cycle IVF and IVM may increase the efficacy of the cycle.37–39 Oocytes from the dominant follicle as small as 12–14 mm diameter can be retrieved 36 hours after an hCG injection. Along

with that mature oocyte from the leading follicle, immature oocytes from the cohort follicles are also retrieved and matured in vitro. Pooled embryos are then transferred 3–4 days later.38

Oocyte collection procedure The immature oocyte collection technique differs from the aspiration of large mature follicles. Based on our clinical experience and the published literature, the key elements in immature oocyte collection procedure are: • • •

a lower aspiration pressure than used in conventional egg retrieval in an IVF cycle immobilization of the ovary by pressure or special holding needle filtering of the aspirate to recover the immature oocytes.7

The aspiration pressure for IVM should be lower than that used in the conventional oocyte retrieval. The range for optimal aspiration pressure is probably wide and depends on the equipment and the type of needle

10 10 5 7 87 132 56 63 91 100 Total 207a

Mikkelsen11 3.7 4.0 4.2 2.4 6.1 3.8 5.1 9.0 6.3 6.5 4.7

76 (36) 85 (36) 71 (48) 71 (48) 60 (28–36) 60 (28–36) 78 (48) 41/72/74 (24/48/56) 67 (30) 55 (30) 56 (29)

62 (ICSI) 65 (ICSI) 61 (ICSI) 61 (ICSI) 77 (ICSI) 73 (ICSI) 73 (ICSI) 73 (IVF, ICSI) 36 (IVF) 67 (ICSI) 67 (ICSI)

54 62 48 59 87 87 93 89 85 86 NA

Percent cleaved embryos 1.8 1.9 1.4 1.1 2 NA 2.6 3.6 1.4 1.5 1.9

Mean no. of embryos

33.3 22.2 20.0 0 NA 17.4 18.0 17.6 31.0 21.0 15.2

Percent PR per ET

18.8 11.8 14.3 0` 8.8 NA 1.5 6.5 22.6 20.0 8.8

Percent IR transferred

9 12 1 6 12 15 7

1

0 19 20 50 33 33 17 24

4

No. of live births 20

Percent Sab

PR, pregnancy rate; ET, embryo transfer; IR, implantation rate; Sab, spontaneous abortion; NA, not available; FSH, follicle-stimulating hormone; hCG, human chorionic gonadotropin; PCO, polycystic ovary; PCOS, polycystic ovary syndrome; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization. a The data are given for all cycles and do not separate between PCOS and non-PCO cycles. Data rearranged and updated from Jurema and Nogueira.7

None FSH × 3 FSH × 3 FSH × 6 None None hCG None None None 169 cycles None, 38 PCOS FSH × 3

Priming

Percent fertilization (type of insemination)

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Mikkelsen33 Mikkelsen20 Child16 Yoon18 SöderströmAnttila13 a Dal Canto14

No. of cycles

Reference

Percent maturation (time)

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Mean no. of oocytes retrieved

Clinical outcome of IVM cycles in women with normal ovaries and regular cycles

158

Table 10.2

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used.6,40 In our program, we use a 17-gauge needle and 30–50 mmHg pressure for IVM and 90–100 mmHg for IVF. Immobilization of the ovary by external pressure may be necessary, because the unstimulated ovary is smaller and potentially more mobile than the stimulated ovary. Alternatively, a special double-lumen holding needle can be used.40 As the immature oocytes do not have a large, expanded cumulus cell complex, they are more difficult to see in the aspirate, which contains more blood than the follicular fluid from large follicles. Therefore, the aspirate is usually filtered, after which the immature oocytes can easily be recovered.5 This means that there is a longer time interval between the oocyte collection and the time the total number of retrieved oocytes is known compared with the oocyte pick-up procedure in conventional IVF. Based on the clinical experience of our program, flushing of the follicles does not improve the total oocyte yield. The average number of oocytes expected to be retrieved from polycystic ovaries is around 14 and in regular cycles from non-PCO ovaries varies between 4 and 9 (see Tables 10.1 and 10.2) Compared with conventional IVF, there are less surplus embryos available for cryopreservation in IVM cycles. However, the oocyte collection is easy enough to be repeated if necessary (Fig 10.1).

In vitro maturation followed by insemination or microinjection In vitro maturation of oocytes means that immature oocytes removed from small antral follicles are allowed to mature in vitro, which takes about 24–48 hours. The oocyte reaches its full size at the time of antrum formation, but the intracellular process of maturation continues until ovulation. Very little is known about the molecular events required in this complex process. Undoubtedly, gonadotropins are the major components directing folliculogenesis, but recent studies have shown that oocytes may independently regulate the rate of follicular growth.41,42 Since the oocytes obtained at immature oocyte collection come from follicles in different developmental stages, it has not been possible to design one culture medium to support oocyte maturation in all stages. Owing to the oocyte collection technique, it has not been possible to identify the exact follicle size from which a specific oocyte is retrieved. Nevertheless, numerous studies have been conducted to define optimal culture conditions.8,15,25,34,43–45 The culture medium has been supplemented with a range of gonadotropins, growth factors, steroids, serum, and proteins from various sources. Many laboratories have used their inhouse-developed culture media and co-culture systems and a few commercial IVM culture medias are currently available.46,47 The in vitro maturation times used have varied between 28 and 52 hours.8,15,25,43 The oocytes that mature within 30 hours after retrieval

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Fig 10.1 Oocyte collection procedure.

have been shown to be developmentally more potent than the ones maturing later.47 It is difficult to draw definite conclusions on the optimal maturation time from these studies because of the variations in culture conditions and study endpoints. For practical purposes a maturation time between 30 and 36 hours is mostly used.7 The quality of the oocytes at the time of immature oocyte collection seems to be the most important singular factor affecting the maturation rate. Oocytes surrounded by intact cumulus cells show better maturation rates than oocytes with scanty or no cumulus cells.15,34,43,48 ICSI has been thought to be necessary for fertilization of in vitro matured oocytes even in conditions where sperm parameters are not impaired. Another reason for using ICSI instead of insemination after IVM is that the assessment of oocyte maturity is more difficult with intact cumulus cells. However, insemination may be a good alternative after IVM when the sperm is suitable for IVF. Our study showed a better overall clinical pregnancy rate per embryo transfer after IVM–IVF (35%) than IVM–ICSI (20%).13 It can be speculated that the intact cumulus cells somehow enhance cytoplasmic maturation and developmental competence of the oocytes.

Embryo transfer and endometrial preparation One of the challenges of IVM is to prepare the uterus for implantation in only a few days between the oocyte retrieval and embryo transfer. Because immature oocytes are usually retrieved before the dominant follicle develops, the endometrium is exposed to relatively low levels of estradiol by the time of oocyte pick-up. As a result, there is a dyssynchrony between the phase of the endometrium and the cleavage-stage embryo. Therefore, adequate preparation of the endometrium

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is of crucial importance in an IVM cycle because of the absent luteinizing hormone (LH) surge at the time of oocyte collection. The most commonly used protocol for endometrial preparation consists of estradiol valerate 2–4 mg/day from the day of IOC and progesterone intravaginal suppositories 200–600 mg/day started 48 hours later at the time of microinjection or insemination.5,6,11,13,25 The start of progesterone administration at the time of insemination coincides with the rise of serum progesterone after ovulation in a natural cycle. Prospective studies comparing different protocols for optimal endometrial preparation in IVM cycles are lacking. In case of pregnancy, it is important to continue the hormone supplementation long enough as there is no endogenous pituitary or corpus luteum support. Most programs continue hormone treatment until 9 weeks of gestation.13,16,49

Clinical outcome and follow-up of children The clinical outcomes of IVM have continued to improve after the modest results of the early studies.4,5,10,11,13,48,51 In other larger studies, the average pregnancy rates in women with PCOS have been reported as between 22 and 30%, and in women with normal ovaries as between 18 and 30% (Tables 10.1 and 10.2). The implantation rates, however, have been modest, leading to a transfer of several embryos at a time. The highest implantation rates published thus far have been 35% for a small group of PCOS patients using insemination of the in vitro matured oocytes, allowing the transfer of only one or two embryos at a time.13 Recently, a pregnancy rate per embryo transfer as high as 52% and an implantation rate of 27% was reported after IVM and blastocyst transfers in highly selected cases.51 There is no data available on the results of frozen–thawed IVM embryo transfers. In our program, significantly fewer IVM (17%) than IVF (65%) cycles include cryopreservation of surplus embryos and the pregnancy rate after frozen–thaw IVM cycle is 7% compared with 27% after IVF.27 To evaluate the true efficacy of IVM compared to conventional IVF, the cumulative delivery or live birth rates of two or three IVM cycles should be compared to one IVF cycle with the transfer of all fresh and cryopreserved and thawed embryos created from one oocyte collection. One of the most important challenges of IVM is the high pregnancy wastage. The miscarriage rate in IVM pregnancies, varying between 25 and 57%, has been higher than that usually published for conventional IVF (Tables 10.1 and 10.2). There are, however, no adequately controlled studies available. Some centers have also observed no difference in miscarriage rates between IVM and conventional IVF in PCOS patients.52 The reasons for increased miscarriage rate could be related to patient population, embryo quality, or

endometrial preparation. The time for hormonal preparation of the endometrium before ET is short, and experience from oocyte donation cycles has shown a higher miscarriage rate after a short estrogen priming compared with a long priming.53 It has been estimated that approximately 1300 IVM babies have been born worldwide (HI Nielsen, pers comm), but the birth and perinatal outcome of only some 400 babies have been reported in the literature.37,49,52,54,55 Reports on obstetric and perinatal outcome of IVM pregnancies show very few complications.49,52,54,55 Prematurity occurs in 4–13% of the pregnancies, which is no different from spontaneous pregnancies. This is thought to be associated with fewer multiple pregnancies compared with standard IVF treatments.54 The birth weight of IVM infants has also been within normal range.49,51,52,54,55 So far there are only two reports on the further development of IVM children, reporting normal neurological development at 2 years of age.54,55

Summary IVM has been introduced for clinical use in several IVF programs worldwide. Despite the good results reported by some clinics, IVM has not yet become a mainstream assisted reproduction technology. The most important reason for this is the lower chance of a live birth per treatment compared with conventional IVF. Several aspects of the clinical IVM need to be improved in order to give IVM the place among assisted reproduction technologies it deserves. Knowledge of the molecular mechanisms of oocyte maturation are still insufficient and the current culture systems designed to mimic the paracrine and endocrine events of the growing follicle–oocyte complex are not yet optimal. Until these systems are improved, the efficacy of IVM will remain lower than that of conventional IVF. Another critical factor for successful IVM is patient selection. The indications for IVM need further clarification. IVM is well documented in patients with increased risk for OHSS, high antral follicle count, and PCOS/PCO, whereas the role of IVM in couples with tubal, unexplained, and male factor infertility remains to be defined. There is also room for improvement in the protocols for endometrial preparation, to better support the early pregnancy and potentially decrease the risk of miscarriage. And last but not least, there is the question of safety of IVM. Ever since ICSI was introduced to human use without extensive basic research on animal models, we have been alerted to possible risks of assisted reproductive technologies. Although the information on the follow-up of the children accumulated so far has been reassuring, there still remains the possibility of some epigenetic changes that may affect the individuals later in life.

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References 1. Pincus G, Enzmann EV. The comparative behaviour of mammalian eggs in vivo and in vitro. J Exp Med 1935; 62: 665–75. 2. Edwards RG. Maturation in vitro of human ovarian oocytes. Lancet 1965; 2: 926–9. 3. Smitz J, Noguiera D, Vanhoutte L, de Matos DG, Cortvrindt R. Oocyte in vitro maturation. In: Gardner DK, Weissman A, Howles CM, Shoham Z, eds. Textbook of Assisted Reproductive Techniques: Laboratory and Clinical Perspectives, 2nd edn. London: Taylor & Francis, 2004: 125–61. 4. Cha KY, Koo JJ, Koo JJ, et al. Pregnancy after in vitro fertilization of human follicular oocytes collected from nonstimulated cycles, their culture in vitro and their transfer in a donor oocyte program. Fertil Steril 1991; 55: 109–13. 5. Trounson A, Wood C, Kausche A. In vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients. Fertil Steril 1994; 62: 353–62. 6. Barnes FL, Crombie A, Gardner DK, et al. Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching. Hum Reprod 1995; 10: 3243–7. 7. Jurema MW, Nogueira D. In vitro maturation of human oocytes for assisted reproduction. Fertil Steril 2006; 86: 1277–91. 8. Wynn P, Picton HM, Krapez JA, et al. Pretreatment with follicle stimulating hormone promotes the number of human oocytes reaching metaphase II by in-vitro maturation. Hum Reprod 1998; 13: 3132–8. 9. De Vos A, Van de Velde H, Joris H, Van Steirteghem A. In-vitro matured metaphase-I oocytes have a lower fertilization rate but similar embryo quality as mature metaphase-II oocytes after intracytoplasmic sperm injection. Hum Reprod 1999; 14: 1859–63. 10. Chian RC, Buckett WM, Tan S-L. In-vitro maturation of human oocytes. Reprod Biomed Online 2004; 8: 148–66. 11. Mikkelsen AL, Smith SD, Lindenberg S. In-vitro maturation of human oocytes from regularly menstruating women may be successful without follicle stimulating hormone priming. Hum Reprod 1999; 14: 1847–51. 12. Hreinsson J, Rosenlund B, Fridén B, et al. Recombinant LH is as effective as recombinant hCG in promoting oocyte maturation in a clinical in-vitro maturation programme: a randomized study. Hum Reprod 2003; 18: 2131–6. 13. Söderström-Anttila V, Mäkinen S, Tuuri T, Suikkari AM. Favourable pregnancy results with insemination of in vitro matured oocytes from unstimulated patients. Hum Reprod 2005; 20: 1534–40. 14. Dal Canto MB, Mignini Renzini M, Brambillisca F, et al. IVM – the first choice for IVF in Italy. Reprod Biomed Online 2006; 13: 159–65. 15. Barnes FL, Kausche A, Tiglias J, et al. Production of embryos from in vitro-matured primary human oocytes. Fertil Steril 1996; 65: 1151–6. 16. Child TJ, Abdul-Jalil AK, Gulekli B, Tan SL. In vitro maturation and fertilization of oocytes from unstimulated normal ovaries, polycystic ovaries, and

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women with polycystic ovary syndrome. Fertil Steril 2001; 76: 936–42. Tan SL, Child TJ, Gulekli B. In vitro maturation and fertilization of oocytes from unstimulated ovaries: predicting the number of immature oocytes retrieved by early follicular ultrasonography. Am J Obstet Gynecol 2002; 186: 684–9. Yoon HG, Yoon SH, Son WY, et al. Pregnancies resulting from in vitro matured oocytes collected from women with regular menstrual cycle. J Assist Reprod Genet 2001; 18: 325–9. Liu J, Lu G, Qian Y, Mao Y, Ding W. Pregnancies and births achieved from in vitro matured oocytes retrieved from poor responders undergoing stimulation in in vitro fertilization cycles. Fertil Steril 2003; 80: 447–9. Mikkelsen AL, Lindenberg S. Benefit of FSH priming of women with PCOS to the in vitro maturation procedure and the outcome: a randomized prospective study. Reproduction 2001; 122: 587–92. Trounson A, Anderiesz C, Jones GM, et al. Oocyte maturation. Hum Reprod 1998; 13(Suppl): 52–70. Suikkari A-M, Tulppala M, Tuuri T, Hovatta O, Barnes F. Luteal phase start of low-dose FSH priming of follicles results in an efficient recovery, maturation and fertilization of immature human oocytes. Hum Reprod 2000; 15: 747–51. Lin YH, Hwang JL, Huang LW, et al. Combination of FSH priming and hCG priming for in-vitro maturation of human oocytes. Hum Reprod 2003; 18: 1632–6. Son WY, Yoon JH, Lim JH. Effect of gonadotrophin priming on in-vitro maturation of oocytes collected from women at risk of OHSS. Reprod Biomed Online 2006; 13: 340–8. Chian RC, Buckett WM, Tulandi T, Tan SL. Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Hum Reprod 2000; 15: 165–70. Nogueira MFG, Buratini J Jr, Price CA, et al. Expression of LH receptor mRNA splice variants in bovine granulosa cells: changes with follicle size and regulation by FSH in vitro. Mol Reprod Dev 2007; 74: 680–6. Suikkari A-M, Söderström-Anttila V. In vitro maturation of eggs – Is it really useful? In: Arulkumaran S, Norman RJ, eds. Best Practice & Research, Clinical Obstetrics & Gynecology, Controversies in Assisted Reproductive Technologies, Vol. 21. Amsterdam: Elsevier, 2007: 145–55. Child TJ, Phillips SJ, Abdul-Jalil AK, Gulekli B, Tan SL. A comparison of in vitro maturation and in vitro fertilization for women with polycystic ovaries. Obstet Gynecol 2002; 100: 665–70. Suikkari A-M. Endometrial preparation for IVM. In: Tan SL, Buckett W, Chian RC, eds. In Vitro Maturation of Human Oocytes. London: Informa, 2006: 263–72. Anderiesz C, Trounson AO. The effect of testosterone on the maturation and developmental capacity of murine oocytes in vitro. Hum Reprod 1995; 10: 2377–81. Baker SJ, Spears N. The role of intra-ovarian interactions in the regulation of dominant follicle. Hum Reprod Update 1999; 5: 153–65.

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32. Barnes FL, Sirard MA. Oocyte maturation. Semin Reprod Med 2000; 18: 123–31. 33. Mikkelsen AL, Smith S, Lindenberg S. Impact of oestradiol and inhibin A concentrations on pregnancy rate in in-vitro oocyte maturation. Hum Reprod 2000; 15: 1685–90. 34. Cobo AC, Requena A, Neuspiller F, et al. Maturation in vitro of human oocytes from unstimulated cycles: selection of the optimal day for ovum retrieval based on follicular size. Hum Reprod 1999; 14: 1864–8. 35. Le Du A, Kadoch IJ, Bourcigaux N, et al. In vitro oocyte maturation for the treatment of infertility associated with polycystic ovarian syndrome: the French experience. Hum Reprod 2005; 20: 420–4. 36. Russell JB. Immature oocyte retrieval with in-vitro oocyte maturation. Curr Opin Obstet Gynecol 1999; 11: 289–96. 37. Chian RC, Buckett WM, Abdul-Jalil AK, et al. Natural-cycle in vitro fertilization combined with in vitro maturation of immature oocytes is a potential approach in infertility treatment. Fertil Steril 2004; 82: 1675–8. 38. Lim J-H, Yang S-H, Chian RC. New alternative to infertility treatment for women without ovarian stimulation. Reprod Biomed Online 2007; 14: 547–9. 39. Kadoch IJ, Fanchin R, Frydman N, Le Du A, Frydman R. Controlled natural cycle IVF: a novel approach for a dominant follicle during an in-vitro maturation cycle. Reprod Biomed Online 2007; 14: 598–601. 40. Hashimoto S, Fukuda A, Murata Y, et al. Effect of aspiration vacuum on the developmental competence of immature human oocytes retrieved using a 20-gauge needle. Reprod Biomed Online 2007; 14: 444–9. 41. Eppig JJ. Oocyte control of ovarian follicular development and function in mammals. Reproduction 2001; 122: 829–38. 42. Thomas FH, Walters KA, Telfer EE. How to make a good oocyte: an update on in-vitro models to study follicle regulation. Hum Reprod Update 2003; 9: 541–55. 43. Goud PT, Goud AP, Qian C, et al. In-vitro maturation of human germinal vesicle stage oocytes: role of cumulus cells and epidermal growth factor in the culture medium. Hum Reprod 1998; 13: 1638–44.

44. Moor RM, Dai Y, Lee C, Fulka J Jr. Oocyte maturation and embryonic failure. Hum Reprod Update 1998; 4: 223–36. 45. Smith SD, Mikkelsen AL, Lindenberg S. Development of human oocytes matured in vitro for 28 or 36 hours. Fertil Steril 2000; 3: 541–4. 46. Hwu YM, Lee RK, Chen CP, et al. Development of hatching blastocysts from immature human oocytes following in-vitro maturation and fertilization using a co-culture system. Hum Reprod 1998; 13: 1916–21. 47. Son WY, Le SY, Lim JH. Fertilization, cleavage and blastocyst development according to the maturation timing of oocytes in in vitro maturation cycles. Hum Reprod 2005; 20: 3204–7. 48. Russell JB, Knezevich KM, Fabian KF, Dickson JA. Unstimulated immature oocyte retrieval: early versus midfollicular endometrial priming. Fertil Steril 1997; 67: 616–20. 49. Mikkelsen AL. Strategies in in-vitro maturation and their clinical outcome. Reprod Biomed Online 2005; 10: 593–9. 50. Cha KY, Han SY, Chung HM, et al. Pregnancies and deliveries after in vitro maturation culture followed by in vitro fertilization and embryo transfer without stimulation in women with polycystic ovary syndrome. Fertil Steril 2000; 73: 978–83. 51. Son WY, Le SY, Yoon JH, Lim JH. Pregnancies and deliveries after transfer of human blastocysts derived from in vitro matured oocytes in in vitro maturation cycles. Fertil Steril 2007; 87: 1491–3. 52. Cha KY, Chung HM, Lee DR, et al. Obstetric outcome of patients with polycystic ovary syndrome treated by in vitro maturation and in vitro fertilization– embryo transfer. Fertil Steril 2005; 83: 1461–5. 53. Navot D, Bergh PA, Williams M, et al. An insight into early reproductive processes through the in vivo model of ovum donation. J Clin Endocrinol Metab 1991; 72: 408–14. 54. Söderström-Anttila V, Salokorpi T, Pihlaja M, Serenius-Sirve S, Suikkari AM. Obstetric and perinatal outcome and preliminary results of development of children born after in vitro maturation of oocytes. Hum Reprod 2006; 21: 1508–13. 55. Shu-Chi M, Jiann-Loung H, Yu-Hung L, et al. Growth and development of children conceived by in-vitro maturation of human oocytes. Early Hum Dev 2006; 82: 677–82.

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11 Equipment and general technical aspects of micromanipulation of gametes and embryos Frank L Barnes

Introduction Over the past 20 years micromanipulation has increased in significance in the livestock and human assisted reproductive technologies (ART) laboratories. Applications of micromanipulation include embryo bisection for embryo twinning,1 the production of chimeras to investigate cell fate and development,2 nuclear transfer to investigate nuclear equivalence,3 pronuclear DNA injection to establish transgenic animals,4 blastomere biopsy for the diagnosis of genetic disease,5 intracytoplasmic sperm injection (ICSI) for the treatment of infertility,6 and cytoplasmic transfer to investigate and improve embryo development.7,8 While all of these procedures have unique characteristics, they all share some fundamental components. This chapter attempts to provide some insights into the general principles of micromanipulation as recorded from my own experiences.

Principle Micromanipulation of ova refers to the reduction and translation of coarse hand movement to microscopic movement at the level of the egg or embryo. There are five critical pieces of a good micromanipulation system: an inverted microscope of sufficient magnification to visualize clearly the microsurgery to be attempted; a micromanipulator of sufficient refinement to provide smooth translation of movement; microscopic glass tools of appropriate design to effect the surgical procedure; a stereomicroscope to prepare eggs and embryos for manipulation; and appropriate environmental control to maintain the temperature and atmosphere as may be required.

Handling conditions Air quality and temperature As with any experimental or clinical procedure, dayto-day variation should be minimized, particularly if

the product of the procedure is to survive throughout subsequent development. Room conditions should be standardized for temperature, particle count, and humidity if possible. Eggs and embryos prefer a warm and moist environment; therefore, maintenance of the laboratory at 25°C with a clean room status of Class 100–1000 and a humidity of 35–45% is recommended. When conditions are not constant, variation in results can occur, as has been experienced with cloning of cattle embryos. Bovine oocytes are extremely sensitive to temperature fluctuation and can activate when chilled.9 The timing of activation can have a significant effect on the subsequent development of an embryo clone.10 The meiotic spindle of the mammalian egg is temperature-sensitive, and manipulation of human oocytes should be performed at 37°C to prevent chromosome disassociation and subsequent aneuploidy. The manner in which a manipulation plate is set up may have a similar impact on subsequent embryo development. Thirty-millimeter plates with 2–3 ml of medium without oil overlay will remain 3–5°C cooler than the controlling heat source, owing to evaporation. When there is no heat source a plate will cool precipitously below room temperature within 5 minutes. Alternatively, microdrops under oil can provide a very stable environment, providing there is a constant heat source, such as a heated microscope stage with very little thermal cycling. The manipulation plate should be designed to handle only a few ova (4– 8) to reduce handling times and exposure to room conditions. Recent studies have demonstrated that microtubule depolymerization may occur during the ICSI procedure. Microtubule depolymerization occurs as a result of specimen cooling. The cooling comes from the room temperature objective and the cool air draft from the microscope stage opening.11 New products such as the Tokai Hit Thermo Plate (Zander Medical Supply) are emerging, which eliminate the stage opening and reduce the cooling effect of the objective. Time will tell if this observation leads to improved ICSI outcomes.

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Media The ionic formulation of handling media can also affect the developmental outcome of an experiment or procedure. Moving eggs and embryos from a complete medium such as Ham’s F10 or TCM-199 into Dulbecco’s phosphate-buffered saline (PBS), a common manipulation medium, can elicit calcium movement within the cell and ultimately affect development.12 Similarly, medium osmolality is an important parameter to consider when manipulating embryos. Current human embryo culture media range in osmolality from 260 to 285 mosmol. At first glance this is seemingly of little significance, but changes of this magnitude can lead to visible swelling and shrinking of oocytes or blastomeres. In situations where a cell membrane is breached by the micromanipulation process, such as ICSI, this may lead to increased cell lyses. It is always preferable to manipulate eggs and embryos in a medium that maintains a similar salt balance while keeping temperature constant from incubation to manipulation.

Fig 11.1 Narishige micromanipulator (left arm) combines motorized coarse adjustment (background) with hydraulic fine adjustment (foreground).

Equipment and materials Manipulators The goal of a good micromanipulation system is the efficient, smooth, and confident translation of hand movement to the clearly visualized specimen. Exaggerated hand movement high above the bench top takes time, and the amount of time spent performing micromanipulation potentially exposes your specimens to room conditions that may affect development. There are two basic types of manipulation systems today, motorized and mechanical. I have not used the manipulators by all vendors, and the discussion provided is not intended as an endorsement but rather to point out some of the pros and cons of each system type. Motorized systems have come a long way since their introduction into the market, and I have recently tested a completely motorized system (Eppendorf and the like) and found their action to be smooth and exact. However, we routinely use the Narishige brand of manipulators on most of our ICSI workstations. These set-ups have a blend of motorized coarse movement with joystick and hydraulic fine movement translated through a separate joystick (Fig 11.1). This system has the advantage that the joysticks are separate from the microscope and thus do not cause any movement of the specimen during manipulation (Fig 11.2). Whether performing microinjection or blastomere biopsy the hydraulic joystick offers good range of motion across a 200–400× field and very smooth movement. Moreover, it is nice to be able to raise and lower tools without dramatic hand movement from the bench top (Fig 11.2). Hand position on the bench top is very comfortable when using the ‘drop down’ joysticks. A disadvantage of this system is the

Fig 11.2 Narishige coarse and fine adjustment joysticks are separate from the microscope and prevent operator-induced vibration of the specimen on the microscope stage. Note the position of the screw-actuated syringe (SAS) and the joysticks, which allows even horizontal movement of the left hand to effect the positioning of the specimen and manipulation. The SAS tool chuck is under the control of the right-handed joystick and allows positioning of the glass microtool while the left hand performs the aspiration or injection.

hydraulic lines (Fig 11.1). If pinched in some way, it may be impossible to fix on a location. Additionally, these systems are not very portable, and require a considerable amount of time to assemble and disassemble. Research Instruments (RI) produce completely mechanical systems. The RI system attaches to the microscope, there are no lines or cords or plugs to deal with, and it provides a very clean and neat workstation. The coarse alignment of the manipulator is adjusted with joysticks that protrude upright from the suspended arms off each side of the microscope (Fig 11.3). The fine-movement joysticks hang down from the suspended arms. They have good threedimensional movement across a 200–400× field; the joysticks are well oriented to the focus knobs of an inverted microscope. The RI system, once set, requires

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almost no coarse adjustment. There are levers on each side of the manipulator above the microscope stage that allow the microtools to be raised and lowered within a fraction of an inch of the bottom of the manipulation plate (Fig 11.3). This manipulation system can be moved very easily without disassembly. The disadvantage is that you must move your hands from the bench top to a position above the microscope stage to raise and lower glass tools. While this is seldom a problem, the ‘drop down’ joysticks can translate some hand vibration to the manipulation plate and specimen.

Optics The optics employed should be sufficient to visualize clearly the ova and any components thereof; some type of contrast adjustment is often preferable to bright-field conditions. Micromanipulation is generally conducted using an inverted microscope between 200 and 400×, and therefore 10, 20, and 40× objectives are essential for set-up and execution of the procedure. Objective focusing rings make it easier to get a crisp par focal adjustment of the scope. Phase contrast, differential interference contrast, or Hoffman modulation contrast can enhance the specimen image; Hoffman contrast is the popular choice for ICSI where plastic dishes are used. Easy, unobstructed movement between the focus adjustment and the objectives is desirable if a change in the objectives during manipulation is required. Look for inverted microscopes that have a 1.5–2.0× slider just beyond the focus adjustment on the right side of the microscope; you can set the Hoffman condenser to 20 and the objective to 20× at the start of your manipulation session and, with very little hand movement, the magnification increases 1.5× (300×) by simply pulling out the slider.

Stereomicroscope Specimens should be quickly moved from the culture plate into the micromanipulation plate, manipulated, and then back again, to reduce the time held at room atmosphere. A stereomicroscope with a magnification range of 10–100× can be valuable for placing specimens into the micromanipulation plate. The ‘set-up’ station should be close to the micromanipulation workstation to avoid unnecessary chair movements (Fig 11.4).

Heated stages Heated stages are required to keep specimens at 37°C. There should be a heated stage on the micromanipulation microscope and on the set-up stereomicroscope. Be aware of hotspots on the stage that may exceed the critical threshold of specimens (greater than 38°C). Thermal cycling can be a problem with some stages; to achieve a 37°C mean temperature the stage may actually cycle between 36 and 38°C.

Fig 11.3 Mechanical micromanipulator from Research Instruments (RI) suspends from the objective pillar of the microscope. Suspension of the joysticks from the mounting arms provides a clean workstation but allows operator-induced vibration of the specimen if bumped. The small lever on the large upright pillar (center of photo) allows the tool chuck and glass microtools to be raised and lowered easily when changing manipulation plates.

Fig 11.4 Set-up station for micromanipulation should have some type of environmental control for the manipulation and culture plates and be in close proximity to the manipulation microscope. Shown are a stereomicroscope with heated stage and a bench-top incubator with temperature and humidified gas atmosphere control.

Bench-top incubators When performing micromanipulation it is important to prevent cellular stress of your specimen as much as possible. Therefore, establishing constant conditions for manipulation and culture (temperature, humidity, and gas atmosphere) can improve survival after manipulation. Bench-top incubation systems are important because: first, all of the specimens may be placed next to the technician at the micromanipulation station so that manipulation plates can be easily and quickly switched, and thus reduce the time required to perform a micromanipulation procedure; and secondly, a mixed gas atmosphere with high humidity allows culture in bicarbonate-buffered

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Fig 11.5 The GE-80 from K-Systems provides temperature regulation and can be set up with humidified mixed gas atmosphere. This system is convenient for short-term incubation at the manipulation bench.

media and limits exposure to 4-(2-hydroxyethyl)-1piperazine-ethanesulfonic acid (HEPES) or phosphate-buffered media to the time that specimens are in the micromanipulation plate. It is important to remember to pass the gas mixture through a water bubbler so the gas atmosphere is stable; anyone who has had the humidity pan in an incubator go dry should be able to aknowledge that one cannot maintain gas atmosphere without humidity. Also, humidity helps to prevent the evaporation of media when working without an oil overlay. Evaporation can decrease the temperature and solution osmolality. There are a variety of bench-top containment systems available that control temperature, humidity, and mixed gas atmosphere (Fig 11.5).

Fig 11.6 Narishige IM 6 syringe commonly used with oil-filled tubing. It is important to use stiff polyethylene tubing to prevent expansion and secondary movement of oil within the line.

Fig 11.7 Screw-actuated syringe (SAS) from Research Instruments uses air-filled tubing to effect aspiration or injection. It is important to use stiff polyethylene tubing to prevent expansion and secondary movement of medium within the glass microtool during manipulation.

Containment systems Micromanipulation systems are sometimes contained completely within a Perspex or Plexiglas cabinet, complete with temperature and atmosphere regulation. Although these systems appear to be the ultimate in control, they often hinder the microsurgical procedure being attempted owing to space limitation.

Microsyringes, tubing, and tool chucks Most micromanipulation procedures that have to do with embryos either inject something, such as sperm, or remove something, such as blastomeres. A microsyringe is required to allow for controlled injection or aspiration. Microsyringes have improved significantly over the past 15 years, progressing from poorly controlled systems that can lead to egg and embryo explosion to highly refined instruments of microliter precision. Microsyringes are usually available from micromanipulator manufacturers and are often quite expensive. There are two basic types of microsyringe systems to choose from: those that require hydraulic

movement of oil within the tubing that connects the glass microtool to the microsyringe, and those that simply contain air within that tubing (Figs 11.6 and 11.7). Both types have their advocates, but I prefer the air-filled systems because I feel I have better control and there is essentially no oily mess around the workstation. It is important to point out that one of the control problems with the oil-filled system is the potential for small air bubbles in the tubing, which can compress and cause unexpected fluid movement in the glass microtool, leading to disastrous results. Possibly more important is what to do when you cannot afford an expensive microsyringe or what to do when your microsyringe is not working properly. One workable ‘homemade’ system is a combination of a three-way valve, 10-ml rubber plunger syringe, and 1-ml rubber plunger syringe (Fig 11.8). Fill the 10-ml syringe with oil and connect it to the threeway valve (Baxter, three-way stopcock, catalog number K75) to which the 1-ml syringe and manipulation tubing are connected. By moving the valve closure

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Fig 11.8 Homemade microsyringe to be used with oil-filled tubing. This device can yield extremely sensitive control and is very useful as a back-up system. Shown with polyethylene tubing and tool chuck.

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appropriate for all types of manipulation. The connection between the micromanipulation line and the syringe can be made with a ureteral catheter connector (French size 3–6, Cook Urological, order number 050010). This is the only connector of its type that I have found, and it works perfectly. The type of tubing used to connect the microsyringe and the glass microtools can be important. Soft tubing allows for too much expansion and ultimately loss of control. Select a hard polythene tubing with little expansion capability. Finally, tool chucks or holders make the connection between the line and the glass microtool. These are usually acquired through the micromanipulator distributor. Some tool chucks require a small silicon gasket to form a tight seal between the glass microtool and the manipulation tubing, while others simply attach with a type of locknut compression fitting. It is wise to have spare parts of all types to troubleshoot these often delicate, but essential parts of the micromanipulation system.

Glass microtools

Fig 11.9 Air-filled 20-ml syringe requires additional skill by the operator but it may be useful as a back-up system. Shown with polyethylene tubing and tool chuck.

Five to seven years ago, this section of a laboratory manual would have been the largest because of the extensive equipment and expertise required to make precision glass tools. Fortunately, today, there are as many microtool vendors as media companies. Generally speaking, they all provide a good product, and will make custom tools to meet your specific needs if given the time. The other consideration with regard to glass microtools is whether they should be straight or angled (30°). I prefer using angled pipettes because one can establish a clear focus on the horizontal section of the tool that provides a straight-on approach to the egg or embryo being manipulated (Fig 11.10).

Vibration between the 1-ml syringe and the tubing, deliberately inject the oil into the system removing all air bubbles. The 1-ml syringe should contain at least 0.5 ml of oil and the tubing should contain oil all the way into the glass microtool; there can be an air space between the oil interface and the medium within the glass microtool. Close off the valve at the 10-ml syringe and control the manipulation by gently moving the plunger in and out of the 1-ml syringe. The result is smooth and controlled injection and aspiration. There is another method which requires a great deal more skill by the technician: simply use a 20-ml rubber plunger syringe filled with air, connected to the manipulation line. There may be some benefit to back-loading a small amount of oil into the glass microtool to improve control (Fig 11.9). The holding pipette also needs some degree of control, and a simple air-filled syringe appears to be

There is a great deal of concern about how vibration can affect the quality of micromanipulation. Certainly, vibration such as slamming doors or moving cattle through a crush in the next room can cause significant disruption to a micromanipulation procedure. However, with just a little cooperation from the staff outside the manipulation room, expensive vibration tables are not required. To minimize vibration, a couple of things have to be borne in mind: set the manipulator on a separate bench or counter that is not connected to the wall or the bench that might be holding the centrifuge or other vibrating equipment, and prepare your micromanipulation plate with small drops (5 µl) overlaid with oil. Place your microscope on a rubber pad; a typewriter pad works well. These simple adjustments are often all that is needed when performing ICSI. Vibration-free micromanipulation is critical, however, when performing embryo biopsy. There, relatively large manipulation drops (50 µl) are

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Fig 11.10 Parallel orientation of angled microtools to the bottom of the manipulation plate provides a straight-on approach to the egg or embryo with clear focus of the tool throughout the observation field (200×). The set-up of the pictured glass microtools are as follows: the tool on the right (acid drilling pipette) is controlled by a joystick on the right, and the injection or aspiration control (microsyringe) is on the left side of the microscope; the tool on the left (holder) is controlled by the joystick on the left, and the aspiration microsyringe is on the right side of the microscope. An example of the convenience of this orientation is that the acid drilling pipette may be moved around inside a secured embryo to allow easy fragment removal without hand crossover at the level of the manipulator.

needed to disperse and dilute the acid Tyrode’s solution required to breach the zona pellucida. After zona drilling, the embryo is moved to a different area of the manipulation drop and then the blastomere is removed. It is at this point that the larger manipulation drop magnifies any vibration; this can significantly slow the procedure, and potentially lead to a more lethal situation (Fig 11.11). There are a great many homemade devices used to stabilize an inverted microscope and manipulator, and most improve the situation. If you are not prone to invention then you should consider a proper vibration table (Fig 11.12) or a bench-top variety of the same (Fig 11.13) if you plan to do embryo biopsy.

Laser-assisted micromanipulation Opening the zona pellucida is common to many manipulation procedures and is performed primarily by three methodologies: mechanical zona dissection, zona drilling with acid Tyrode’s solution, or laser. All of these methods have their supporters and critics but are effective and do little harm to the developing embryo if performed by an experienced technician. The use of a 1.48-µm diode laser beam (Zylos-TK Laser, Hamilton Thorne Research, Beverly, MA) to open the zona pellucida for assisted hatching or embryo biopsy offers some significant advantages in terms of both efficiency and pregnancy outcomes.13–15 Mechanical zona dissection and zona drilling with acid Tyrode’s calls for set up of specific micromanipulation plates and microtools while assisted hatching with a laser may be performed in an open well or drop without microtools of any kind or additional handling, media steps, and rinses. Also, with the advent of blastocyst vitrification protocols, a single laser

Fig 11.11 Embryo biopsy. Photograph courtesy of Reproductive Specialties Medical Center, Newport Beach, CA, USA.

Fig 11.12 Newport Elite 3 series active vibration isolation workstation. Photograph courtesy of Newport Corporation, Irvine, CA, USA, and Newbury, UK.

pulse can be used to shrink the blastocoel which has been demonstrated to improve blastocyst survival following warming.16 The net outcome is faster manipulation, which can translate into improved outcomes.14

Procedure stepwise Micromanipulator set-up The manipulation workstation is oriented such that the holding pipette is on the left and the manipulation pipette or biopsy pipette is on the right. Their respective controls (syringes) are on the opposing side to avoid hand crossover during the procedure. The holding pipette is attached to a 10-ml syringe via approximately 1 m of polyethylene tubing. The manipulation pipette is attached to a microsyringe via 1 m of polyethylene tubing obtained from the

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patient or procedure. Care should be taken to keep plates warm. The time that microtools are exposed to air should be minimized when changing plates. Microtools become sticky when exposed to air.

Micromanipulation technique 1. 2. 3. 4.

5. 6. 7.

Fig 11.13 Zander bench-top antivibration table: (a) from above; (b) from below. Photograph courtesy of Zander Medical Supplies and Zander IVF, Inc., Vero Beach, FL, USA.

microsyringe distributor. The microtools should be oriented such that they are perpendicular to the microdrop interface at the 9 o’clock and 3 o’clock positions and parallel to the bottom of the manipulation plate (Fig 11.10).

Manipulation plate set-up 1. 2.

3.

4.

Label a Falcon 1006 plate with the identity and/or ownership of the specimen to be manipulated. Place small microdrops in the center of the plate of sufficient size to contain the specimens (5–10 µl). The drops are overlaid with 4–5 ml of mineral oil and placed into a GE-80 or other suitable incubator to equilibrate the temperature. The micromanipulation plate set-up should be performed at least 1 hour prior to manipulation so that the temperature is equilibrated at 37°C. It is important that the drops be close together so they fit easily within the objective opening of the microscope stage. Prepare enough manipulation plates such that any given plate is only used once for a given

At 200× magnification, focus on the zona pellucida of the oocyte or embryo. Bring the manipulation pipette into focus. Lower the holding pipette into the drop until it is in focus. With gentle suction, aspirate the oocyte or embryo until it is held firmly without causing distortion of the zona pellucida. When held, the oocyte or embryo should be resting gently on the bottom of the plate with the lumen of the holding pipette and the manipulation pipette and the zona pellucida in sharp focus. If performed exactly in the order described, the microtools will be aligned with the equator of the oocyte or embryo. Change magnification to 400× and focus on the area to be manipulated. Bring the manipulation pipette into focus. Adjust the range of motion of the manipulation pipette. The manipulation pipette should have a range of motion over the area of the oocyte or embryo. Perform the desired manipulation procedure.

References 1. Willadsen SM, Lehn-Jensen H, Fehilly CB, Newcomb R. The production of monozygotic twins of preselected parentage by micromanipulation of non-surgically collected cow embryos. Theriogenology 1981; 15: 23–7. 2. Fehilly CB, Willadsen SM, Tucker EM. Interspecific chimaerism between sheep and goat. Nature (London) 1984; 307: 634–6. 3. Willadsen SM. Nuclear transplantation in sheep embryos. Nature (London) 1986; 320: 63. 4. Brinster RL, Chen HY, Trumbauer ME, Yagle MK, Palmiter RD. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci USA 1985; 82: 4438–42. 5. Munne S, Magli C, Cohen J, et al. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum Reprod 1999; 14: 2191–9. 6. Palermo P, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992; 340: 17–18. 7. Muggelton-Harris A, Whittingham DG, Wilson L. Cytoplasmic control of preimplantation development in vitro in the mouse. Nature (London) 1982; 299: 460–2. 8. Cohen J, Scott R, Schimmel T, et al. Birth of infant after transfer of anucleate donor oocyte cytoplasm into recipient eggs. Lancet 1997; 350: 186–7.

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9. Powell R, Barnes FL. The kinetics of oocyte activation and polar body formation in bovine embryo clones. Mol Reprod Dev 1992; 33: 53–8. 10. Barnes FL, Collas P, Powell R, et al. Influence of recipient oocyte cell cycle stage on DNA synthesis, nuclear envelope breakdown, chromosome constitution and development in nuclear transplant bovine embryos. Mol Reprod Dev 1993; 36: 33–41. 11. Keefe D. New morphologic criteria for imaging viable eggs and embryos. Presented at the 13th Annual In Vitro Fertilization and Embryo Transfer, a Comprehensive Update – 2000, Mini-symposium on the IVF laboratory, UCLA School of Medicine, Santa Barbara, CA, 2000: 83–8. 12. Collas P, Fissore R, Robl JM, Sullivan EJ, Barnes FL. Electrically-induced calcium elevation, activation and parthenogenetic development of bovine oocytes. Mol Reprod Dev 1992; 34: 212–23.

13. Jones AE, Wright G, Kort H, Straub RJ, Nagy ZP. Comparison of laser-assisted hatching and acidified Tyrode’s hatching by evaluation of blastocyst development rates in sibling embryos: a prospective randomized trial. Fertil Steril 2006; 85(2): 487–91. 14. Makrakis E, Angeli I, Agapitou K, et al. Laser versus mechanical assisted hatching: a prospective study of clinical outcomes. Fertil Steril 2006; 86(6): 1596–600. 15. Lanzendorf SE, Ratts VS, Moley KH, et al. A randomized, prospective study comparing laser-assisted hatching and assisted hatching using acidified medium. Fertil Steril 2007; 87(6): 1450–7. 16. Mukaida T, Oka C, Goto T, Takahashi K. Artificial shrinkage of blastocoeles using either a micro-needle or a laser pulse prior to the cooling steps of vitrification improves survival rate and pregnancy outcome of vitrified human blastocysts. Hum Reprod 2006; 21(12): 3246–52.

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12 Intracytoplasmic sperm injection: technical aspects Gianpiero D Palermo, Queenie V Neri, Takumi Takeuchi, Simon J Hong, Zev Rosenwaks

Introduction Spermatozoa sometimes fail to fertilize even when they are artificially placed in close proximity to eggs during conventional in vitro fertilization (IVF). Fertilization failure in IVF is particularly common where there are grossly abnormal semen parameters or when the number of spermatozoa is insufficient. Gamete micromanipulation is the only way to overcome this problem in most cases. The different techniques developed in this regard focused initially on the obstacle to sperm penetration represented by the zona pellucida (ZP), by thinning it through exposure to enzymes or creating an opening through localized chemical digestion, mechanical breach, or even photoablation.1–3 The placing of the spermatozoon beneath the zona has yielded consistent results, achieving a fertilization rate of ∼20%.4 However, these techniques have been almost abandoned because of limiting factors such as the need for many functional spermatozoa with good progressive motility, and complications such as multiple sperm penetration.5 The intracytoplasmic sperm injection (ICSI) procedure entails the deposition of a single spermatozoon directly into the cytoplasm of the oocyte, thus bypassing the ZP and the oolemma. The ability of ICSI to achieve higher fertilization and pregnancy rates regardless of sperm characteristics makes it the most powerful micromanipulation procedure yet with which to treat male factor infertility. In fact, the therapeutic possibilities of ICSI go from cases in which, after sperm selection, the spermatozoa show poor progressive motility, to its application to azoospermic men where spermatozoa are microsurgically retrieved from the epididymis and the testis.6–8 Retrieval of a low number of oocytes represents a further indication for this procedure, because only after cumulus cell removal is it possible to identify the oocytes that have extruded the first polar body and then inseminate them accordingly. ICSI is also suggested when oocytes are to be considered for preimplantation genetic diagnosis (PGD). When PGD is to be performed on oocytes,

the removal of the polar body requires the stripping of cumulus corona cells, thus leaving ICSI the only option to avoid polyspermy. When embryos need to be analyzed for gene defects, the avoidance of contaminating spermatozoa on the zona pellucida reduces the chance of false positivity with the polymerase chain reaction (PCR).

Materials and methods Semen collection When possible, semen samples are collected by masturbation after ≥3 days of abstinence and allowed to liquefy for at least 20 minutes at 37°C before analysis. When the semen has high viscosity, this can be reduced within 3–5 minutes usually by adding it to 2–3 ml of 4(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES)-buffered human tubal fluid (HTF-HEPES) containing 200–300 IU of chymotrypsin (Sigma Chemical Co., St Louis, MO, USA). Electroejaculation is applied to cases of spinal cord injury or psychogenic anejaculation.9 In the case of irreparable obstructive azoospermia, a condition which is often caused by a congenital bilateral absence of the vas deferens (CBAVD) and is associated with a cystic fibrosis gene mutation, spermatozoa are retrieved by microsurgical epididymal sperm aspiration (MESA) or percutaneous epididymal sperm aspiration (PESA).10–12 Azoospermic patients undergo testicular sperm retrieval either when the epididymal approach is unsuccessful because of impaired sperm production or transport, or in nonobstructive situations. Variable volumes of fluid (1–500 µl) are collected from the epididymal lumen by a glass micropipette or metal needle. Since spermatozoa are highly concentrated, only microliter quantities are needed. Open biopsy or the more recent fine-needle aspiration technique is used for testicular sampling.13 The biopsy specimen of approximately 500 mg is rinsed in medium to remove red blood cells and is divided into small pieces with sterile tweezers on a stereomicroscope.14 Motility or twitching is then assessed on a microscope at

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100–200×, and a second biopsy specimen is obtained if spermatozoa are not found. Four different approaches can then be used to release spermatozoa from the testicular tissue. The tissue is roughly shredded using two glass slides in a Petri dish. This procedure produces unraveled and broken tubules. An alternative method consists of mincing the tissue with two fine tweezers in a Petri dish until free tubular fragments are obtained. Other methods to treat testicular tissue include vortexing or crushing in a tissue homogenizer.

Cryopreservation of epididymal and testicular sperm When in excess, epididymal spermatozoa and testicular tissue are cryopreserved in order to avoid repeated microsurgery in case of need for later use.15 The sperm suspension (adjusted to a concentration of ∼ 30 × 106/ml) is diluted with an equal amount of cryopreservation medium (Freezing Medium-Test Yolk Buffer with Glycerol; Irvine Scientific, Irvine, CA, USA), and up to 1 ml aliquots of the final solution are placed in 1 ml cryogenic vials (Nalgene Brand Products, Rochester, NY, USA). The vials are then kept at −20°C for 35 minutes, exposed to liquid nitrogen (N2) vapor at −70°C for 10 minutes, and then plunged into liquid N2 at −196°C. Vials are thawed at room temperature when required. Epididymal and testicular samples are processed similarly to fresh semen and, when necessary, may be exposed to a motility enhancer (3.5 mmol/l pentoxifylline) to allow selection of the most viable spermatozoa.7

Semen analysis, classification, and selection Semen concentration and motility are assessed in a Makler counting chamber (Sefi Medical Instruments, Haifa, Israel). Morphologic characterization of sperm has a significant correlation with male infertility, and is performed using the strict criteria of Kruger et al.16 Classification is usually made after spreading 5 ml of semen or sperm suspension on prestained slides (Testsimplets; Boeringher), which can provide rapid results. The specimen is examined microscopically, and at least 100–200 spermatozoa are categorized. Semen parameters are considered to be impaired when the sperm concentration is <20 × 106/ml, the progressive motility is <40%, or a normal morphology is exhibited by <5% of the spermatozoa. For selection of spermatozoa, the sample is washed by centrifugation at 500g for 5 minutes in HTF medium supplemented with 6% human serum albumin (HSA, Vitrolife, Englewood, CO, USA). Semen samples with <5 × 106/ml spermatozoa or <20% motile spermatozoa are washed in HTF medium by a single centrifugation at 500–1800g for 5 minutes. The resuspended pellet is layered on a discontinuous Isolate gradient (Irvine Scientific) on three layers (90%, 70%, and 50%), and centrifuged at 300g for 20 minutes. An Isolate gradient in two layers (95% and 47.5%) is

used when samples have a sperm density <5 × 106/ml spermatozoa and <20% motile spermatozoa. The sperm-rich Isolate fraction is washed twice by adding 4 ml of HTF medium and centrifuged at 1800g for 5 minutes to remove the silica gel particles. For spermatozoa with poor kinetic characteristics, the sperm suspension is exposed to a 3 mmol/l solution of pentoxifylline and is washed again. The concentration of the assessed sperm suspension is adjusted to 1–1.5 × 106/ml, when necessary, by the addition of HTF medium, and subsequently incubated at 37°C in a gas atmosphere of 5% CO2 in air.

Collection and preparation of the oocytes Oocyte retrieval is performed after pituitary desensitization with a gonadotropin-releasing hormone agonist, with ovulation induction carried out by administering a combination of human menopausal gonadotropins (hMGs) (Pergonal; Serono, Waltham, MA, USA; Humegon; Organon Inc., West Orange, NJ, USA) and follicle-stimulating hormone (FSH) (GonalF; Serono; Follistim, Organon Inc.). Human chorionic gonadotropin (hCG) is administered when criteria for oocyte maturity are met, and oocyte retrieval by vaginal ultrasound-guided puncture is performed 35 hours later. Under the inverted microscope at 100×, the cumulus corona cell complexes are scored as mature, slightly immature, completely immature, or slightly hypermature. Thereafter, the oocytes are incubated for more than 4 hours. Immediately prior to micromanipulation, the cumulus corona cells are removed by exposure to HTF-HEPES-buffered medium containing 40 IU/ml of Cumulase (Halozyme Therapeutics Inc., San Diego, CA, USA). The removal is necessary for observation of the oocyte and effective use of the holding and/or injecting pipette during micromanipulation. For final removal of the residual corona cells, the oocytes are repeatedly aspirated in and out of a hand-drawn Pasteur pipette with an inner diameter of ~200 mm. Each oocyte is then examined under the microscope to assess the maturation stage and its integrity, metaphase II (MII) being assessed according to the absence of the germinal vesicle and the presence of an extruded polar body. ICSI is performed only in oocytes that have reached this level of maturity.

Setting for the microinjection The holding and injecting pipettes are both made from borosilicate glass capillary tubes (Drummond Scientific, Broomall, PA) with a 0.97 mm external diameter, 0.69 mm internal diameter, and 78 mm length. Drawing of the thin-walled glass capillary tubes is performed on a horizontal microelectrode puller. The holding pipette is cut and fire-polished on a microforge (Narashige Co. Ltd, Tokyo, Japan) to obtain a final outer diameter of 60 mm and an inner

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one of 20 mm. The injection pipette is prepared by opening and sharpening the pulled capillary on a grinder; the bevel angle is 30°, and the outer and inner diameters are approximately 7 mm and 5 mm, respectively. On the microforge, a spike is made on the injection pipette and both pipettes are bent to an angle of approximately 35° at 1 mm from the tip, to be able to perform the injection procedure with the tips of the tools horizontally positioned in a plastic Petri dish (model 1006, Falcon; Becton and Dickinson, Lincoln Park, NJ, USA). Immediately before injection, 1 µl of the sperm suspension is diluted with 4 µl of a 7% polyvinyl pyrrolidone (PVP) solution with HSA (90121, Irvine Scientific) in HTF-HEPES medium placed in the middle of the plastic Petri dish. It is necessary to use the viscous solution during the procedure in order to slow down the aspiration and prevent the sperm from sticking to the injection pipette. When there are <500 000 spermatozoa per sample, the sperm suspension is concentrated to approximately 3 µl and transferred directly to the injection dish. Each oocyte is placed in a 8 µl droplet of medium surrounding the central drop containing the sperm suspension. HTF-HEPES medium supplemented with 6% HSA is used in the injection dish. The droplets are covered with lightweight oil (Sage Medical, Trumbull, CT, USA). Spermatozoa are aspirated from the central droplet or the concentrated 3 µl sperm suspension drop, and transferred into the droplet containing PVP in order to remove debris and gain better aspiration control. The procedure is carried out on a heated stage (Easteach Laboratory, Centereach, NY, USA) fitted on a Nikon Diaphot inverted microscope at 400× using Hoffman modulation contrast optics. This microscope is equipped with two motor-driven coarse control manipulators and two hydraulic micromanipulators (MM-188 and MO-109; Narishige Co. Ltd). The micropipettes are inserted into a tool holder controlled by two IM-6 microinjectors (Narishige Co. Ltd).

Selection of the spermatozoon At a magnification of 400×, it is not easy to select spermatozoa according to morphologic characteristics while they are in motion. However, selection of a normal spermatozoon can be accomplished by observing its shape, its light refraction, and its motion pattern in the viscous medium. Preference goes to the spermatozoa that swim at the droplet edge.

Sperm immobilization Although ICSI does not require any specific spermatozoa pretreatment, gentle immobilization achieved through mechanical pressure is needed. This sperm immobilization is a membrane permeabilization process that may allow the release of a sperm cytosolic factor which activates the oocyte, and it has been

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demonstrated to improve fertilization rates.17–19 Owing to physiologic differences in their membrane characteristics, a more aggressive technique is necessary when using epididymal and/or testicular spermatozoa that are considered immature. In fact, human spermatozoa undergo important modifications in the nuclear chromatin and several tail organelles during the epididymal transit. These modifications include the formation of disulfide bonds, a change in the membrane surface charge, a profound qualitative and quantitative modification in their lipid composition, and the absorption of specific proteins secreted by the epithelium of the epididymis.20,21 The lack of all these changes is associated with a decreased ability of epididymal sperm to bind and penetrate the oocyte.22 When the immobilization procedure is performed in a standard fashion, spermatozoa are positioned at 90° to the tip of the pipette, which is then lowered gently to compress the sperm flagellum. The correctly immobilized sperm should maintain the shape of its tail. If during the process the latter is damaged or kinked, that spermatozoon is discarded and the procedure repeated with another sperm. An alternative procedure is aggressive immobilization, where the sperm tail is rolled over the bottom of the Petri dish in a location posterior to the mid-piece. This induces a permanent crimp in the tail section, making it kinked, looped, or convoluted (Fig 12.1). When these two distinct immobilization methods were applied to immature spermatozoa and the fertilization rates after ICSI were compared, the more extensive sperm tail disruption prior to oocyte injection appeared to improve the outcome. When the fertilization rate was compared with that of ejaculated spermatozoa, the difference was less remarkable.23 A possible explanation of the variation in fertilization rate after aggressive immobilization may lie in the structural membrane differences between mature and immature spermatozoa. Immature gametes probably require additional manipulation to promote membrane permeabilization that enhances the post-injection events involved in nuclear decondensation.

Penetration into the ooplasm The oocyte is held in place by the suction applied to the holding pipette. The inferior pole of the oocyte touching the bottom of the dish allows better grip of the egg during the injection procedure. The injection pipette is lowered and focused in accordance with the outer right border of the oolemma on the equatorial plane at 3 o’clock. The spermatozoon is then brought in proximity to the beveled opening of the injection pipette (Fig 12.2). The latter is pushed against the zona, permitting its penetration and thrusting forward to the inner surface of the oolemma. As the point of the pipette reaches the approximate center of the egg, a break in the membrane should occur. This is reflected by a sudden quivering of the convexities (at

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Fig 12.1 Aggressive immobilization of the spermatozoon for intracytoplasmic sperm injection (ICSI). The correctly immobilized spermatozoon has its tail permanently kinked (a), convoluted (b), or looped (c).

the site of invagination) of the oolemma above and below the penetration point, as well as the proximal flow of the cytoplasmic organelles and the spermatozoon back up into the pipette (Fig 12.3). These are then slowly ejected back into the cytoplasm, where the aspiration of the cytoplasm becomes an additional stimulus to activate the egg. To optimize the interaction with the ooplasm, the sperm cell should be ejected past the tip of the pipette to insure an intimate position among the organelles that will help to maintain the sperm in place while withdrawing the pipette. When the pipette is approximately at the center of the egg, some surplus medium is re-aspirated, with the result that the cytoplasmic organelles tighten around the sperm, thereby reducing the size of the breach produced during penetration. Once the pipette is removed, the breach area is observed, and the order of the opening should maintain a funnel shape with a vertex into the egg (Fig 12.4). If the border of the oolemma becomes inverted, ooplasmic organelles can leak out.24

Evaluation of fertilization, embryo development, culture conditions, and embryo replacement Around 12–17 hours after injection, oocytes are analyzed with regard to the integrity of the cytoplasm as well as the number and size of pronuclei. First-day cleavage is assessed 24 hours after fertilization, and the number and size of blastomeres recorded for each embryo. After an additional 24 hours, embryos are screened as to their need for assisted hatching. At 72 hours after microinjection (the afternoon of day 3), those with good morphology are transferred into the uterine cavity. The number of embryos transferred depends on maternal age, even though this main criterion is influenced by embryo availability and quality.

Fig 12.2 Intracytoplasmic sperm injection (ICSI) procedure. Prior to penetrating the oolemma, the spermatozoon is brought in proximity to the beveled opening of the injection pipette.

When the patient is ≤30 years old, two or three embryos are usually transferred, whereas in the 31–34, 35–41, and ≥42 years old age groups, the number of embryos increases to three, four, and five or over, respectively.

Blastocyst transfer The association between the increased incidence of multiple pregnancies after IVF and the occurrence of maternal and neonatal complications is well documented.25,26 Interest in blastocyst culture and transfer, as a strategy to overcome this problem, has been recently transformed by the introduction of more sophisticated culture media. The extended culture of embryos to the blastocyst stage allows a ‘self-embryo selection,’ indicating the fast-cleaving embryos, and

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Fig 12.3 Intracytoplasmic sperm injection (ICSI) procedure. After the injection pipette has reached the approximate center of the oocyte, a break in the oolemma is visible as a quivering of the convexities of the membrane above and below the site of penetration.

thus permitting the transfer of a lower number of them. The embryonic endometrial synchronization as well as the possibility of assessing the viability of the blastocyst, since genomic activation most likely occurs at day 4 after fertilization, may explain a higher chance of implantation. Patients who are considered candidates for blastocyst culture are young women (<35 years old) with a good ovarian reserve, or older patients with an adequate number (≥4) of pronuclear embryos. The number of embryos observed on day 3 and the capacity of embryo cleavage are also important criteria to select cases suitable for this procedure. Sequential culture media that meet changing physiologic requirements of the embryos are used, thus supporting viability of the blastocyst. Injected oocytes are rinsed and placed in a culture medium that is a variation of G1 medium, previously described by Barnes et al and Gardner et al, until assessment of fertilization.27–29 Resulting two-pronuclear embryos are further cultured in the same conditions. On day 3, after evaluation of embryo cell number and morphology, all embryos are transferred to a modified G2 medium and cultured for ≥48 hours.27–29 Thereafter, blastocyst formation is assessed and blastocysts selected according to the established criteria for subsequent transfer.30

Development of children born through ART Consenting parents of 3-(IRB # 0399-613) and 5-(IRB # 0299-581) year-old ICSI, IVF, and naturally conceived (NC) children completed a general questionnaire, Aged and Stages Questionnaire (ASQ), Social Skills Rating System (SSRS), and Parenting Stress Index (PSI). According to the child’s score and normative guidelines specified by each instrument, questionnaires

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Fig 12.4 Intracytoplasmic sperm injection (ICSI) procedure. After withdrawal of the needle from the oocyte, the breach in the oolemma should be observed as a cone-shaped opening with its vertex toward the center of the oocyte.

were classified as typically developing, or ‘at risk’ as needing further evaluation.

Outcomes From September 1993 through March 2007 at our Center, ICSI was performed in 12 578 cycles with ejaculated spermatozoa, and in 1458 cycles with surgically retrieved sperm. The mean maternal age was 37.0 ± 5 years old for the ejaculated group, and 34.3 ± 5 years old in the couples undergoing surgical retrieval of the sperm. Clinical pregnancy was defined as the presence of a gestational sac as well as at least one fetal heartbeat on ultrasonographic examination.

ICSI with ejaculated spermatozoa A total of 12 578 ICSI cycles were performed with ejaculated spermatozoa, consisting of 1615 with normal and 10 963 with abnormal semen parameters. A total of 103 631 MII oocytes were obtained from 12 578 oocyte retrievals. After ICSI, 93.8% (97 228/103 631) of these oocytes survived, and 78 016 were fertilized and displayed two pronuclei (2PN). Fertilization and clinical pregnancy rates were not influenced by the condition (fresh or cryopreserved) and collection method (masturbation, electroejaculation, or bladder catheterization) of the spermatozoa used (Table 12.1). However, fertilization was clearly superior with ejaculated spermatozoa (p = 0.0001), whereas clinical pregnancy favored the surgically retrieved (p = 0.0001) (Table 12.2).

ICSI with surgically retrieved spermatozoa A total of 707 cycles were performed with epididymal spermatozoa and 751 cycles with testicular samples.

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When the fertilization and pregnancy characteristics were analyzed according to the origin of the spermatozoa, it was observed that cryopreservation of epididymal samples clearly impaired motility parameters (p <0.0001) and pregnancy outcome (p = 0.0001), though without affecting the fertilization rate. When testicular samples were used for ICSI, the situation was reversed, with a better fertilization seen in the fresh specimens (p = 0.001) but without consequences on the pregnancy rate (Table 12.3).

Blastocyst transfer after ICSI From July 1999 through March 2007, blastocyst transfer was performed in a total of 1194 ICSI cycles, 1117 with ejaculated spermatozoa, 47 cycles with epididymal, and

Table 12.1

30 with testicular samples. Out of 14 654 injected oocytes at MII stage, 11 930 were successfully fertilized and showed two pronuclei, thus giving a fertilization rate of 81.4%. The cleaving embryos observed at day 3 were 10 311 (86.4%). On day 5, a total of 2926 (24.5%) blastocysts were obtained. Of these, 2578 (88.1%) blastocysts were replaced into the uterine cavity. Additional good-quality blastocysts (n = 348) were cryopreserved at this stage for later use. A total of 708 patients presented with a positive β-hCG (59.2%). Of these, 99 (14.0%) pregnancies were biochemical, 42 (5.9%) had a blighted ova, while the remaining 561 cases (79.2%) were clinical pregnancies with a positive fetal heartbeat detected by ultrasound, achieving an implantation rate per embryo of over 35% (Table 12.4). Among these pregnancies, 67% were singleton pregnancies, 32% twins,

Fertilization rates according to collection method and specimen condition

Semen origin

Cycles

Oocyte fertilized/oocyte inseminated (%)

Clinical pregnancies (%)

Fresh ejaculate Frozen ejaculate Electroejaculate Frozen electroejaculate Retrograde ejaculate

11 470 1012 51 21 24

70 960/94 277 (75.3) 6346/8432 (75.3) 394/502 (78.5) 121/187 (64.7) 195/233 (83.7)

4545 (39.6) 397 (39.2) 26 (51.0) 8 (38.1) 9 (37.5)

Table 12.2

Fertilization and pregnancy rates according to semen origin Spermatozoa

No. of (%)

Ejaculated

Cycles Fertilization Clinical pregnancies ∗ †

Surgically retrieved

12 578 78 016/103 631 (75.3)∗ 4985 (39.6)†

1458 8947/13 888 (64.4)∗ 678 (46.5)†

χ2, 2 × 2, 1 df, effect of spermatozoal source on fertilization rate, p = 0.0001. χ2, 2 × 2, 1 df, effect of spermatozoal source on clinical pregnancy rate, p = 0.0001.

Table 12.3 Spermatozoal parameters and intracytoplasmic sperm injection (ICSI) outcome according to retrieval sites and specimen condition Spermatozoa Epididymal No. of (%) Cycles Density (106/ml ± SD) Motility (% ± SD) Morphology (% ± SD) Fertilization Clinical pregnancies ∗

Testicular

Fresh

Frozen/thawed

Fresh

Frozen/thawed

266 37.9 ± 44 19.2 ± 17∗ 1.0 ± 2 1946/2701(72.0) 163 (61.28)†

441 25.7 ± 29 3.1 ± 6∗ 0.6 ± 1.6 2873/4034 (71.2) 214 (48.53)†

601 0.2 ± 0.7 2.9 ± 8 0 3412/5821 (58.6)‡ 251 (41.76)

150 0.2 ± 0.6 0.9 ± 3.3 0 716/1332 (53.8)‡ 50 (33.33)

Student’s t-test, two independent samples, effect of epididymal cryopreservation on sperm motility, p = 0.0001. χ2, 2 × 2, 1 df, effect of epididymal cryopreservation on clinical pregnancy rate, p = 0.001. ‡ χ2, 2 × 2, 1 df, effect of testicular cryopreservation on fertilization rate, p = 0.001. †

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and 1% triplets (Table 12.5). As expected, the higherorder gestation were more common in the day 3 embryo replacement (p = 0.01).

Development of children born through ART A total of 678 IVF and 1659 ICSI families were invited to participate in the study. Of those, 54.4% IVF and 59.1% ICSI families agreed to complete at least one questionnaire. The average maternal age was similar in both ART procedures (35.2 ± 5 years old). The rate of congenital malformation for IVF was 1.3% (3/223) and for ICSI was 1.1% (5/459). According to the ASQ, 90.6% of 223 IVF and 89.8% of 459 ICSI children displayed normal development, while 21 IVF and 47 ICSI were ‘at risk.’ The children ‘at risk’ were found to have psychomotor delays in at least two developmental areas. The commonest developmental defect in both IVF and ICSI children involved gross-motor (48.5%), fine-motor (51.5%), and problem-solving (50.0%) skills. Among the at-risk group, a total of 51% (35/68) of the children came from multiple gestations. In addition, the fine-motor and problem-solving skills were the most recurrent deficiency in the twins and triplets. On the SSRS, both IVF and ICSI groups had very positive social and interactive skills. Not surprisingly, parents rearing twins or triplets showed significantly higher levels of stress. No symptoms could be identified from the childrens’ behavior that would link them to a specific epigenetic disorder such as Beckwith–Wiedemann syndrome, Prader–Willi syndrome, and Angelman syndrome.

Pregnancy and delivery characteristics The pregnancy outcome of 14 036 ICSI cycles is described in Table 12.6. Of a total of 7420 patients presenting with a positive β-hCG (52.9%), 1162 were biochemical (8.3%) and 538 were blighted ova (3.8%). Among 5663 patients in whom a viable fetal heartbeat was observed, 520 had a miscarriage or were therapeutically aborted, and 57 had an ectopic pregnancy. The final ongoing pregnancy rate was 36.2% per retrieval (5086/14 036), and 38.7% per replacement (5086/13 146). A total of 5450 babies were born from 3891 deliveries, including 2504 being female and 2504 being male, with an overall frequency of multiple deliveries of 36.4% (1416/3891): 1251 twins (32.2%), 162 triplets (4.2%), and three quadruplets (0.08%). Of the 5450 newborns, 191 (3.5%) exhibited congenital abnormalities at birth: 104 (1.9%) were major and 87 (1.6%) were minor. Compared with the frequency of malformations in offspring born after standard IVF, ICSI newborns experienced a similar rate of congenital malformation (Table 12.7).

Table 12.4

Implantation rate according to embryo culture Embryo replacement

No. of (%)

Day 3

Embryos replaced Sacs implanted Positive fetal heartbeats †

Day 5

38 079 8713 (22.9)∗ 7620 (20.0)†

2578 934 (36.2)∗ 827 (32.0)†

χ2, 2 × 2, 1 df, effect of extended in vitro culture on implantation, p = 0.0001.

Table 12.5

Influence of embryonic stage on pregnancy outcome and gestational order Embryo replacement

No. of (%) ICSI cycles Replacements Embryos replaced (mean) Clinical pregnancies (+FHB) Deliveries Singleton births Twin births Triplet births Quadruplet births

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Day 3

Day 5

12 842 11 952 (93.1) 3.0 5102 (39.7)∗ 3560 (27.7) 2246 1153 158 3

1194 1194 (100) 2.2 561 (47.0)∗ 307 (25.7) 205 98 4 0

∗ 2 χ , 2 × 2, 1 df, effect of embryonic stage on pregnancy rate, p = 0.0001. FHB, fetal heartbeat.

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Table 12.6

Evolution of intracytoplasmic sperm injection (ICSI) pregnancies in 14 036 cycles No. of

ICSI cycles Embryo replacements Positive hCGs Biochemical pregnancies Blighted ova Ectopic pregnancies Positive fetal heartbeats Miscarriages/therapeutic abortions Deliveries

Positive outcomes

14 036 13 146 7420 1162 538 57 5663 520 3891

Pregnancy 52.9% (7420/14 036)

Clinical pregnancy 40.3% (5663/14 036) Delivery rate 27.7% (3891/14 036)

hCG, human chorionic gonadotropin.

Table 12.7

Occurrence of congenital abnormalities with assisted reproductive technologies

No. of (%) Cycles Offspring delivered Newborns with major malformations Newborns with minor malformations Total malformations

ICSI

IVF

14 035 5450 104 (1.9) 87 (1.6) 191 (3.5)

8664 4054 77 (1.9) 61 (1.5) 138 (3.4 )

Conclusions ICSI is the newest and, to date, most successful technique used to overcome fertilization failure. In addition, it has helped us to understand better some important key steps of the fertilization process. The results demonstrate that the injection of mechanically immobilized spermatozoa achieves fertilization at a higher rate than the injection of motile spermatozoa. This is the result of destabilization and consequent permeabilization of the sperm plasma membrane, which is responsible for the release of an oocyte-activating factor.23,31,32 These profound physiologic changes induced on the sperm membrane by its interaction with the injection needle seem to be critically important for immature, surgically retrieved spermatozoa.8 It has been demonstrated that the positive outcome of ICSI is largely independent of the three basic sperm parameters – motility, morphology, and concentration – in couples in whom these characteristics are severely impaired, and even when no spermatozoa are present in the initial ejaculate.6 In the latter case, its successful application to surgically retrieved sperm proves that this micromanipulation technique is able to achieve fertilization regardless of the maturation of the gametes. The possibility to bypass the steps of testicular and epididymal sperm maturation, acrosome reaction, binding to the zona pellucida, and fusion with the oolemma now permits infertility due to a male factor to be addressed successfully. However, it should be considered that subfertile men have a higher frequency of

chromosomal abnormalities.8,33 Therefore, the earlier concern focused on the ICSI procedure itself has been shifted to the subfertile man who may transmit his genetic defects to his offspring. However, our experience suggests that these conditions, in spite of being associated with a higher frequency of genetic anomalies, can be treated with ICSI without a significant increase in adverse outcome of offspring.8 The more recent practice of long-term embryo culture, made possible by the advent of new sequential media, seems to be a promising treatment option in conjunction with ICSI, especially for infertile couples where a multifetal pregnancy would be of particular risk. Data from 1194 ICSI cycles with blastocyst transfer showed a significant increase in implantation and pregnancy rates. The reason for this improvement in implantation may lie in a more physiologic environment for the conceptus at this stage, while the earlier embryo finds its environment in the fallopian tube. Furthermore, the rate of multiple gestation was lowered. Complete fertilization failure after ICSI is very unusual, and in most cases is probably due to either failed oocyte activation or incomplete decondensation of the spermatozoon.34–36 The only factor that obviously impacts on ICSI-related pregnancy rates is maternal age, impairing oocyte/embryo quality. This issue is currently the focus of many embryologists, with micromanipulation of the oocyte seeming to offer some solution to this female-related aspect of the infertility picture.37

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The potential effects of ART on child development should not be underestimated and, in order to confirm the safety and the well-being of such children, cognitive and motor testing is essential. This study indicates that the Ages and Stages Questionnaire provides a cost- and time-effective test through which to monitor the development of ART children, particularly for centers where pediatric and psychological follow-up would be inconvenient. In both the ICSI and IVF groups, the 3 year olds assessed are developing well without significant delays in their cognitive abilities, socioemotional development, and motor skills. Although the health and development of these children are normal, continuous and vigilant screening should be conducted.

Acknowledgments We thank the clinical and scientific staff of the Center for Reproductive Medicine and Infertility. We are grateful to Professor J Michael Bedford for his scientific support and Maryanne Williams-Pitman for kindly recruiting patients for the child study.

References 1. Gordon JW, Grunfeld L, Garrisi GJ, et al. Fertilization of human oocytes by sperm from infertile males after zona pellucida drilling. Fertil Steril 1988; 50: 68–73. 2. Malter HE, Cohen J. Partial zona dissection of the human oocyte: a non-traumatic method using micromanipulation to assist zona pellucida penetration. Fertil Steril 1989; 51: 139–48. 3. Feichtinger W, Strohmer H, Fuhrberg P, et al. Photoablation of oocyte zona pellucida by erbium– YAG laser for in vitro fertilization in severe male infertility. Lancet 1992; 339: 811. 4. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Induction of acrosome reaction in human spermatozoa used for subzonal insemination. Hum Reprod 1992; 7: 248–54. 5. Cohen J, Alikani M, Malter HE, et al. Partial zona dissection or subzonal sperm insertion: microsurgical fertilization alternatives based on evaluation of sperm and embryo morphology. Fertil Steril 1991; 56: 696–706. 6. Palermo GD, Cohen J, Alikani M, Adler A, Rosenwaks Z. Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil Steril 1995; 63: 1231–40. 7. Palermo GD, Cohen J, Rosenwaks Z. Intracyto-plasmic sperm injection: a powerful tool to overcome fertilization failure. Fertil Steril 1996; 65: 899–908. 8. Palermo GD, Schlegel PN, Hariprashad JJ, et al. Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men. Hum Reprod 1999; 14: 741–8. 9. Bennet CJ, Ayers JWT, Randolph JF Jr, et al. Sexual dysfunction and electroejaculation in men with spinal cord injury: review. J Urol 1988; 139: 453–7.

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10. Schlegel PN, Berkley A, Goldstein M, et al. Epididymal micropuncture with in vitro fertilization and oocyte micromanipulation for the treatment of unreconstructable obstructive azoospermia. Fertil Steril 1994; 61: 895–901. 11. Schlegel PN, Cohen J, Goldstein M, et al. Cystic fibrosis gene mutations do not affect sperm function during in vitro fertilization with micromanipulation for men with bilateral congenital absence of vas deferens. Fertil Steril 1995; 64: 421–6. 12. Tsirigotis M, Pelankos M, Yazdani N, et al. Simplified sperm retrieval and intracytoplasmic sperm injection in patients with azoospermia. Br J Urol 1995; 76: 765–8. 13. Friedler S, Raziel A, Strassburger D, et al. Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia. Hum Reprod 1997; 12: 1488–93. 14. Silber SJ, Van Steirteghem AC, Liu J, et al. High fertilization and pregnancy rates after intracytoplasmic sperm injection with spermatozoa obtained from testicular biopsy. Hum Reprod 1995; 10: 148–52. 15. Verheyen G, Pletinck I, Van Steirteghem AC. Effect of freezing method, thawing temperature and postthaw dilution/washing on motility (CASA) and morphology characteristics of high-quality human sperm. Hum Reprod 1993; 8: 1678–84. 16. Kruger TF, Menkveld R, Stander FSH, et al. Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril 1986; 46: 1118–23. 17. Dozortzev D, Rybouchkin A, De Sutter P, Quian C, Dhont M. Human oocyte activation following intracytoplasmic sperm injection; the role of the sperm cell. Hum Reprod 1995; 10: 403–7. 18. Palermo GD, Joris H, Derde MP, et al. Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertil Steril 1993; 63: 1231–40. 19. Fishel S, Lisi F, Rinaldi L, et al. Systematic examination of immobilizing spermatozoa before intracytoplasmic sperm injection in the human. Hum Reprod 1995; 10: 497–500. 20. Bedford JM, Calvin HI, Cooper GW. The maturation of spermatozoa in the human epididymis. J Reprod Fertil 1973; 18 (Suppl): 199. 21. Kirchoff C, Osterhoff C, Habben I, Ivell R. Cloning and analysis of mRNAs expressed specifically in the human epididymis. Int J Androl 1990; 13: 155–67. 22. Moore HD, Hartman TD, Pryor JP. Development of the oocyte-penetrating capacity of spermatozoa in the human epididymis. Int J Androl 1983; 6: 310–18. 23. Palermo GD, Schlegel PN, Colombero LT, et al. Aggressive sperm immobilization prior to intracytoplasmic sperm injection with immature spermatozoa improves fertilization and pregnancy rates. Hum Reprod 1996; 11: 1023–9. 24. Palermo G, Alikani M, Bertoli M, et al. Oolemma characteristics in relation to survival and fertilization patterns of oocytes treated by ICSI. Hum Reprod 1996; 11: 172–6. 25. Society for Assisted Reproduction and Technology. Assisted reproductive technology in the United States and Canada. Results generated from the

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26.

27.

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30.

31.

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Textbook of Assisted Reproductive Technologies American Society for Reproductive Technology Registry. Fertil Steril 1998; 69: 389–98. Gardner DK, Schoolcraft WB. Elimination of high order multiple gestations by blastocyst culture and transfer. In: Shoham Z, Howles C, Jacobs H, eds. Female Infertility Therapy: Current Practice. London: Martin Dunitz, 1998: 267–74. Barnes FL, Crombie A, Gardner DK, et al. Blastocyst development and birth after in vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching. Hum Reprod 1995; 10: 3243–7. Gardner DK, Lane M. Culture and selection of viable blastocysts: a feasible proposition for human IVF? Hum Reprod Update 1997; 3: 367–82. Gardner DK, Vella P, Lane M, et al. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 1998; 69: 84–8. Schoolcraft WB, Gardner DK, Lane M, et al. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril 1999; 72: 604–9. Takeuchi T, Tsai MC, Hariprashad JJ, Rosenwaks Z, Palermo GD. Ultrastructure of immobilized spermatozoa used for ICSI. Fertil Steril 1999; 72: S118–19 (abstr).

32. Wolny YM, Fissore RA, Wu H, et al. Human glucosamine-6-phosphate isomerase, a homologue of hamster oscillin, does not appear to be involved in Ca2+ release in mammalian oocytes. Mol Reprod Dev 1999; 52: 277–87. 33. de Krester DM, Burger HGG, Fortune D, et al. Hormonal, histological, and chromosomal studies in adult males with testicular disorders. J Clin Endocrinol Metab 1972; 35: 392–401. 34. Sousa M, Tesarik J. Ultrastructural analysis of fertilization failure after intracytoplasmic sperm injection. Hum Reprod 1994; 9: 2374–80. 35. Flaherty SP, Payne D, Swann NJ, et al. Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection. Hum Reprod 1995; 10: 2623–9. 36. Moomjy M, Colombero LT, Veeck LL, Rosenwaks Z, Palermo GD. Sperm integrity is critical for normal mitotic division and early embryonic development. Mol Hum Reprod 1999; 5: 836–44. 37. Silber SJ, Nagy Z, Devroey P, Camus M, Van Steirteghem AC. The effect of female age and ovarian reserve on pregnancy rate in male infertility: treatment of azoospermia with sperm retrieval and intracytoplasmic sperm injection. Hum Reprod 1997; 12: 2693–700.

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13 Assisted hatching Anna Veiga, Irene Boiso, Itziar Belil

Introduction The zona pellucida The zona pellucida (ZP) of mammalian eggs and embryos is an acellular matrix composed of sulfated glycoproteins with different roles during fertilization and embryo development.1 Three distinct glycoproteins have been described both in mice and in humans (ZP1, ZP2, ZP3).2 Acrosome-reacted spermatozoa bind to ZP receptors, and biochemical changes have been observed after fertilization3 responsible for the prevention of polyspermic fertilization. The main function of the ZP after fertilization is protection of the embryo and the maintenance of its integrity.4 It has been postulated that blastomeres may be weakly connected, and that the ZP is needed during the migration of embryos through the reproductive tract to maintain the embryo structure. Implantation has been observed after replacement of zona-free mouse morulas or blastocysts, while the transfer of zona-free precompacted embryos results in the adherence of transferred embryos to the oviductal walls or to one another. A possible protective role against hostile uterine factors has also been described.4 Degeneration of sheep eggs after complete or partial zona pellucida removal that could be ascribed to an immune response was described by Trounson and Moore.5

Hatching Once in the uterus, the blastocysts must get out of the ZP (hatching) so that the trophectoderm cells can interact with endometrial cells and implantation can occur. The loss of the ZP in utero is the result of embryonic and uterine functions. Zona hardening occurs after zona reaction, subsequent to fertilization, and is evidenced by an increased resistance to dissolution by different chemical agents. A loss of elasticity is also observed. This physiological phenomenon is essential for polyspermy block and for embryo protection during transport through the reproductive tract. It has been postulated that additional ZP hardening may occur both in mice and in humans as a consequence

of in vitro culture.4,6,7 Hatching could be inhibited in some in vitro cultured human embryos owing to the inability of the blastocysts to escape from a thick or hardened ZP.8 Schiewe et al performed a study to characterize ZP hardening in unfertilized and abnormal embryos and to correlate it with culture duration, patient age, and ZP thickness.9 Dispersion of ZP glycoproteins and the time needed for complete digestion after α-chymotrypsin treatment were assessed. The results obtained proved that zona hardening of fertilized eggs was increased, compared with inseminated unfertilized eggs. Wide patient-to-patient variation in zona hardness was observed, but no correlation between zona hardness or thickness and patient age was established. Furthermore, the data obtained did not support the concept that additional ZP hardening occurs during extended culture. Expansion and ZP thinning occurs in mammalian blastocysts prior to hatching. Cycles of contraction and expansion have been described in mice, sheep, cattle, and human blastocysts in vitro prior to ZP hatching. As a result of several cycles of contraction and expansion and because of its elasticity, the ZP thins. Contraction–expansion cycles as well as cytoplasmic extensions of trophectoderm (trophectoderm projections, TEPs) have been documented by time-lapse video recording10 in human blastocysts. TEPs could be a component of zona escape in cultured embryos. It is not clear whether TEPs are needed in vivo for ZP hatching, but they seem to have a role in attachment, implantation, and possibly embryo locomotion.11 Lysins of embryonic and/or uterine origin are involved in ZP thinning and hatching. Gordon and Dapunt showed that, in mice, hatching is predominantly the result of zona lysis, and that the pressure exerted against the zona by the expanding blastocyst plays little or no part in the escape of the embryo from the ZP.12 Schiewe et al demonstrated, with the use of a mouse antihatching model, the involvement of zona lysins in the mechanism of hatching;13 physical expansion of the blastocyst, even though involved in hatching, does not seem to be the primary mechanism. Their results also show that trophectoderm

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cells are responsible for secreting the zona lysins required for hatching. Recent data on mouse blastocysts indicate that hatching in vitro is dependent on a sufficient number of cells constituting the embryo. Hatching in vivo must be different from that in vitro, the difference involving uterine and/or uterine-induced trophectoderm lytic factors.14

Assisted hatching The first report of the use of assisted hatching (AH) in human embryos was published by Cohen et al in 1990.7 These authors documented an important increase of implantation rates with mechanical AH in embryos from unselected in vitro fertilization (IVF) patients.

Why perform assisted hatching? The ratio of lysin production to ZP thickness could determine whether the embryo will lyse the zona and undergo hatching. Embryos with thick zonae or those that present extensive fragmentation or cell death after freezing and thawing may benefit from assisted hatching.15 Both quantitative and qualitative deficiencies in lysin secretion could result in hatching impairment. Suboptimal culture conditions may cause such deficiencies. The trophectoderm of some embryos may not be able to secrete the ‘hatching factor,’ and lysin production could be influenced by a patient’s age.8,13 Uterine lysin action could also be impaired in some patients or cycles.16 It is believed that ZP hardening may be exacerbated at any stage of embryo development after long-term in vitro culture and cryopreservation of embryos.17 Furthermore, experiments on mouse embryos have demonstrated that damaged blastomeres have a toxic effect, reducing dramatically the rate of hatching.18 However, embryo viability was restored after microsurgical removal of the degenerating material. Removal of necrotic blastomeres from frozen–thawed partially damaged human embryos significantly increased the implantation rate.19 It has been stated that overall zona thickness varies between age groups and types of infertility.20 The variability of zona thickness in the same embryo is one of the most significant morphologic predictive factors of implantation.21 Palmstierna et al demonstrated that human embryos with zona thickness variation of >20% resulted in a 76% pregnancy rate with two embryos transferred.22 Zona-assisted thinning of a substantial area may favor complete hatching in embryos with invariable zona thickness.23 Khalifa et al have shown that ZP thinning significantly increases the complete hatching of mouse embryos.24 Gordon and Dapunt demonstrated the usefulness of ZP thinning with acid Tyrode’s to improve hatching

in hatching-defective mouse embryos created by the destruction of one-quarter of the blastomeres.15 They reported normal implantation rates in pseudopregnant female mice after the transfer of assisted-hatched embryos that had cell numbers reduced. The mechanism by which assisted hatching promotes embryo implantation remains unclear. The implantation window is the critical period when the endometrium reaches its ideal receptive state for implantation. Precise synchronization between the embryo and the endometrium is essential. In a randomized study, Liu et al demonstrated that implantation occurred significantly earlier in patients whose embryos were submitted to AH when compared with the control group, possibly by allowing earlier embryo– endometrium contact.25 Furthermore, although most molecules are able to cross the ZP, the rate of transport may be related to zona thickness. The presence of an artificial gap may alter the two-way transport of metabolites and growth factors across the ZP, permitting earlier exposure of the embryo to vital growth factors.8

Methods When breaches are made in the ZP of early-cleavage IVF embryos, embryonic cell loss may occur through the zona as a result of uterine contractions after replacement of the embryos. It is advisable to manipulate embryos for AH after the adherence between blastomeres has increased, just before compaction.26 Artificial opening of the ZP of blastocysts can also be performed to promote complete blastocyst hatching.27,28 Embryos at the 6–8-cell stage, at day 3 after insemination, or at the blastocyst stage, at day 5 or 6 after insemination, can be manipulated with different methods for the performance of AH. Microtools for AH can be made by means of a pipette puller and microforge, but are also commercially available. Micropipettes are mounted on micromanipulators. It is very important to minimize the time that the embryo is out of the incubator, and to optimize methodologies to reduce pH and temperature variations that can be detrimental for embryo development. To reduce environmental variations, AH has to be performed in microdrops of 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES)-buffered medium covered with oil, under an inverted microscope with Nomarski or Hoffman optics, on a heated microscope stage, at 37°C. It is important that the size of the hole created in the zona is large enough to avoid trapping of the embryo during hatching, but not so large that it permits blastomere loss.29–32 Monozygotic twinning has been described as a consequence of AH.33 The adequate size of the hole seems to be 30–40 µm. Different protocols have been described, but a minimum 30-minute culture period seems to be sufficient before the transfer of the manipulated embryos.

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Embryo transfer to the uterus has to be performed as atraumatically as possible to avoid damage of ZPmanipulated embryos. Treatment during 4 days, starting on the day of oocyte retrieval, with broad-spectrum antibiotics and corticosteroids (methylprednisolone, 16 mg daily) has been postulated. Cohen et al suggested that such treatment may be useful for patients whose embryos have been assisted-hatched, to avoid infection and immune cell invasion of the embryos.7

Partial zona dissection The method of partial zona dissection (PZD) is similar to that described for oocytes, to assist oocyte zona pellucida penetration by spermatozoa34 with no preincubation of the embryos in sucrose. Embryos denuded of corona cells are micromanipulated in microdrops of HEPES-buffered medium under paraffin oil. As mentioned above, the procedure is performed at 37°C, under an inverted microscope. The embryo is held with a holding pipette, and the zona pellucida is tangentially pierced with a microneedle from the 1 o’clock to the 11 o’clock position. The embryo is released from the holding pipette and the part of the ZP between the two points is rubbed against the holding pipette until a slit is made in the zona. The embryo is washed twice in fresh culture medium and placed in the transfer dish. Three-dimensional partial zona dissection (3DPZD) in the shape of a cross has been described.32 The procedure starts as conventional PZD, and a second cut is made in the ZP under the first slit. A crossshaped cut can be seen on the surface of the ZP. This method allows the creation of larger openings while permitting protection of the embryo by the ZP flaps during embryo transfer. A new technique called ‘controlled zona dissection’ (CDZ) has been recently described as a variation of PZD.28 The embryo is held at the 8 o’clock position by a beveled opened holding pipette and a thin angled hatching needle with a blunted tip pierces the ZP at the 5 o’clock position. The hatching needle is inserted deeply into the holding pipette until the embryo is pushed to the angle of the hatching needle. The curve of the needle is then pressed against the bottom of the dish to cut the pierced ZP. A large slit (two-thirds of the embryo’s diameter) created by CDZ enhances significantly the rate of complete in vitro hatching of blastocysts compared to 3D-PZD.

Acid Tyrode’s assisted hatching It has been described that zona hardening and the increase in volume of the perivitelline space in zygotes and embryos allows efficient and safe use of acid Tyrode’s solution (AT) in human embryos for ZP drilling, compared with oocytes. Nevertheless, it has to be taken into account that the use of acidic

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solutions for AH may be detrimental for the blastomere(s) adjacent to the drilled portion of the ZP. Limiting embryo exposure to AT by adequate and quick manipulation is necessary to avoid harmful effects on embryo development. Acid Tyrode’s solution can be prepared in the laboratory with the protocol of Hogan et al35 and adjusted to a pH of 2.5, or can be purchased commercially. One advantage of AT drilling compared with PZD is the possibility of increasing the size of the hole in the ZP. Large holes have proved to be more efficient for enhancing hatching and avoiding embryo entrapment.7,31,36 The embryo is held with a holding pipette in such a way that the micropipette containing acid Tyrode’s (internal diameter 3–5 µm) at the 3 o’clock position faces a large perivitelline space or an area with cytoplasmic fragments of the embryo. The acidic solution is gently delivered with the help of a microinjector over a small area of the ZP, with the tip of the pipette positioned very close to the zona. Accumulation of AT in a single area must be avoided. Extracellular fragments can also be removed during the procedure.8 As soon as a hole in the ZP is created, suction is applied to avoid excess AT entering the perivitelline space. If the inner region of the ZP is difficult to breach, creation of the hole can be facilitated by pushing the AT micropipette against the ZP.37 It is necessary to rinse the embryo several times in fresh culture medium.

Laser-assisted hatching The use of laser techniques in the field of assisted reproduction for application in gametes or embryos was first described by Tadir et al.38,39 For fast and efficient clinical use of laser systems in assisted hatching, it is important that the laser is accurately controlled and produces precise ZP openings without thermal or mutagenic effects. The application of a laser on the ZP for AH results in photoablation of the zona pellucida. Contact lasers The procedure is performed on a microscope slide, and the embryo is placed in a drop of medium covered with paraffin oil. The embryo is held with a holding pipette, and the laser is delivered through a microscopic laser glass fiber, fitted to the manipulator by a pipette holder, in direct contact with the ZP. Several pulses are necessary to penetrate the ZP. Because each laser pulse removes only small portions of the ZP, the fiber tip has to be continuously readjusted to guarantee close contact with the remaining zona. The first use of a laser for ZP drilling was reported by Palanker et al with an ArF excimer laser (ultraviolet [UV] region, 193 nm wavelength).40 This laser system makes it necessary to touch the ZP with the laser-delivering pipette (contact mode laser). The erbium:yttrium–aluminum–garnet (Er:YAG) laser (2940 nm radiation), also working in contact

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mode, has been used for ZP assisted hatching and thinning, and its safety and efficacy have been demonstrated in clinical practice.41,42 Obruca et al performed a study to evaluate the ultra structural effects of the Er:YAG laser on the ZP and membrane of oocytes and embryos.43 No degenerative alterations were observed using light and scanning electron microscopy after ZP drilling with such a system. Antinori et al44 described the method for ZP thinning with the use of an Er:YAG laser. Five to eight pulses were needed to ablate 50% of the ZP thickness in a length of 20 µm. The necessity of sterile micropipettes and optical fibers to deliver the laser beam to the target are the main disadvantages of contact mode lasers.45 Noncontact lasers Noncontact laser systems allow microscope objective-delivered accessibility of laser light to the target. Laser propagation is made through water and, as it avoids the UV absorption peak of DNA, no mutagenic effect on the oocyte or embryo is expected. Blanchet et al first reported the use of a noncontact laser system (248 nm KrF excimer) for mouse ZP drilling.46 Neev et al described the use of a noncontact holmium:yttrium–scandium–gallium–garnet (Ho:YSGG) laser (2.1 µm wavelength) for AH in mice.47 The study showed a lack of embryotoxic effects as well as improved blastocyst hatching. Similar results were reported by Schiewe et al.48 Rink et al designed and introduced a noncontact infrared diode laser (1.48 µm) that delivers laser light through the microscope objective.49 The drilling mechanism is explained by a thermal effect induced at the focal point by absorption of the laser energy by water and/or ZP macromolecules, leading to thermolysis of the ZP matrix.50 Laser absorption by the culture dish and medium is minimal. The effect on the ZP is greatly localized, and the holes are cylindrical and precise. Exposure time (1–40 ms) can be minimized. The safety and usefulness of the system were demonstrated in mice and humans.51–53 Its use for polar body as well as blastomere and blastocyst biopsy has also been reported.54–56 The system is compact and easily adapted to all kinds of microscopes. The size of the hole is related to the laser exposure time, and thus the system is simple, quick, and easy to use. Fig 13.1 shows an 8-cell embryo in which laser assisted hatching has been performed. Antinori et al have reported the use of a compact, noncontact ultraviolet (337 nm wavelength) laser microbeam system to create holes in the zona pellucida of human embryos.57,58 This equipment requires the manipulation of the oocytes and embryos in Petri dishes with a membrane bottom. Depending on the laser equipment, different methods are used, varying in energy, time, and number of pulses needed to open the zona pellucida. Two studies have reported the immediate effects of localized heating after the use of noncontact infrared

Fig 13.1 Day 3 embryo in which the zona pellucida (ZP) has been drilled with two laser shots (Fertilase, MTM, Montreaux, Switzerland).

lasers in animal models.59,60 The diode laser beam produces superheated water approaching 200°C on the beam axis. The action of the laser must be strictly limited to the targeted region of the ZP, since focused laser irradiation on a specific cell would cause damage and would probably be lethal to that cell. Following irradiation, the heat is conducted away from the target and is dissipated into the surrounding medium. The potential to damage blastomeres adjacent to the hole created by the laser is minimized by using pulse durations of ≤5 ms and laser power ∼100 mW at a safe distance from the blastomeres. In a recent study made on the murine model to determine the optimal technical settings for laser AH – changing laser intensity, pulse duration, number of pulses, as well as the depth of disruptions – the highest hatching rate seemed to be achieved when laser intensity was reduced.61 There are studies that compare laser AH to other AH methods.62,63 Sister embryos of patients undergoing preimplantation genetic diagnosis (PGD), randomly assigned on day 3 to acid Tyrode’s zona drilling or to laser zona drilling, showed similar blastocyst development rates.62 However, implantation rates of laser ZP drilled embryos were significantly higher than those of mechanically treated embryos, when the embryos of women of advanced age (≥39 years old) underwent AH.63

Zona pellucida thinning The aim of ZP thinning is to thin the ZP without complete lysis and perforation. By not breaching the zona, the potential risk of blastomere loss and embryonic infection is minimized. Zona pellucida thinning with AT has been described in mice and in humans.24,64 It involves bidirectional thinning of a cross-shaped area of the ZP over about one-quarter of the embryo circumference. Care has to be taken not to rupture the ZP

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Fig 13.2 Laser-drilled assisted hatching (Fertilase, MTM Montreaux, Switzerland) in an expanded blastocyst. A trophectoderm cell is protruding through the thin zona pellucida (ZP).

completely. Embryos are washed in fresh droplets of medium and cultured before transfer. This methodology has proved useful for hatching enhancement in mice but not in humans, probably because of differences observed in both the morphologic and the biophysical characteristics of the ZP between the two species. The mouse ZP has a monolayer structure, whereas the human ZP, as shown by electron microscopy, is composed of a less dense, easily digestible, thick outer layer and a more compact but resilient inner layer.64 The use of laser technology for ZP thinning at the cleavage stage seems to be beneficial for embryo implantation for certain authors.23,44,65–67 Antinori et al demonstrated a significant increase in implantation and pregnancy rate when 50% of the zona thickness from 2-day-old embryos was thinned for a length of 20 µm using a YAG contact laser.44 Diode laser ZP thinning enhances the variation of zona thickness in human embryos, allows natural zona thinning, and increases significantly the rate of blastocyst hatching.23 Acceptable clinical pregnancy rates were obtained after transfer of frozen–thawed blastocysts that underwent laser-assisted thinning at the day 3 cleaving stage before freezing.65 Laser partial zona thinning has been associated with higher implantation and pregnancy rates than total laser AH, especially in women who suffer from recurrent implantation failure.68 The enzymatic action of pronase to thin the ZP of human early-cleaving embryos yields similar benefit to other AH methods.69 Nevertheless, zona thinning for cryopreserved–thawed embryos, using pronase action or laser methodology, has failed to show improvement of the implantation rate.70–72

Blastocyst assisted hatching Even though AH is usually performed on early-cleavage embryos (day 3, 6–8-cell stage), it can also be applied to blastocysts to increase implantation rates.

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A monozygotic twin pregnancy was achieved after transfer of a frozen–thawed human blastocyst, on which zona pellucida rubbing with a microneedle was applied.73 The size of the hole made on the human blastocyst’s ZP during AH seems to be important for the final hatching development. A large slit created on the ZP of human blastocysts after mechanical AH with CZD enhanced significantly total blastocyst hatching in vitro compared to a moderate-size slit (two-fifths of ZP diameter).28 Zona opening of small or moderate size induced the hatching blastocyst into an ‘8’ shape and often trapped the inner cell mass (ICM). AH can be also applied on frozen–thawed blastocysts. Artificial opening of the ZP by 3D-PZD after warming of vitrified blastocysts significantly improved the implantation and pregnancy rates.27 Fong et al described a method for enzymatic treatment of the zona pellucida of blastocysts.74 Culture to the blastocyst stage was achieved with the use of sequential media; early and expanding blastocysts were treated with 10 IU/ml pronase for 1 minute at 37°C. Just before complete disappearance of the ZP in the pronase solution, the blastocysts were placed in fresh medium and washed twice. They were cultured for a few hours before transfer. The results obtained showed that ZP-manipulated blastocysts have a high implantation rate (33%), and there is a need to limit the number of AH blastocysts to be transferred to one or two to reduce multiple pregnancies. Park et al reported the use of a 1.48 µm noncontact diode laser for assisted hatching of in vitro matured/in vitro fertilized/in vitro cultured (IVM/ IVF/IVC) blastocysts.75 Short irradiation exposure times (3–5 ms) were applied, and a significant increase in the hatching rate was observed. We have described the use of a 1.48 mm diode laser for AH in human blastocysts.76 Even though no statistically significant differences were observed, a trend towards higher pregnancy and implantation rates was obtained when laser-drilled AH blastocysts were replaced, compared with nondrilled blastocysts (44.4% vs 23.8% and 30.6% vs 11.6%) (Fig 13.2).

Conclusions Several studies have been performed to demonstrate the usefulness and efficacy of AH in different groups of patients using the various methods described. Most of the studies have been done in patients with poor prognosis, including advanced-age patients, patients with elevated concentrations of folliclestimulating hormone (FSH), patients with previous implantation failures, or with embryos with thick ZP, some of them with contradictory results. Only one study included women with endometriosis, not showing improvement after AH.77 Assisted hatching has been applied to fresh early cleavage-stage embryos and blastocysts and also to frozen–thawed

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Table 13.1

Assisted hatching (AH) results reported by different authors (1992–1999)

Author

Study method

Population

Randomized

Pregnancy rate increase

Cohen 19928

AT

Tucker 199364

Yes Yes Yes

Yes, NS Yes, sig. No

Olivennes 199480

AT ZP thinning PZD

Normal FSH ≥15 µm ZP All IVF

Obruca 199442 Tucker 199481

Er:YAG laser AT, CC

No (no control) Yes No Yes (control: AT)

– Yes, sig. Yes, sig. Yes, sig.

Schoolcraft 199437

AT

No

Yes, sig.

Schoolcraft 199582 Stein 199583 Hellebaut 199666 Antinori 199644 Check 199684 Antinori 199657

AT PZD PZD UV laser AT Er:YAG laser

Tucker 199685

AT

No, retrosp. Yes Yes No No Yes Yes No

Yes, sig. Yes Sig. ≥38 years No Yes, sig. Yes, NS Yes, sig. Yes, sig. Yes, sig.

Bider 199786 Chao 199787

AT PZD

Impl. failures Day 3, FSH >15 Impl. failures Age ≥38 years old Impl. failures Elevated FSH 39 years old Impl. failures ≥40 years old ≥3 impl. failures 1st cycle Impl. failures Frozen ET 1st cycle Impl. failures ICSI ≥35 years old ≥38 years old Impl. failures

No Yes

Hurst 199888 Magli 199889

AT AT

Lanzendorf 199890 Meldrum 199891 Antinori 199958

AT AT Er:YAG laser

Edirisinghe 199992

PZD

Yes No Yes Yes No

Baruffi 199953

Yes

No

Veiga 199976

Diode laser ZP thinning Diode laser

Cieslak 199932

3D PZD

1st cycle ≥38 years old ≥3 impl. failures Both ≥36 years old ≥35 years old 1st cycle ≥6 impl. failures ≥38 years old ≥1 impl. failure ZP ≥15 <37 years old 1st cycle Impl. failure, CC 1st cycle All IVF

No Yes, IVF No, TET No Yes, sig. Yes, sig. No No Yes, NS Yes (?) Yes (?) No

Yes, NS No (unpubl.) Yes, NS

Alikani 199993 Nakayama 199994

PZD + frag. removal Piezomicromanipulator

≥6% frag. ≥2 impl. failures

Yes Yes Yes (control: conv. PZD) No, retrosp. Yes

Yes No

Yes, sig. Yes, sig.

AT, acid Tyrode’s solution; ZP, zona pellucida; Er:YAG, erbium:yttrium–aluminum–garnet; CC, co-culture; PZD, partial zona dissection; UV, ultraviolet; frag., fragment/ed; blast., blastomere; D, day; FSH, follicle-stimulating hormone; IVF, in vitro fertilization; impl., implantation; ET, embryo transfer; ICSI, intracytoplasmic sperm injection; retrosp., retrospective; conv., conventional; NS, not significant; sig., significant; TET, thawed embryo transfer; unpubl., unpublished.

embryos. Removal of necrotic blastomeres from partially damaged cryopreserved–thawed embryos may help to maintain their development potential.19,78 Tables 13.1 and 13.2 show the results reported by different authors. A recent Cochrane meta-analysis concluded that there is insufficient evidence to determine any effect of AH on live birth rates.79 From the published results

and taking into account the variability in methods and study designs, the conclusions concerning AH benefits are: 1. 2.

AH does not increase the pregnancy/implantation rate in patients in their first IVF attempt. AH increases the pregnancy/implantation rate in patients with previous implantation failures.

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Assisted hatching (AH) results reported by different authors (2000–2007)

Author

Study method

Population

Randomized

Pregnancy rate increase

Mansour 200095

ZP removal, AT

Yes Yes

No Yes, sig.

Mantoudis 200196

Diode laser Total AH Partial AH ZP thinning Diode laser vs AT PZD AT Diode laser Pronase thinning Diode laser Necrotic blast. removal Diode laser vs AT AH D+3 vs CC PZD blastocysts AT Diode laser ZP thinning Diode laser ZP thinning Diode laser ZP thinning Diode laser ZP thinning Diode laser Pronase ZP thinning Diode laser ZP thinning Diode laser

1st cycle ≥40 years old ≥2 impl. failures Poor responders ≥38 years old ≥2 impl. failures Frozen ET All IVF/ICSI All IVF/ICSI

No

Yes, sig. (for ZP thinning)

Yes (control: AT) No, retrosp.

Malter 200197 Balaban 200269

Rienzi 200219 Hsieh 200298 Milki 200299 Vanderzwalmen 200327 Gabrielsen 2004100 Petersen 200567 Ng 200571 Nadir 200577 Frydman 2006101 Balaban 2006102 Sifer 200670 Petersen 200672 Sagoskin 2007103

Frozen ET

Yes

No No No No No Yes, sig.

≥38 years old 40–43 years old Frozen (Vitrif.) ET Frozen ET ≥2 impl. failures

Yes No, retrosp. No, retrosp Yes Yes

Yes, sig. (for laser) No Yes, sig. No Yes, NS

Frozen ET

Yes

No

Endometriosis

Yes

No

≥37 years old

Yes

No

Frozen ET Frozen ET

Yes Yes

Yes, sig. No

Frozen ET from OHSS IVF cycles Good prognosis

Yes

Yes, NS

Yes

No

AT, acid Tyrode’s solution; ZP, zona pellucida; CC, co-culture; PZD, partial zona dissection; blast., blastomere; D, day; IVF, in vitro fertilization; impl., implantation; ET, embryo transfer; ICSI, intracytoplasmic sperm injection; retrosp., retrospective; NS, not significant; sig., significant; Vitrif., vitrification; OHSS, ovarian hyperstimulation syndrome.

3.

4.

It is not clear whether AH is beneficial for patients of an advanced age, for patients with a thick ZP, or for frozen–thawed embryos. Currently, there is insufficient evidence to recommend assisted hatching as a routine technique in patients undergoing assisted reproduction technologies (ART).

It is questionable whether different methods of AH yield similar outcomes. Large randomized studies comparing AH methods with regard to embryo implantation rate and live birth rate are needed. Follow-up of obstet and postnatal outcome is highly recommended. Mechanical hatching by PZD is limited by the difficulty of creating a hole of consistent size. The variability and possible embryotoxicity remain as potential problems with the use of AT for zona drilling. Enzymatic methods to dissolve or thin the zona seem to be effective and safe. Although the equipment may

be expensive, the use of a 1.48 µm diode infrared laser system for zona drilling offers a low potential risk, it is quick and relatively simple to perform with high consistency between operators, and appears to be the most suitable method for AH in the IVF laboratory.

References 1. Dean J. Biology of mammalian fertilization: the role of the zona pellucida. J Clin Invest 1992; 89: 1055–9. 2. Shabanowitz RB, O’Rand MG. Characterization of the human zona pellucida from fertilized and unfertilized eggs. J Reprod Fertil 1988; 82: 151–61. 3. Ducibella T, Kurasawa S, Ramgarajan S, Kopf GS, Schultz RM. Precocious loss of cortical granules during oocyte meiotic maturation and correlation with an egg-induced modification of the zona pellucida. Dev Biol 1990; 137: 46–55. 4. Cohen J. Assisted hatching of human embryos. J In Vitro Fert Embryo Transf 1991; 8: 179–90.

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5. Trounson AO, Moore NW. The survival and development of sheep eggs following complete or partial removal of the zona pellucida. J Reprod Fertil 1974; 41: 97–105. 6. De Felici M, Siracusa G. Spontaneous hardening of the zona pellucida of mouse oocytes during in vitro culture. Gamete Res 1982; 6: 107–13. 7. Cohen J, Elsner C, Kort H, et al. Impairment of the hatching process following IVF in the human and improvement of implantation by assisted hatching using micromanipulation. Hum Reprod 1990; 5: 7–13. 8. Cohen J, Alikani M, Trowbridge J, Rosenwaks Z. Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod 1992; 7: 685–91. 9. Schiewe MC, Araujo JR, Asch RH, Balmaceda JP. Enzymatic characterization of zona pellucida hardening in human eggs and embryos. J Assist Reprod Genet 1995; 12: 2–7. 10. Gonzales D, Bavister B. Zona pellucida escape by hamster blastocysts in vitro is delayed and morphologically different compared with zona escape in vivo. Biol Reprod 1995; 52: 470–80. 11. Gonzales DS, Jones JM, Pinyopumintr P, et al. Trophectoderm projections: a potential means for locomotion, attachment and implantation of bovine, equine and human blastocysts. Hum Reprod 1996; 11: 2739–45. 12. Gordon J, Dapunt U. A new mouse model for embryos with a hatching deficiency and its use to elucidate the mechanism of blastocyst hatching. Fertil Steril 1993; 59: 1296–301. 13. Schiewe MC, Hazeleger NL, Sclimenti C, Balmaceda JP. Physiological characterization of blastocyst hatching mechanisms by use of a mouse antihatching model. Fertil Steril 1995; 63: 288–94. 14. Montag M, Koll B, Holmes P, Van der Ven H. Significance of the number of embryonic cells and the state of the zona pellucida for hatching of mouse blastocysts in vitro versus in vivo. Biol Reprod 2000; 62: 1738–44. 15. Gordon J, Dapunt U. Restoration of normal implantation rates in mouse embryos with a hatching impairment by use of a new method of assisted hatching. Fertil Steril 1993; 59: 1302–7. 16. Mandelbaum J. The effects of assisted hatching on the hatching process and implantation. Hum Reprod 1996; 11: 43–50. 17. Ludwig M, Al-Hasani S, Felderbaum DK. New aspects of cryopreservation of oocytes and embryos in assisted reproduction and future perspectives. Hum Reprod 1999; 14 (Suppl 1): 162–85. 18. Alikani M, Olivennes F, Cohen J. Microsurgical correction of partially degenerate mouse embryos promotes hatching and restores their viability. Hum Reprod 1993; 8: 1723–8. 19. Rienzi L, Nagy ZP, Ubaldi F, et al. Laser-assisted removal of necrotic blastomeres from cryopreserved embryos that were partially damaged. Fertil Steril 2002; 77: 1196–201. 20. Loret de Mola JR, Garside WT, Bucci J, et al. Analysis of the human zona pellucida during culture: correlation with diagnosis and the preovulatory hormonal environment. Assist Reprod Genet 1997; 14: 332–6.

21. Cohen J, Wiker SR, Inge KL, et al. Videocinematography of fresh and cryopreserved embryos: a retrospective analysis of embryonic morphology and implantation. Fertil Steril 1989; 51: 821–7. 22. Palmstierna M, Murkes D, Csemiczdy G, et al. Zona pellucida thickness variation and occurrence of visible mononucleated blastomeres in preembryos are associated with a high pregnancy rate in IVF treatments. J Assist Reprod Genet 1998; 15: 70–5. 23. Blake DA, Forsberg AS, Johansson BR, Wikland M. Laser zona pellucida thinning – an alternative approach to assisted hatching. Hum Reprod 2001; 16: 1959–64. 24. Khalifa EAM, Tucker MJ, Hunt P. Cruciate thinning of the zona pellucida for more successful enhancement of blastocyst hatching in the mouse. Hum Reprod 1992; 7: 532–6. 25. Liu HC, Cohen J, Alikani M, Noyes N, Rosenwaks Z. Assisted hatching facilitates earlier implantation. Fertil Steril 1993; 60: 871–5. 26. Dale B, Talevi R, Gualtieri R, et al. Intercellular communication in the early human embryo. Mol Reprod Dev 1991; 29: 22–8. 27. Vanderzwalmen P, Bertin G, Debauche Ch, et al. Vitrification of human blastocysts with the HemiStraw carrier: application of assisted hatching after thawing. Hum Reprod 2003; 18: 1504–11. 28. Lyu QF, Wu LQ, Li YP, et al. An improved mechanical technique for assisted hatching. Hum Reprod 2005; 20: 1619–23. 29. Talansky BE, Gordon JW. Cleavage characteristics of mouse embryos inseminated and cultures after zona pellucida drilling. Gamete Res 1998; 21: 277–8. 30. Nichols J, Garner RL. Effect of damage of the zona pellucida on development of preimplantation embryos in the mouse. Hum Reprod 1989; 4: 180–7. 31. Cohen J, Feldberg D. Effects of the size and number of zona pellucida openings on hatching and trophoblast outgrowth in the mouse embryo. Mol Reprod Dev 1991; 30: 70–8. 32. Cieslak J, Ivakhnenko V, Wolf G, Sheleg S, Verlinsky Y. Three-dimensional partial zona dissection for preimplantation genetic diagnosis and assisted hatching. Fertil Steril 1999; 71: 308–13. 33. Alikani M, Noyes N, Cohen J, Rosenwaks Z. Monozygotic twinning in the human is associated with the zona pellucida architecture. Hum Reprod 1994; 9: 1318–21. 34. Malter HE, Cohen J. Partial zona dissection of the human oocyte: a nontraumatic method using micromanipulation to assist zona pellucida penetration. Fertil Steril 1989; 51: 139–48. 35. Hogan B, Constantini F, Lacy E. Manipulating the Mouse Embryo: a Laboratory Manual. New York: Cold Spring Harbor Laboratory Press, 1986. 36. Malter H, Cohen J. Blastocyst formation and hatching in vitro following zona drilling of mouse and human embryos. Gamete Res 1989; 24: 67–80. 37. Schoolcraft W, Schlenker T, Gee M, Jones GS, Jones HW. Assisted hatching in the treatment of poor prognosis in vitro fertilization candidates. Fertil Steril 1994; 62: 551–4. 38. Tadir Y, Wright WH, Vafa O, et al. Micromanipulation of sperm by a laser generated optical trap. Fertil Steril 1989; 52: 870–3.

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Assisted hatching 39. Tadir Y, Wright WH, Vafa O, et al. Micromanipulation of gametes using laser microbeams. Hum Reprod 1991; 6: 1011–16. 40. Palanker D, Ohad S, Lewis A, et al. Technique for cellular microsurgery using the 193 nm excimer laser. Laser Surg Med 1991; 11: 589–6. 41. Strohmer H, Feichtinger W. Successful clinical application of laser for micromanipulation in an in vitro fertilization program. Fertil Steril 1992; 58: 212–14. 42. Obruca A, Strohmer H, Sakkas D, et al. Use of lasers in assisted fertilization and hatching. Hum Reprod 1994; 9: 1723–6. 43. Obruca A, Strohmer H, Blaschitz A, et al. Ultrastructural observations in human oocytes and preimplantation embryos after zona opening using an Er:YAG laser. Hum Reprod 1997; 12: 2242–5. 44. Antinori S, Panci C, Selman HA, et al. Zona thinning with the use of laser: a new approach to assisted hatching in humans. Hum Reprod 1996; 11: 590–4. 45. Neev J, Tadir Y, Ho P, et al. Microscope-delivered ultraviolet laser zona dissection: principles and practices. J Assist Reprod Genet 1992; 9: 513–23. 46. Blanchet GB, Russell JB, Fincher CR, Portman M. Laser micromanipulation in the mouse embryo: a novel approach to zona drilling. Fertil Steril 1992; 57: 1337–41. 47. Neev J, Schiewe M, Sung VW, et al. Assisted hatching in mouse embryos using a noncontact Ho:YSGG laser system. J Assist Reprod Genet 1995; 12: 288–93. 48. Schiewe M, Neev J, Hazeleger NL, et al. Developmental competence of mouse embryos following zona drilling using a non-contact Ho:YSGG laser system. Hum Reprod 1995; 10: 1821–4. 49. Rink K, Delacretaz G, Salathe RP, et al. Non-contact microdrilling of mouse zona pellucida with an objective-delivered 1.48-microns diode laser. Laser Surg Med 1996; 18: 52–62. 50. Rink K, Delacretaz G, Salathe RP, et al. Non-contact microdrilling of mouse zona pellucida with an objective-delivered 1.48 µm diode laser. Laser Surg Med 1996; 18: 52–62. 51. Germond M, Nocera D, Senn A, et al. Microdissection of mouse and human zona pellucida using a 1.48 µm diode laser beam: efficacy and safety of the procedure. Fertil Steril 1995; 64: 604–11. 52. Germond M, Nocera D, Senn A, et al. Improved fertilization and implantation rates after non touch zona pellucida microdrilling of mouse oocytes with a 1.48 µm diode laser beam. Hum Reprod 1996; 11: 1043–8. 53. Baruffi R, Mauri AL, Petersen C, et al. Assisted hatching with a laser diode in patients <37 years old with no previous failure of implantation: a prospective randomized study. Hum Reprod 1999; 14(abstr book 1) [Abstracts of the 15th Annual meeting of the ESHRE, Tours, France]. 54. Montag M, Van der Ven K, Delacretaz G, et al. Laserassisted microdissection of the zona pellucida facilitates polar body biopsy. Fertil Steril 1998; 69: 539–42. 55. Boada M, Carrera M, de la Iglesia C, et al. Successful use of a laser for human embryo biopsy in preimplantation genetic diagnosis: report of two cases. J Assist Reprod Genet 1998; 15: 302–7. 56. Veiga A, Sandalinas M, Benkhalifa M, et al. Laser blastocyst biopsy for preimplantation diagnosis in the human. Zygote 1997; 5: 351–4.

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57. Antinori S, Selman HA, Caffa B, et al. Zona opening of human embryos using a non-contact UV laser for assisted hatching in patients with poor prognosis of pregnancy. Hum Reprod 1996; 11: 2488–92. 58. Antinori S, Versaci C, Dani L, et al. Laser assisted hatching at the extremes of the IVF spectrum: first cycle and after 6 cycles: a randomized prospective trial. Hum Reprod 1999; 14(abstr book 1) [Abstracts of the 15th Annual Meeting of the ESHRE, Tours, France]. 59. Douglas-Hamilton DH, Conia J. Thermal effects in laser-assisted pre-embryo zona drilling. J Biomed Optics 2001; 6: 205–13. 60. Chatzimeletiou K, Picton HM, Handyside AH. Use of a non-contact, infrared laser for zona drilling of mouse embryos: assessment of immediate effects on blastomere viability. Reprod Biomed Online 2001; 2: 178–87. 61. Tinney GM, Windt ML, Kruger TF, Lombard CJ. Use of a zona laser treatment system in assisted hatching: optimal laser utilization parameters. Fertil Steril 2005; 84: 1737–41. 62. Jones AE, Wright G, Kort HI, et al. Comparison of laser-assisted hatching and acidified Tyrode’s hatching by evaluation of blastocyst development rates in sibling embryos: a prospective randomized trial. Fertil Steril 2006; 85: 487–91. 63. Makrakis E, Angeli I, Agapitou K, et al. Laser versus mechanical assisted hatching: a prospective study of clinical outcomes. Fertil Steril 2006; 86: 1596–600. 64. Tucker MJ, Luecke NM, Wiker SR, Wright G. Chemical removal of the outside of the zona pellucida of day 3 human embryos has no impact on implantation rate. J Assist Reprod Genet 1993; 10: 187–91. 65. Kung FT, Lin YC, Tseng YJ, et al. Transfer of frozen– thawed blastocysts that underwent quarter laserassisted hatching at the day 3 cleaving stage before freezing. Fertil Steril 2003; 79: 893–9. 66. Hellebaut S, De Sutter P, Dozortsev D, et al. Does assisted hatching improve implantation rates after in vitro fertilization or intracytoplasmic sperm injection in all patients? A prospective randomized study. J Assist Reprod Genet 1996; 13: 19–22. 67. Petersen CG, Mauri AL, Baruffi RL, et al. Implantation failures: success of assisted hatching with quarter-laser zona thinning. Reprod Biomed Online 2005; 10: 224–9. 68. Ghobara TS, Cahill DJ, Ford WCL, et al. Effects of assisted hatching method and age on implantation rates of IVF and ICSI. Reprod Biomed Online 2006; 13: 261–7. 69. Balaban B, Urman B, Alatas C, et al. A comparison of four different techniques of assisted hatching. Hum Reprod 2002; 17: 1239–43. 70. Sifer C, Sellami A, Poncelet C, et al. A prospective randomized study to assess the benefit of partial zona pellucida digestion before frozen–thawed embryo. Hum Reprod 2006; 21: 2384–9. 71. Ng EHY, Naveed F, Lau EYL, et al. A randomized double-blind controlled study of the efficacy of laser-assisted hatching on implantation and pregnancy rates of frozen–thawed embryo transfer at the cleavage stage. Hum Reprod 2005; 20: 979–85. 72. Petersen CG, Mauri AL, Baruffi RL, et al. Laserassisted hatching of cryopreserved–thawed embryos by thinning one quarter of the zona. Reprod Biomed Online 2006; 13: 668–75.

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73. Nijs M, Vanderzwalmen P, Segal-Berti G, et al. A monozygotic twin pregnancy after application of zona rubbing on a frozen–thawed blastocyst. Hum Reprod 1993; 8: 127–9. 74. Fong CY, Bongso A, Ng SC, et al. Blastocyst transfer after enzymatic treatment of the zona pellucida: improving in vitro fertilization and understanding implantation. Hum Reprod 1998; 13: 2926–32. 75. Park S, Kim EY, Yoon SH, Chung KS, Lim JH. Enhanced hatching rate of bovine IVM/IVF/IVC blastocysts using a 1.48 µm diode laser beam. J Assist Reprod Genet 1999; 16: 97–101. 76. Veiga A, Torelló MJ, Ménézo Y, et al. Use of co-culture of human embryos on Vero cells to improve clinical implantation rate. Hum Reprod 1999; 14: 112–20. 77. Nadir H, Bener F, Karagenc L, et al. Impact of assisted hatching on ART outcome in women with endometriosis. Hum Reprod 2005; 20: 2546–9. 78. Rienzi L, Ubaldi F, Iacobelli M, et al. Developmental potential of fully intact and partially damaged cryopreserved embryos after laser-assisted removal of necrotic blastomeres and post-thaw culture selection. Fertil Steril 2005; 84: 888–94. 79. Seif MM, Edi-Osagie EC, Farquhar C, et al. Assisted hatching on assisted conception (IVF & ICSI). Cochrane Database Syst Rev 2005; 1: CD001894. 80. Olivennes F, Bergere M, Fanchin R, et al. [Assisted embryonal hatching]. Contracept Fertil Sex 1994; 22: 493–7. 81. Tucker M, Ingargiola P, Massey JB, et al. Assisted hatching with or without bovine oviductal epithelial cell co-culture for poor prognosis in vitro fertilization patients. Hum Reprod 1994; 9: 1528–31. 82. Schoolcraft WB, Schlenker T, Jones GS, Jones HW. In vitro fertilization in women age 40 and older: the impact of assisted hatching. J Assist Reprod Genet 1995; 12: 581–4. 83. Stein A, Rufas O, Amit S, et al. Assisted hatching by partial zona dissection of human pre-embryos in patients with recurrent implantation failure after in vitro fertilization. Fertil Steril 1995; 63: 838–41. 84. Check JH, Hoover L, Nazari A, O’Shaughnessy A, Summers D. The effect of assisted hatching on pregnancy rates after frozen embryo transfer. Fertil Steril 1996; 65: 254–7. 85. Tucker MJ, Morton PC, Wright G, et al. Enhancement of outcome from intracytoplasmic sperm injection: does co-culture or assisted hatching improve implantation rates? Hum Reprod 1996; 11: 2434–7. 86. Bider D, Livshits A, Yonish M, et al. Assisted hatching by zona drilling of human embryos in women of advanced age. Hum Reprod 1997; 12: 317–20. 87. Chao KH, Chen SU, Chen HF, et al. Assisted hatching increases the implantation and pregnancy rate of in vitro fertilization (IVF)–embryo transfer (ET), but not that of IVF–tubal ET in patients with repeated IVF failures. Fertil Steril 1997; 67: 904–8. 88. Hurst BS, Tucker KE, Awoniyi CA, Schlaff WD. Assisted hatching does not enhance IVF success in good-prognosis patients. J Assist Reprod Genet 1998; 15: 62–4. 89. Magli MC, Gianaroli L, Ferraretti AP, et al. Rescue of implantation potential in embryos with poor

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prognosis by assisted zona hatching. Hum Reprod 1998; 13: 1331–5. Lanzendorf SE, Nehchiri F, Mayer JF, Oehninger S, Muasher SJ. A prospective, randomized, doubleblind study for the evaluation of assisted hatching in patients with advanced maternal age. Hum Reprod 1998; 13: 409–13. Meldrum DR, Wisot A, Yee B, et al. Assisted hatching reduces the age-related decline in IVF outcome in women younger than age 43 without increasing miscarriage or monozygotic twinning. J Assist Reprod Genet 1998; 15: 418–21. Edirisinghe WR, Ahnonkitpanit V, Promviengchai S, et al. A study failing to determine significant benefits from assisted hatching: patients selected for advanced age, zonal thickness of embryos, and previous failed attempts. J Assist Reprod Genet 1999; 16: 294–301. Alikani M, Cohen J, Tomkin G, et al. Human embryo fragmentation in vitro and its implications for pregnancy and implantation. Fertil Steril 1999; 71: 836–42. Nakayama T, Fujiwara H, Yamada S, et al. Clinical application of a new assisted hatching method using a piezo-micromanipulator for morphologically low-quality embryos in poor-prognosis infertile patients. Fertil Steril 1999; 71: 1014–18. Mansour RT, Rhodes CA, Aboulghar MA, Serour GI, Kamal A. Transfer of zona-free embryos improves outcome in poor prognosis patients: a prospective randomized controlled study. Hum Reprod 2000; 15: 1061–4. Mantoudis E, Podsiadly BT, Gorgy A, Venkat G, Craft IL. A comparison between quarter, partial and total laser assisted hatching in selected infertility patients. Hum Reprod 2001; 16: 2182–6. Malter H, Schimmel T, Cohen J. Zona dissection by infrared laser: developmental consequences in the mouse, technical considerations, and controlled clinical trial. Reprod Biomed Online 2001; 3: 117–23. Hsieh YY, Huang CC, Cheng TC, et al. Laserassisted hatching of embryos is better than the chemical method for enhancing the pregnancy rate in women with advanced age. Fertil Steril 2002; 78: 179–82. Milki AA, Hinckley MD, Behr B. Comparison of blastocyst transfer to day 3 transfer with assisted hatching in the older patient. Fertil Steril 2002; 78: 1244–7. Gabrielsen A, Agerholm I, Toft B, et al. Assisted hatching improves implantation rates on cryopreserved–thawed embryos. A randomized prospective study. Hum Reprod 2004; 19: 2258–62. Frydman N, Madoux S, Hesters L, et al. A randomized double-blind controlled study on the efficacy of laser zona pellucida thinning on live birth rates in cases of advanced female age. Hum Reprod 2006; 21: 2131–5. Balaban B, Urman B, Yakin K, Isiklar F. Laser-assisted hatching increases pregnancy and implantation rates in cryopreserved embryos that were allowed to cleave in vitro after thawing: a prospective randomized study. Hum Reprod 2006; 21: 2136–40. Sagoskin AW, Levy MJ, Tucker MJ, et al. Laser assisted hatching in good prognosis patients undergoing in vitro fertilization–embryo transfer: a randomized controlled trial. Fertil Steril 2007; 87: 283–7.

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14 Human embryo biopsy procedures Alan R Thornhill, Alan H Handyside

Introduction In the mid-1980s, the development of polymerase chain reaction (PCR) strategies for amplification of specific fragments of DNA from single cells1–3 facilitated preimplantation genetic diagnosis (PGD) of inherited disease using one or more cells biopsied from embryos at preimplantation stages after in vitro fertilization (IVF).4 Currently, PGD requires the removal of one or more cells from each embryo, making embryo biopsy comparable to amniocentesis or chorionic villus sampling (CVS) at fetal stages, since the primary aim is the removal of sufficient embryonic tissue to allow diagnosis. Embryo biopsy is a two-step micromanipulation process involving the penetration or removal of part of the zona pellucida surrounding the oocyte or embryo followed by removal of one or more cells. Theoretically, this can be accomplished at any developmental stage between the mature oocyte and blastocyst, but to date only three discrete stages have been proposed: polar body, cleavage stage, and blastocyst. Clearly, each of these stages is biologically different, and thus the strategic considerations have both advantages and disadvantages (Table 14.1). Furthermore, the different biopsy strategies, both between and within developmental stages, require different technical approaches (Table 14.2), each providing varying prospects of success. Many of the biopsy techniques currently in use for human embryos5 were pioneered in animal models, notably the mouse,6,7 rabbit,8 cow,9 and marmoset.10 While the total number of human embryos biopsied in clinical cases is vast, relatively little work has been published to define the relative merits of different biopsy methods and their safety and efficacy in clinical application. This chapter focuses on cleavagestage embryo biopsy, since the majority of clinical cases and PGD centers have employed this technique.11

Penetration of the zona pellucida Until the advent of noncontact lasers for use in micromanipulation (see below), two basic methods

were employed for penetrating the zona. Both of these were pursued initially as a means to enhance fertilization rates with oligozoospermic men, and have now been overtaken for this purpose by the use of intracytoplasmic sperm injection (ICSI). The first approach, partial zona dissection (PZD), involves using a fine needle to penetrate through the zona and, avoiding damage to the oocyte or embryo, penetrating out through the zona again at a distance around the circumference.12 The embryo can then be detached from the holding pipette as it is effectively held on the needle, and a gentle rubbing action against the side of the holding pipette used to make a slit between the two apertures generated by the needle. Although a narrow-diameter micropipette can be pushed through such a slit, it is difficult to use one large enough to aspirate cleavage-stage blastomeres, and, with the human embryo, pressure on the zona, can lead to lysis of blastomeres and/or, where a slit has been made, force blastomeres out through the slit. The latter approach is used for embryo biopsy in some centers, but requires highly skilled micromanipulation, can be difficult to control, does not allow precise selection of blastomeres, and the risk of lysis can be high. A modification is to make two slits to create a ‘flap’ or ‘cross’ in the zona that can be flipped open, allowing more flexibility in the size of the opening created. This method is effective for both polar body and blastomere biopsy.13 In general, mechanical methods for zona penetration are time-consuming and require skillful micromanipulation, possibly making them inaccessible to some IVF laboratories. As an alternative, zona drilling using acid Tyrode’s solution (pH 2.2–2.4) to dissolve the zona glycoproteins has been extensively used and is commercially available from most culture media manufacturers. Again, this method was developed in the mouse embryo model, as a possible means to improve fertilization rates with low sperm densities.14 However, its use with human oocytes, while increasing the incidence of fertilization, arrested the further development of the zygote, presumably consequent to changes in intracellular pH.15 With zona drilling, the effect of the acid Tyrode’s solution is localized to a

ADO, allele drop-out; TE, trophectoderm; ICM, inner cell mass.

Sample multiple cells on day 5/6 Increased amplification efficiency and accuracy (reduce ADO) Increased scope for diagnostic testing (genes + chromosomes) TE sampled rather than ICM Embryo quality preselected (higher implantation rate) Reduced diagnostic burden and cost Low template analysis required (not single cell sensitivity)

One or two cells removal still results in viable development

6–10 cell

Blastocyst (trophectoderm)

95% embryo cohort available for analysis

2–4 cell

Biopsied embryos develop into normal blastocysts

Diagnosis of maternally and paternally inherited disease Gender determination possible Large body of clinical data available 1–3 cells available for analysis

TE cells may not represent embryo proper Fewer embryos for analysis

Time for analysis may be limited Blastocysts may need cryopreservation

Reduced embryo cohort on day of biopsy Possible selected cell allocation to TE/ICM

Detrimental effects of acid/reduced cell mass Possible selected cell allocation to TE/ICM

Chromosomal mosaicism compromises accuracy Choice of blastomere is critical Time for analysis may be limited Most cells in interphase (no karyotypic data) Single cell sensitive analysis required Reduced embryo implantation potential post-biopsy

Maternally inherited disease only Gender determination not possible Simultaneous biopsy (first polar body may degenerate) Sequential biopsy (extra manipulation required)

Only one cell available for analysis Increased risk of diagnostic error Gender determination not possible Maternally inherited disease only Fewer embryos for transfer (crossover at metaphase I)

Disadvantages

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Cleavage stage (blastomeres)

Two cells for analysis (greater accuracy/reliability) Removal has no effect on embryo development Increased time to perform diagnosis prior to transfer Can transfer between day 2 and beyond

~80% aneuploidy originates in maternal metaphase I

Removal has no effect on embryo development Increased time to perform analysis prior to transfer Can transfer between pronucleus stage and day 2 or beyond

Advantages

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first and second polar bodies

Oocyte first polar body

Developmental stage

Strategic considerations for preimplantation genetic diagnosis (PGD) biopsy at different developmental stages

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Table 14.1

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Difficult to learn Operator dependent Time consuming Operator dependent Effect on cryopreservation? Difficult to limit aperture size Cost (US $30 000 to 60 000) Not all systems portable Invisible thermal damage/stress

Least invasive to embryo (safer) Inexpensive Improved survival after freeze–thaw? Relatively inexpensive Widespread clinical use

Rapid and reproducible Simple to use Documentation/measurement software

Mechanical

Chemical (acid Tyrode’s)

Laser (1.48 µm noncontact diode)

Laser hatching + aspiration/laser ablation

Mechanical/chemical/laser + stitch and pull

Pre-empt spontaneous hatching – saves time Otherwise – as above Rapid (more time for analysis) Some control over cell numbers sampled Rapid

Noninvasive (cells undisturbed in zona)

(ii) Trophectoderm sampling Spontaneous hatching/herniation

Mechanical/chemical/laser + herniation

Limited ability to select cell Limited ability to select cell Damage to non-biopsied cells?

Aspiration pipette does not contact cells Aspiration pipette does not contact cells

Fluid displacement Mechanical displacement

Biopsied/non-biopsied cell damage Operator skill essential Biopsied/non-biopsied cell damage

No control over timing or cell numbers Time for analysis very limited

Cell lysis during aspiration

Ability to select cell

High blastocyst development rate

Appropriate microtools needed Sensitive suction device Operator skill essential Operator skill essential

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(i) Cleavage stage blastomere biopsy Aspiration

Laser alignment and validation Consider pulse time carefully Distance between laser and zona

Acid Tyrode’s solution (pH 2.2–2.4) Sensitive control of acid flow Rinse acid from embryos Double tool holder required

Operator skill essential Appropriate microtools needed

Factors critical to success

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Cell removal method

Limitations

Benefits

Embryo biopsy methods – benefits, limitations, and factors critical to success

Zona penetration method

Table 14.2

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small area of the zona using a fine micropipette, with an inner diameter of 5–10 µm. The micropipette filled with acid Tyrode’s solution is brought into direct contact with the zona at the appropriate position, and a combination of slight pulling away and ‘stroking’ movements used to control the flow of acid and the area to be drilled, respectively. Medium pH was originally maintained by employing phosphate-buffered saline but is now routinely maintained using modified culture medium buffered with either 4-morpholinopropanesulfonic acid (MOPS) or 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (HEPES). When the drilling is complete, the micropipette is immediately withdrawn. More recently, there has been a shift across centers worldwide from zona drilling using acid Tyrode’s solution16 to laser ablation of the zona pellucida. Indeed, according to a recent survey of PGD centers, the laser has overtaken acid Tyrode’s solution as the most popular form of zona ablation, accounting for 60% cleavage-stage embryo biopsies.11 This shift may be more to do with ease of use and the elimination of the need for a double tool holder rather than any measurable improvement in safety or efficacy. The preferred model of laser is the near-infrared (NIR) solid-state compact diode 1.48-µm laser. The advantage of using light as a cutting tool is that it obviates the need for disposable or reusable cutting tools, it is extremely precise, and, if used appropriately it provides consistent, repeatable, and rapid results. Moreover, since neither microtools nor reagents are required to dissect the zona, the opportunity for introducing contamination or pH changes in the medium surrounding the embryo is greatly reduced. The 1.48-µm diode laser is small but at the appropriate pulse duration can emit light at power levels sufficient to cause selective thermal disruption of the zona pellucida glycoproteins and is not absorbed by water. This noncontact laser can be inserted into the body of the microscope on which the manipulations take place or it can be integrated in a special objective and the beam delivered to the target through the dish. Since the laser beam travels up through an objective which lies below the sample, localized heating causes denaturation of the zona proteins in a cylindrical spot where the laser beam is focused, and the size of the aperture created is controlled by adjusting the duration of the laser pulse. The thermal energy created produces a groove in the zona perpendicular to the microscope stage, rather than a circular aperture. However, an ‘aperture’ is produced in the zona at the point at which the zona is perpendicular to the microscope stage. The size of the aperture (or more accurately the width of the groove at its widest point) created in the zona ranges from 5 to 20 µm and is governed by the pulse irradiation time (ranging from 3 to 100 ms) or the accumulation of pulses along the length of the zona margin. The precision of the laser is

illustrated by the fact that drilled mouse and human embryos show no sign of extraneous thermal damage under light or scanning microscopy.17 Many clinics are now using this equipment for assisted hatching as well as PGD18 and there appears to be no detrimental effect of the laser itself on development to the blastocyst stage or pregnancy rates in animal and human studies.19–23 However, studies of the immediate effects at the blastomere level in a mouse model have shown that the laser can cause damage if used inappropriately.24 Certainly, if the laser beam is fired in an area in direct contact with a blastomere, its viability is always compromised. However, as the pulse length and therefore localized heating is increased, the distance between the laser beam and blastomere required to avoid damage increases. Hence, care is required to drill the zona away from underlying blastomeres and from as far away as possible, and also to use minimum pulse lengths to restrict any damaging effects. Several practical guidelines have emerged to ensure safe and effective use of the laser for human embryo biopsy as follows. A single aperture is used for cellular aspiration as double or multiple apertures may cause problems during embryo hatching as the embryo will attempt to hatch out of multiple openings, which could compromise further inner cell mass (ICM) development or lead to an increased monozygotic twinning rate. To generate the desired aperture, it is preferable to use several pulses of short duration rather than a single pulse of long duration and higher energy, which could cause thermal damage. During laser use, it is imperative to maintain the oocyte or embryo as close to the bottom of the biopsy dish as possible to allow a focused beam to ablate the zona pellucida. As the embryo is raised above the dish surface, the beam energy is diffused and can create localized heating or simply prevent effective ablation of the zona.

Polar body biopsy Neither the first nor second polar body is required for successful fertilization or normal embryonic development. Thus, removal of either the first or second polar body or both for the purposes of genetic diagnosis should have no deleterious effect per se on the developing embryo. Originally, it was suggested that biopsy and genetic analysis of the first polar body would allow PGD of maternal defects prior to conception.25 Apart from some arguable practical advantages (see below), this concept was also attractive as it involves manipulation of only the human egg and not the fertilized embryo, and would therefore be more acceptable to those with moral or ethical objections to screening embryos, as is the legal situation in several European countries including Italy, Germany, and Switzerland.26 For preconception diagnosis, either the

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first polar body alone or both the first and second polar bodies may be biopsied to provide genetic information relating to a particular embryo. Initially, preconception diagnosis focused on the former approach. However, biopsy of the first polar body has limited applicability for PGD for a number of reasons. The procedure only allows the detection of maternal genetic defects, and crossing-over of homologous chromosomes during meiosis I can prevent identification of the maternal allele remaining in the oocyte, leading to a reduction in the number of embryos available for transfer.27 Also there is only the possibility of a single cell for analysis, leading to a lower overall reliability (in contrast to cleavage-stage biopsy in which 2 cells may be taken for independent analysis). As a consequence, it was suggested that more misdiagnoses would result from polar body analyses when compared with blastomere analysis28 and, to overcome these disadvantages, both the first and second polar bodies were removed for analysis29 after first assessing the safety of removing the second polar body in a mouse model.30 This approach has been successfully applied to PGD for the detection of a large number and variety of different single gene disorders, chromosomal aneuploidies, and maternal chromosome translocations.31 The first polar body can be removed from the oocyte on the day of the oocyte collection between 36 and 42 hours after injection of human chorionic gonadotropin (hCG), as long as the oocyte has entered metaphase II and fully extruded the first polar body.25 To perform polar body biopsy by mechanical means, a holding pipette and a beveled micropipette (12–15 µm in diameter) are needed. The oocyte is held in place with the polar body at the 12 o’clock position. The beveled micropipette is passed through the zona and into the perivitelline space tangentially towards the polar body. The polar body may then be aspirated into the pipette. Alternatively, after mechanical zona dissection to form a flap or cross or laser ablation, an aspiration micropipette is introduced into the perivitelline space, and the polar body removed. If the polar body is still attached to the ooplasm, further incubation may be required to permit complete extrusion.25 Most approaches to polar body biopsy have adopted mechanical or laser techniques rather than chemical methods. While live offspring resulted after treating the zona pellucida of mouse oocytes with acid Tyrode’s solution, studies using human oocytes showed that, despite fertilization, there was an inhibitory effect on embryonic development15 due to a direct effect of acid on the oocyte spindle, possibly as a result of the difference in thickness of the human and mouse zona pellucida. The first and second polar body can be removed simultaneously29 from the zygote between 18 and 22 hours post-insemination, but the first polar body may have degenerated by this time, leading to possible diagnostic failure. Simultaneous biopsy of the two

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polar bodies is acceptable for fluorescent in situ hybridization (FISH) analysis, since they can provide distinguishable results.32 Moreover, the polar bodies are morphologically distinguishable: the first polar body tends to have a crinkled surface and may fragment; the second polar body is generally smooth and may have a visible interphase nucleus under interference contrast. However, sequential biopsy of polar bodies, where the first polar body is removed on day 0 and the second polar body on day 1, is recommended for PCR analysis to determine recombination events between the first and second polar body. In addition to the obvious advantages of not damaging the embryo and allowing a maximum time for genetic analysis, at a technical level, analysis of both polar bodies allows detection of allele drop-out (ADO). ADO is the random amplification failure of one parental allele after PCR from single cells,33 and is therefore a significant source of potential error in PGD. Despite the removal of both polar bodies, in many cases, cleavagestage biopsy is also required to confirm the polar body diagnosis. Despite the large number of cycles reported using polar body biopsy and analysis, relatively few centers have used the approach. This may be the result of a number of factors. First, the approach can only be applied to maternally inherited diseases. Secondly, diseases that are detected by assessing changes in gene product34 would not be candidates for this approach. Thirdly, polar body biopsy cannot be used for gender determination. Finally, biopsy of both the first and second polar bodies is required for optimal diagnostic efficiency and, although this can be achieved by either sequential or simultaneous biopsy with successful results, it is labor-intensive and may involve oocytes and zygotes, which, ultimately, do not develop into therapeutic-quality embryos. If polar bodies are sampled sequentially and cleavage-stage blastomere confirmation is required, three independent manipulations are required, with the possibility of ICSI in between (for PCR-based cases), making a total of four manipulations on the same oocyte and embryo. However, in experienced hands, the three independent biopsy manipulations appear to have no deleterious effect on development.35,36

Cleavage-stage embryo biopsy The first PGD cycles were carried out in late 1989 in a series of couples at risk of X-linked disease and involved cleavage-stage embryo biopsy.37 Cleavagestage biopsy has remained the most widely practiced form of embryo biopsy worldwide (according to ESHRE PGD Consortium, it accounts for around 90% of all reported PGD cycles11). However, there have been a number of modifications and improvements since 1989 (see Appendix for a typical clinical protocol). In the first cases, a tapered micropipette with a

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Fig 14.1 Human cleavage-stage embryo from which a single blastomere with a single visible interphase nucleus is being removed by micromanipulation after laser ablation of the zona pellucida.

narrow lumen (internal diameter 5–7 µm) containing acidified Tyrode’s solution (pH 2.2–2.4) was used to drill relatively large apertures (20–30 µm) in the zona, The pipette is placed close to the zona pellucida and the acidified solution gently expelled from the pipette until the zona thins and an aperture is drilled (in some cases, the zona can be seen to ‘pop’ as an aperture is made). The flow can be controlled via an oil-filled syringe (hydraulic), air-filled syringe (pneumatic), or by using a mouth pipette. The human zona is bilayered and the zona drilling process must be carefully monitored as the outer layer dissolves more rapidly than the inner layer. Moreover, there is great variation in zona pellucidae, both between and within cohorts of human oocytes and embryos. The final diameter of the aperture made will be determined by a combination of the above factors. An excessively large aperture may result in the unwanted loss of blastomeres but, more significantly, may indicate that the blastomeres were exposed to potentially damaging quantities of acid, which could compromise further development. A second micropipette, filled with biopsy medium and held in a double holder alongside the acid Tyrode’s pipette, can be used to aspirate single cells.38– 40 It is possible to use a single micropipette for both drilling and aspiration, but care is needed to prevent overexposure to acid.41,42 Any advantage accrued in terms of speed of the procedure may be offset by potential damage as a result of overexposure to acid. A typical procedure for cleavage-stage biopsy using laser and blastomere aspiration is provided in the Appendix and is illustrated in Fig 14.1. Briefly, following laser ablation of the zona pellucida adjacent to the blastomere selected for analysis, the blastomere is aspirated by gentle suction using a polished pipette. The aperture may be sited adjacent to either a selected blastomere or a subzonal space between blastomeres. A finely polished ‘sampling’ pipette (internal diameter of 30–40 µm, depending on the cell size) is used to aspirate the blastomere. The pipette is placed through the aperture, close to the blastomere to be aspirated. By gentle suction, the blastomere is drawn into the

pipette while the pipette is withdrawn from the aperture. The aperture of the sampling pipette is critical for successful biopsy. If the internal diameter is too large for the cell being removed, the pipette will have little purchase on that cell and may result in unwanted suction on nonbiopsied cells. Conversely, an undersized pipette will cause the biopsied cell to be squeezed unnecessarily, resulting in blebbing on the cell membrane and ultimately lysis, which will probably reduce the chances of a successful diagnosis in that embryo. Similarly, use of a holding pipette with an internal diameter of 30 µm (i.e. larger than a regular ICSI holding pipette) ensures safe and reliable suction on the zona, particularly during difficult biopsies. Once the blastomere is free of the embryo, it is gently expelled from the sampling pipette. Following biopsy, the embryo should be rinsed in culture medium at least twice to remove residual embryo biopsy medium and acid Tyrode’s solution (if applicable) before returning to culture. The blastomere should be washed extensively in handling medium before proceeding to the analysis. The most frequently used method of blastomere removal is aspiration, but other methods have been described and used clinically, although no studies have been conducted to compare their relative safety and efficacy. In the extrusion method, after zona pellucida drilling, the blastomere is extruded through the aperture by pushing against the zona at another site (usually at 90° to the aperture) using a blunt pipette.43 The slit in the zona pellucida can be introduced using mechanical means, chemical (acid Tyrode’s solution) exposure, or laser ablation. Another variation in the method of cell removal involves fluid displacement, whereby culture medium surrounding the embryo is used to displace individual cells following a zona breach. This method was pioneered in mouse embryos by introducing a slit in the zona with a sharpened needle and, through a second puncture site, injecting medium to dislodge the blastomere through the first puncture site. 44 This method requires the production of two separate apertures and considerable skill to displace the blastomere of choice, but has since been modified for clinical application.45 Challenges common to both of these methods are to ensure the selected cell is removed and the difficulties encountered when two different cells are required for analysis.

Practical considerations for embryo biopsy Preparation before biopsy ICSI is recommended for all PCR cases to reduce the chance of paternal contamination from extraneous sperm attached to the zona pellucida or nondecondensed sperm within blastomeres. Similarly, all

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cumulus cells should be removed before biopsy, as these cells can contaminate both FISH and PCR diagnosis. Embryo and blastomere identity (individual drops or dishes) should be checked throughout the procedure so that diagnostic results can be reliably linked to specific embryos.46,47 The use of standard IVF culture medium during biopsy is acceptable, but its effectiveness may be highly dependent upon the developmental stage of the embryo biopsied. Commercially produced calcium- and magnesiumfree (Ca2+/Mg2+-free) medium is widely available and is used by many centers for routine clinical biopsy, with the benefit of reducing the frequency of cell lysis combined with a shorter time needed to perform the biopsy procedure.46,48

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of cells to the ICM and trophectoderm and abnormal postimplantation development,50 whereas human embryos biopsied on day 2 show cleavage rate retardation and smaller blastocysts.49 Conversely, four-cell stage human embryos surviving freeze–thaw procedures with the loss of one or more blastomeres can develop, implant, and result in live birth, albeit at a reduced rate compared with nonfrozen embryos. Stringent biopsy policies have the benefits that fewer embryos need to be biopsied and fewer cells prepared and tested, with only developmentally competent embryos considered. On the down side, an opportunity to identify genotypes on a full cohort of embryos may be lost.

Timing of biopsy

Number of cells to remove during cleavage-stage biopsy

Since the first clinical application of PGD, culture media have been improved and optimized, and the new generation of media are designed, tested, and manufactured to high-quality control standards specifically for clinical use. Although embryos developed to the blastocyst stage, pregnancy rates after transfer were very low and, importantly for embryo biopsy, most embryos did not appear to compact. With the newer media, compaction on day 3 is much more pronounced, which has necessitated the use of calciumand magnesium-free (Ca2+/Mg2+-free) medium to reverse the initial calcium-dependent adhesion.48 The use of Ca2+/Mg2+-free medium also facilitates later biopsy (i.e. beyond the 8-cell stage), making the timings more flexible. Most cleavage-stage biopsy takes place on the third morning following insemination, although the exact timing varies according to timings of procedures in different laboratories. One variation is to alter the timing of ICSI to allow cleavage-stage biopsy at the same embryonic stage, but late on day 2 (biopsy at earlier cleavage stages on day 2 may adversely affect embryo development49), allowing more time for genetic analysis. In cases where retarded development is observed, the possibility of delaying the biopsy procedure to allow diagnosis of a larger proportion of the embryo cohort should be considered. Furthermore, as a result of increased use of sequential media and experience with blastocyst culture and transfer, most groups routinely delay transfer until day 4 or 5, allowing more time for analysis and with the additional aim of improving pregnancy and implantation rates, because developing embryos that have undergone further cleavage divisions following biopsy can be preferentially selected for transfer. Most laboratories exclude very poor-quality embryos or those not reaching a predefined cell stage from the embryo biopsy procedure. Of centers surveyed, most will consider only embryos at the fivecell stage and beyond for biopsy.16 Biopsy at the four-cell stage in mice results in a distorted allocation

In deciding how many cells to biopsy from cleavagestage embryos, it is necessary to balance diagnostic accuracy with potential to implant and develop, which is progressively compromised as a greater proportion of the embryo is removed.51 There is no consensus on the number of blastomeres that can be safely removed during cleavage-stage embryo biopsy. In many centers, a second blastomere is removed from embryos having seven or more cells, regardless of the type of analysis involved, but this approach has been criticized as compromising the implantation potential of the biopsied embryo based on extrapolation from frozen–thaw embryo implantation rates.52 The decision to remove one or two cells is based on many factors, including the embryo cell number and the accuracy and reliability of the diagnostic test used. If removal of two cells is considered, it is recommended to be undertaken only on embryos with six or more cells.53 While removal of two blastomeres decreases the likelihood of blastocyst formation, compared with removal of one blastomere, day 3 in vitro developmental stage is a stronger predictor for day 5 developmental potential than the removal of one or two cells. The biopsy of only one cell significantly lowers the efficiency of a PCR-based diagnosis, whereas the efficiency of the FISH PGD procedure remains similar whether one or two cells are removed. However, a recent trial demonstrated that live birth rate was compromised at a level of one birth for every 33 cycles of two-cell embryo biopsy, suggesting that, ideally, onecell biopsy should always be performed unless the diagnostic test is suboptimal.54 In the case of lost or anucleate blastomeres and failed diagnosis, rebiopsy of embryos is possible but embryo cell number and timing of rebiopsy should be considered to avoid excessive harm to the embryo. Although technically challenging, the original zona breach site should be accessed to prevent later problems with embryos hatching via multiple hatching sites. No specific recommendations for time limits for embryos out of the incubator are available but,

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ideally, biopsy should be performed as quickly as possible to ensure pH, temperature, and osmolality are maintained. A documented record for biopsy timings is recommended for quality control/quality assurance purposes.46,55

Safety and success rates after biopsy The reliability of cleavage-stage biopsy has now been established in many centers, and in the latest ESHRE PGD Consortium report the efficiency of successful embryo biopsy is 98% in over 70 000 cleavage-stage embryos in clinical PGD cycles.11 Pregnancy rates after PGD are notoriously difficult to assess between different indications and centers. Nevertheless, in the largest series analyzed in detail to date, mostly following cleavage-stage biopsy, pregnancy rates are only 18% per oocyte retrieval and 25% per embryo transfer on average.11 The reasons for the apparently low success rates are many-fold, but unsurprising considering that a proportion of embryos cannot be transferred because they are diagnosed as affected, and in many countries the number of embryos transferred is limited to a maximum of two. To demonstrate the possible detrimental effects of embryo biopsy alone, one would need to conduct a clinical trial involving biopsied and nonbiopsied embryos which would be transferred after selection on purely morphological grounds following biopsy (i.e. without any genetic selection). Such a trial would be considered unethical. However, data from a recent trial provide some insight into the possible detrimental effects of biopsy, with a reduction in implantation potential evident in undiagnosed biopsied embryos compared with nonbiopsied control embryos.56,57 It is well established in mammalian embryos that as an increasing proportion of the embryo is removed or destroyed before transfer, implantation and fetal development rates decline, suggesting a lower limit of embryo mass compatible with implantation and development.58 Reduction of 50% or more of the cell mass frequently results in cell proliferation in the absence of normal differentiation; thus, it is important to minimize the cellular mass removed at biopsy. However, cell reduction within this limit is compatible with normal embryo metabolism, blastocyst development, and fetal growth, while cell numbers in the trophectoderm (TE) and inner cell mass (ICM) of blastocysts were in proportion to the cellular mass removed at biopsy, making cleavage-stage biopsy for PGD a viable option.38 Hence, human cleavage-stage biopsy is delayed until just before the beginning of compaction, the process of intercellular adhesion and junction formation, which progressively makes removal of blastomeres more difficult and eventually impossible without causing damage to the embryo. Generally, cells identified as having completed the third cleavage division (on the basis of their size) are selected for biopsy. Theoretically, therefore, each blastomere removes only

one-eighth of the cellular mass of the embryo. As zona drilling for assisted hatching may be beneficial, it is also possible that this offsets to some extent the adverse effects of reducing the cell mass of the embryo. In frozen embryo transfer (FET) cases, viable pregnancies can be achieved and no increase in fetal abnormalities has been reported following transfer of cryopreserved embryos in which some cells have been destroyed by freezing and subsequent thawing.59 Indeed, estimates of the loss of implantation potential have been made based on outcomes following FET involving embryos with one or more nonviable cells after thawing.52 It is now apparent that cleavage-stage biopsy should be considered a ‘cost’ to the embryo, and this must always be weighed against the potential benefit to the embryo of any diagnostic testing.

Selection of cells in the cleavage-stage embryo Biopsy at cleavage stages is based on the principle that at these stages the blastomeres remain totipotent and equivalent, such that the removal of a single blastomere will (1) provide a representative sample of the entire embryo and (2) compromise the embryo only to the extent of one-eighth of the embryo mass rather than removal of a developmentally important blastomere. The importance of selecting a blastomere with a single visible interphase nucleus cannot be stressed enough (Fig 14.1). It is probably the most challenging aspect of cleavage-stage biopsy, and time spent in careful examination of the embryo and orienting it to selectively remove specific blastomeres is essential to attain the high diagnostic efficiencies required for clinical effectiveness. The reasons for this are that, first, an interphase nucleus is essential for FISH analysis, since the nucleus is prepared on a slide by a process of cell lysis in which individual chromosomes will not be visible and are likely to be lost.60 Secondly, postzygotic chromosomal mosaicism arising during cleavage is known to be associated with nuclear abnormalities.61 The exception is binucleate blastomeres, in which there are two normalsized nuclei. In most cases, these are generated through failure of cytokinesis, and both nuclei contain the normal diploid chromosomal complement for that embryo.62 In general, multinucleate cells should not be selected at biopsy if FISH analysis for aneuploidy detection follows, and the removal of mononucleate cells only is recommended.46,61 For accuracy during FISH-based diagnosis, it is advisable to only use bi- or multinucleated cells as a backup to biopsied mononucleated cells. This may be less critical for PCR-based testing in which presence or absence of a specific parental chromosome is important rather than copy number per se. However, even with careful blastomere selection, diagnostic efficiency is not 100%, and aneuploid results are common even in mononucleate blastomeres primarily as a result of

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chromosomal mosaicism.62 Biopsy of two nucleated blastomeres is only possible in good-quality embryos at a sufficiently advanced stage, such that even with a two-cell biopsy policy, a mixture of embryos with one or two blastomeres for analysis is common.53 Where possible, one of the smaller blastomeres should be selected to minimize the reduction in mass and the relative sizes of cells may provide an indication of recent mitosis. This may also reduce the risk that a cell in metaphase will be taken, the chromosomes of which could be lost during the fixation process.

Concerns surrounding cleavage-stage embryo biopsy As with any micromanipulation procedure involving human gametes or embryos, every reasonable precaution should be taken to minimize damage and stress during the procedure. General precautions include the correct installation, calibration, and maintenance of all micromanipulation equipment (particularly the laser). In advance of all clinical procedures, one should ensure that all appropriate reagents and micromanipulation tools are available, sterile, and within their expiration date. Biopsy should be performed by a suitably qualified and trained person. Regular reviews of biopsy efficiency, postbiopsy morphology, and cell numbers of untransferred embryos provide an indication of the possible harm as a result of biopsy, as do pregnancy rates after biopsy (particularly those not developing beyond the biochemical stage).55 Clearly, effects on postimplantation development should also be closely monitored, as any increase in fetal malformations or congenital abnormalities would be unacceptable. To date, studies of pregnancies and children born after PGD have identified no significant increase in abnormalities above the rate seen in routine IVF.11,63-65 The main problem in terms of diagnostic efficiency with cleavage-stage biopsy is the presence of chromosomal mosaicism, which is reported to occur in up to 80% embryos.66–68 A full discussion of the impact of chromosomal mosaicism on the accuracy of PGD is beyond the scope of this review, but its impact can be significant. Mosaicism is thought to be the primary reason for the high rate of false positives depleting the pool of chromosomally ‘normal’ embryos for transfer and hence significantly lowering the chance of live birth following preimplantation genetic screening (PGS) for chromosomal aneuploidy compared with controls in a recent randomized controlled trial.56

Blastocyst-stage biopsy (trophectoderm biopsy) The number of cells present at the blastocyst stage make blastocyst biopsy more akin to early prenatal diagnosis and therefore, to some, more ethically

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acceptable. In theory, TE cells, which form the spherical outer epithelial monolayer of the blastocyst, can be removed without harming or depleting the ICM from which the fetus is derived. For blastocyst biopsy, it is therefore possible to remove more than 10 TE cells for analysis, which would overcome many of the problems encountered in single-cell PCR and FISH. FISH analysis would be more successful with a virtual guarantee of a result for each sample and the problems of split signal, signal overlap, or probe failure would be significantly less misleading. In the case of PCR, the problems of amplification failure and ADO or preferential amplification would be much reduced. Indeed, when more than two cells are present in the same sample tube, these problems have been shown to virtually disappear,69 particularly if using whole genome amplification (WGA) techniques.70 With or without WGA, the availability of more cells automatically increases the diagnostic possibilities (more chromosomes analyzed with FISH or more specific sequences with PCR, or both). In the mouse, TE biopsy is easily achieved by partial zona dissection using mechanical means, followed by a period in culture during which the expansion of the blastocele cavity forces the TE to herniate out of the slit.71 The herniating TE vesicle can then be excised on a bed of agarose by using a needle (which can be hand-held or attached to a micromanipulator) and a cutting action close to the zona, which causes the embryo to roll. Both the biopsied embryo and TE vesicles often remain expanded, since they appear to be resealed, possibly as a consequence of twisting at the constriction. Furthermore, to some extent, the size of the TE biopsy can be controlled by the size of the slit and the length of incubation. A similar approach was used to biopsy human blastocysts on day 5 or 6 postinsemination72 and was later used to examine effects on viability following biopsy, with the finding that hCG production was equivalent for biopsied and nonbiopsied controls.73 Another more aggressive technique to remove trophectoderm cells during blastocyst biopsy is the mechanical stitch and pull method.74 The best technique seems to be to stabilize the blastocyst by gentle suction and make an incision at the pole opposite to the ICM mass using a 2-µm beveled pipette. The pipette is pushed in and out through the zona and pulled upwards to make the incision. The blastocysts are then left for 6–24 hours until some trophectoderm herniates through the slit. When herniation involves about 10–25% of the blastocyst (10–30 cells), the trophectoderm is excised using a glass needle. More recently, noncontact infrared lasers have been used not only to create an opening to assist hatching but also to excise the herniating trophectoderm.75 Originally, pregnancy rates following blastocyst-stage transfers were too low to consider biopsy at this stage. With the development of sequential media, the proportion of embryos developing to the blastocyst stage has

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increased, and implantation rates per blastocyst transferred are significantly better than at cleavage stages.76 Another concern was the effect that removal of a proportion of the TE cells and damage of additional cells in the process would have on implantation. However, skilled practitioners are able to biopsy 8–10 TE cells (up to 20) from blastocysts on day 5 using a noncontact infrared laser for zona drilling and excision of herniating TE cells, and achieve pregnancy and implantation rates that are comparable to those for nonbiopsied controls.77 The high incidence of multiple pregnancies in PGD demands efforts to reduce the number of unaffected embryos transferred, and transfer of blastocysts with high implantation potential is an effective strategy.78

Challenges associated with blastocyst biopsy Currently, the main limitation of blastocyst biopsy is the low number of embryos that reach the blastocyst stage in vitro for unselected patients, even with improved culture conditions. Since a high number of embryos are needed for successful PGD to allow for sufficient embryo selection from the desired genotype, blastocyst culture may not produce enough embryos for diagnosis and transfer to make PGD at this stage effective for all patients and may only be suitable for younger, good prognosis patients. An additional problem is that TE cells may have diverged genetically from the ICM as, in approximately 2% of human conceptions, confined placental mosaicism (CPM) is observed79 in which the chromosome status of the embryo is different from the placenta. In a mouse model, abnormal cells were shown to be preferentially allocated to the trophectoderm80 but the situation is less clear in the human. The level of mosaicism in the human blastocyst is lower than that in cleavage-stage embryos81 and, where present, often takes the form of polyploidy in the trophectodermal lineage82 with no obvious preferential allocation of aneuploid cells to the TE lineage.83 Considering polyploidy cells, for PGD analyses using FISH, if enough chromosomes are analyzed then any underlying abnormality (such as trisomy 21) may be recognized within the polyploidy (HC Kuo and AH Handyside, unpublished observation). Similarly, for PCR-based diagnoses, the presence of multiple copies of each chromosome in polyploid cells should pose few problems, so long as both parental copies of the chromosome are represented. Clearly, chromosomal differences between the ICM and TE as a consequence of high levels of mosaicism at the cleavage stage even in younger women84 will reduce the accuracy of diagnosis even when multiple biopsied TE cells are available. As an alternative to blastocyst biopsy, it is possible to co-culture blastomeres biopsied at cleavage stages with the biopsied embryo.85 Over a period of 3 days, division and development of the biopsy significantly mirrors the behavior of the parent embryo. Hence, if

the embryo reached the blastocyst stage, in most cases the blastomere divided and developed into a small TE vesicle. On average, those blastomeres that divided and formed these vesicles divided two or three times, resulting in an average of 5.6 ± 0.6 (n = 13) cells for single eight-cell-stage blastomeres, and 9.1 ± 1.1 (n = 11) cells where two blastomeres were biopsied and encouraged to form a single morula. In this approach the behavior of the biopsy in vitro could predict the potential for the biopsied embryo,86 and avoid the difficulties and damage of biopsy at the blastocyst stage itself. Furthermore, in the mouse, it is possible to derive trophectoderm stem (TS) cell lines from single blastomeres biopsied from eightcell-stage embryos by culturing the cells on mouse embryo fibroblast feeder cell layers in the presence of fibroblast growth factor 4 and heparin (M Al Badr and AH Handyside, unpublished observations). So far, it has not been possible to isolate TS cells from human embryos under the same conditions (J Rossant, pers comm). However, if it became possible to derive either TS or embryonic stem cells, it would provide an unlimited source of cells for diagnosis as well as providing stem cells which might be useful for therapeutic purposes. In summary, whereas blastocyst biopsy has been performed successfully in the clinical arena18,87,88 and is particularly promising for younger patients aiming for single embryo transfer following PGD,89 its widespread use awaits large-scale clinical assessment. Furthermore, the logistics of blastocyst biopsy dictate a limited time in which to perform diagnosis which might necessitate cryopreservation of biopsied blastocysts for transfer in a later unstimulated thaw cycle.90,91

Future developments A major challenge at present is to develop an effective standardized method for cryopreservation of biopsied embryos. Attempts to use established protocols either in the mouse model or in humans have shown extensive damage after thawing, presumably because of the loss of protection from ice crystals in the medium provided by an intact zona pellucida.92,93 However, recently, several improved slow-freezing protocols have been reported in which damage is much reduced.94,95 However, following successful application in animal models,96–98 vitrification looks set to replace slow freezing for both cleavage- and blastocyststage embryos after polar body or embryo biopsy. With the high rate of multiple pregnancies reported after PGD, it is imperative to develop effective methods of cryopreservation that will (1) allow storage of unaffected embryos for later transfer so that the numbers transferred can be limited to two- or even single-embryo transfers and (2) provide additional time to perform more extensive diagnostic tests.

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With the introduction of quality management systems and accreditation in IVF laboratories,46,47,55 safer biopsy can be anticipated through agreed definitions of successful and safe biopsy, standardized training and procedures, validation of new techniques, as well as calibration of new and existing instruments such as the laser. It has become clear that embryo biopsy, as with any form of invasive testing or manipulation, exacts a cost to the embryo in the form of either cellular depletion, metabolic stress, or both. Thus, it is imperative to assess the potential benefit to the embryo itself in terms of improved selection or disease-free status before performing embryo biopsy. However, in the future it may be possible to diagnose inherited diseases or chromosomal imbalance in early human embryos by noninvasive analysis of the secretome or metabolome in spent culture medium,99 which would shift the cost–benefit ratio heavily towards potential benefit. For the time being, it is critical that the diagnostic laboratory optimizes the use of the single biopsied blastomere. One promising method allows testing of multiple loci, repeat testing, sample sharing for external quality assessment and archiving for later assessment of additional loci, involving WGA.70 A sufficiently large amount of DNA is generated following this process such that even microarray-based testing is possible from a single cell100 and, beyond this approach, the opportunities for additional molecular analysis appear to be almost unlimited.

Appendix Typical clinical protocol for cleavage-stage embryo biopsy A. Preparation prior to biopsy 1.

2. 3. 4.

5.

Prepare 1 blastocyst culture dish per embryo on the day prior to biopsy labeled with the patient’s name and embryo number on the base of the dish and on the front panel. Allow to equilibrate in the patient allocated incubator section. This dish should contain 5×10 µl drops of SAGE (or other) blastocyst media overlaid with SAGE (or other) culture oil. Turn on workstation micromanipulators and heated stage (check temperature). Ensure anti-vibration table is inflated and functioning. Set up biopsy dishes with SAGE ‘biopsy medium’ (Ca2+/Mg2+-free) + 10% SSR and pre-warmed SAGE culture oil. These should be kept on a heated stage at 37OC for 5 min prior to the procedure to ensure that they are warm. 3×10 µl drops of media are aliquoted into a Falcon 50×9 mm petri dish (code 351006) and covered with 4 ml oil. Turn on ‘SATURN’ laser and workstation computer. Open ‘CRONUS’ software and follow RI instruction

6.

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booklet for alignment of laser using ‘pilot’ to check target is correct (refer to Saturn laser manual for additional guidance). Pipette set up is essentially the same as that done for ICSI; however, in place of the injection pipette, a blastomere aspiration pipette is inserted into the holder. These are available in a range of diameters and can be bevelled if required. Current diameters used are between 25 and 35 µm and are available commercially from a number of different providers.

B. Biopsy 1. Transfer one embryo to the central drop of media in a new biopsy dish. The embryo should be ‘rinsed’ in the extra ‘wash’ drops so that excess culture media does not dilute the biopsy medium. This also acts to remove any remaining cumulus cells. 2. Place the dish on the heated stage of the micromanipulation workstation. 3. Carefully lower the holding and aspiration pipettes into the central drop taking care to avoid damaging the embryo. 4. Ensure both pipettes have equilibrated and are offering good control. 5. Position the embryo in order to give a clear view of the blastomere to be aspirated and secure the embryo by suction with the holding pipette. 6. Select appropriate laser duration for desired hole size and begin to make an opening in the embryo zona with a series of laser pulses working inwards from the outer surface of the zona. Take care to avoid damaging neighboring blastomeres. There are three pre-set pulse times (0.9 ms. 1.2 ms, and 1.5 ms) that are adequate in the majority of cases. Zona thickness variation between embryos will mean that pulse time and number of pulses will vary. Pulse duration can be altered manually if required. 7. As soon as the opening is wide enough to accommodate the aspiration pipette, carefully insert it through the opening and remove the selected blastomere with gentle suction. 8. Release the blastomere and embryo from suction and remove both pipettes from the drop. 9. Take a picture of the embryo and blastomere. 10. Make a note of the presence of a nucleus in the blastomere and time taken to ‘biopsy’. 11. Label a pre-equilibrated post-biopsy culture dish clearly with the embryo number on both the lid and base (dish is pre-labeled with patient name). 12. Return the biopsied embryo to this dish, ensuring that wash drops are used to minimize any carry-over of Ca2+/Mg2+-free medium into the culture drop. 13. Return the dish to the patient incubator section and culture at 37oC and 5% CO2 until embryo transfer or disposal.

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14. Once the embryo is safely back in culture, the biopsy dish containing the blastomere should be given to the scientist who is spreading or tubing the blastomer for genetic analysis. 15. Occasionally, it may be necessary to re-biopsy an embryo. In such cases a new biopsy dish should be made with pre-warmed culture oil and biopsy medium. Re-biopsy should be attempted using the original zona aperture. 16. Witnessing must be performed and noted for every embryo and blastomere that is moved between dishes or fixed. 17. Proceed with the next embryo. 18. Biopsy all embryos that have reached the appropriate cleavage stage. This is normally set at 6–8 cells. 19. Exceptions may be made in certain cases after review with the Scientific Director. This may mean that embryos of a lower cell number are biopsied.

C. Selection of embryos for transfer 1.

2. 3.

4.

5. 6.

7.

8. 9.

Results of the genetic analysis are normally ready within 24–48 hours. This is dependent upon the type of testing. The Scientific Director or suitable designee must approve results. If several embryos are available, selection should be made using normal morphology grading and rate of development. In some cases it may be appropriate to delay the ET to day 5 to allow further embryo development before selection. Never perform ET without an official diagnostic report signed by the scientist and Scientific Director. Never perform ET without confirming the embryo(s) for transfer with the clinician. Confirm with the clinician and patients if surplus embryos are to be cryopreserved immediately or after further culture. Confirm with the clinician and patients if followup genetic analysis is to be performed and by whom. If embryos are to be fixed for FISH analysis, refer to blastomere fixation SOP. If embryos are to be tubed for PCR analysis, refer to tubing for PCR SOP.

D. Special considerations Timing: If the majority of embryos have not reached 6– 8 cell stage the case should be reviewed with the Scientific Director. Considerations such as need to courier cells to another genetic analysis center (this may be overseas) and delaying the procedure to allow further embryo cleavage have to be taken into account. It may be necessary to review with the patient’s clinician before electing to proceed or delay the biopsy.

References 1. Li A, Gyllenstein UB, Cui X, et al. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature (London) 1988; 335: 414–19. 2. Coutelle C, Williams C, Handyside A, et al. Genetic analysis of DNA from single human oocytes: a model for preimplantation diagnosis of cystic fibrosis. Br Med J 1989; 299: 22–4. 3. Holding C, Monk M. Diagnosis of β-thalassaemia by DNA amplification in single blastomeres from mouse preimplantation embryos. Lancet 1989; 2: 532–5. 4. Handyside AH, Delhanty JD. Preimplantation genetic diagnosis: strategies and surprises. Trends Genet 1997; 13: 270–5. 5. Tarin JJ, Handyside AH. Embryo biopsy strategies for preimplantation diagnosis. Fertil Steril 1993; 59: 943–52. 6. Monk M, Muggleton-Harris AL, Rawlings E. Whittingham DG. Pre-implantation diagnosis of HPRT-deficient male and carrier female mouse embryos by trophectoderm biopsy. Hum Reprod 1988; 3(3): 377–81. 7. Wilton LJ, Shaw JM, Trounson AO. Successful single-cell biopsy and cryopreservation of preimplantation mouse embryos. Fertil Steril 1989; 51(3): 513–17. 8. Yang X, Foot RH. Production of identical twin rabbits by micromanipulation of embryos. Biol Reprod 1987; 37: 1007–14. 9. Ozil JP. Production of identical twins by bisection of blastocysts in the cow. J Reprod Fertil 1983; 69: 463–8. 10. Summers PM, Campbell JM, Miller MW, Normal in vivo development of marmoset monkey embryos after trophectoderm biopsy. Human Reprod 1988; 3(3): 389–93. 11. Harper JC, de Die-Smulders C, Goossens V, ESHRE PGD consortium data collection VII: cycles from January to December 2004 with pregnancy followup to October 2005. Hum Reprod 2008; 23: 741–55. 12. Cohen J, Malter H, Wright G, et al. Partial zona dissection of human oocytes when failure of zona pellucida penetration is anticipated. Hum Reprod 1989; 4: 435–42. 13. Cieslak J, Ivakhnenko V, Wolf G, Sheleg S, Verlinsky Y. Three-dimensional partial zona dissection for preimplantation genetic diagnosis and assisted hatching. Fertil Steril 1999; 71: 308–13. 14. Gordon JW, Talansky BE. Assisted fertilization by zona drilling: a mouse model for correction of oligospermia. J Exp Zool 1986; 239: 347–54. 15. Malter HE, Cohen J. Partial zona dissection of the human oocyte: a nontraumatic method using micromanipulation to assist zona pellucida penetration. Fertil Steril 1989; 51: 139–48. 16. Geraedts J, Handyside A, Harper J, et al. ESHRE Preimplantation Genetic Diagnosis (PGD) Consortium: preliminary assessment of data from January 1997 to September 1998. ESHRE PGD Consortium Steering Committee. Hum Reprod 1999; 14(12): 3138–48.

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Human embryo biopsy procedures 17. Germond M, Nocera D, Senn A, et al. Microdissection of mouse and human zona pellucida using a 1.48-microns diode laser beam: efficacy and safety of the procedure. Fertil Steril 1995; 64(3): 604–11. 18. Boada M, Carrera M, De La Iglesia C, et al. Successful use of a laser for human embryo biopsy in preimplantation genetic diagnosis: report of two cases. J Assist Reprod Genet 1998; 15: 302–7. 19. Montag M, Van der Ven H. Laser-assisted hatching in assisted reproduction. Croat Med J 1999; 40: 398–403. 20. Montag M, Van der Ven K, Delacretaz G, Rink K, Van der Ven H. Laser-assisted microdissection of the zona pellucida facilitates polar body biopsy. Fertil Steril 1998; 69: 539–42. 21. Park S, Kim EY, Yoon SH, Chung KS, Lim JH. Enhanced hatching rate of bovine IVM/IVF/IVC blastocysts using a 1.48-micron diode laser beam. J Assist Reprod Genet 1999; 16: 97–101. 22. Han TS, Sagoskin AW, Graham JR, Tucker MJ, Liebermann J. Laser-assisted human embryo biopsy on the third day of development for preimplantation genetic diagnosis: two successful case reports. Fertil Steril 2003; 80: 453–5. 23. Joris H, De Vos A, Janssens R, et al. Comparison of the results of human embryo biopsy and outcome of PGD after zona drilling using acid Tyrode medium or a laser. Hum Reprod 2003; 18: 1896–902. 24. Chatzimeletiou K, Picton HM, Handyside AH. Use of a non-contact, infrared laser for zona drilling of mouse embryos: assessment of immediate effects on blastomere viability. Reprod BioMed Online 2001; 2: 178–87. 25. Verlinsky Y, Ginsberg N, Lifchez A, et al. Analysis of the first polar body: preconception genetic diagnosis. Hum Reprod 1990; 5: 826–9. 26. Corveleyn A, Morris MA, Dequeker E, et al. Provision and quality assurance of preimplantation genetic diagnosis in Europe. Eur J Hum Genet 2008; 16(3): 290–9. 27. Dreesen JC, Geraedts JP, Dumoulin JC, Evers JL, Pieters MH. RS46(DXS548) genotyping of reproductive cells: approaching preimplantation testing of the fragile-X syndrome. Hum Genet 1995; 96(3): 323–9. 28. Navidi W, Arnheim N. Using PCR in preimplantation genetic disease diagnosis. Hum Reprod 1991; 6(6): 836–49. 29. Verlinsky Y, Rechitsky S, Cieslak J, et al. Preimplantation diagnosis of single gene disorders by two-step oocyte genetic analysis using first and second polar body. Biochem Mol Med 1997; 62: 182–7. 30. Kaplan B, Wolf G, Kovalinskaya L, Verlinsky Y. Viability of embryos following second polar body removal in a mouse model. J Assist Reprod Genet 1995; 12(10): 747–9. 31. Verlinsky Y, Cohen J, Munne S, et al. Over a decade of experience with preimplantation genetic diagnosis: a multicenter report. Fertil Steril 2004; 82(2): 292–4. 32. Verlinsky Y, Cieslak J, Ivakhnenko V, et al. Preimplantation diagnosis of common aneuploidies

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by the first- and second-polar body FISH analysis. J Assist Reprod Genet 1998; 15: 285–9. Ray P, Winston R, Handyside A. Reduced allele dropout in single-cell analysis for preimplantation genetic diagnosis of cystic fibrosis. J Assist Reprod Genet 1996; 13(2): 104–6. Eldadah ZA, Grifo JA, Dietz HC. Marfan syndrome as a paradigm for transcript-targeted preimplantation diagnosis of heterozygous mutations. Nat Med 1995; 1(8): 798–803. Magli MC, Gianaroli L, Ferraretti AP, et al. The combination of polar body and embryo biopsy does not affect embryo viability. Hum Reprod 2004; 19(5): 1163–9. Cieslak-Janzen J, Tur-Kaspa I, Ilkevitch Y, et al. Multiple micromanipulations for preimplantation genetic diagnosis do not affect embryo development to the blastocyst stage. Fertil Steril 2006; 85(6): 1826–9. Handyside AH, Kontogianni EH, Hardy K, Winston RM. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990; 344: 768–70. Hardy K, Martin KL, Leese HJ, Winston RML, Handyside AH. Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage. Hum Reprod 1990; 5: 708–14. Ao A, Handyside AH. Cleavage stage human embryo biopsy. Hum Reprod Update 1995; 1: 3. Handyside AH, Thornhill AR. Cleavage stage embryo biopsy for preimplantation genetic diagnosis. In: Kempers RD, Cohen J, Haney AF, Younger JB, eds. Fertility and Reproductive Medicine. Amsterdam: Elsevier, 1998; 223–9. Chen SU, Chao KH, Wu MY, et al. A simplified twopipette technique is more efficient than the conventional three-pipette method for blastomere biopsy in human embryos. Fertil Steril 1998; 69: 569–75. Inzunza J, Iwarsson E, Fridstrom M, et al. Application of single-needle blastomere biopsy in human preimplantation genetic diagnosis. Prenat Diagn 1998; 8(13): 1381–8. Levinson G, Fields RA, Harton GL, et al. Reliable gender screening for human preimplantation embryos, using multiple DNA target-sequences. Hum Reprod 1992; 7(9): 1304–13. Roudebush WE, Kim JG, Minhas BS, Dodson MG. Survival and cell acquisition rates after preimplantation embryo biopsy: use of two mechanical techniques and two mouse strains. Am J Obstet Gynecol 1990; 162(4): 1084–90. Pierce KE, Michalopoulos J, Kiessling AA, Seibel MM, Zilberstein M. Preimplantation development of mouse and human embryos biopsied at cleavage stages using a modified displacement technique. Hum Reprod 1997; 12(2): 351–6. Thornhill AR, deDie-Smulders CE, Geraedts JP, et al. ESHRE PGD Consortium ‘Best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS)’. Hum. Reprod 2005; 20: 35–48. Preimplantation Genetic Diagnosis International Society (PGDIS). Guidelines for good practice in PGD: programme requirements and laboratory

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Textbook of Assisted Reproductive Technologies quality assurance. Reprod Biomed Online 2008; 16(1): 134–47. Dumoulin JC, Bras M, Coonen E, et al. Effect of Ca2+/Mg2+-free medium on the biopsy procedure for preimplantation genetic diagnosis and further development of human embryos. Hum Reprod 1998; 13: 2880–3. Tarin JJ, Conaghan J, Winston RM, Handyside AH. Human embryo biopsy on the 2nd day after insemination for preimplantation diagnosis: removal of a quarter of embryo retards cleavage. Fertil Steril 1992; 58: 970–6. Tsunoda Y, McLaren A. Effect of various procedures on the viability of mouse embryos containing half the normal number of blastomeres. J Reprod Fertil 1983; 69: 315–22. Liu J, Van den Abbeel E, Van Steirteghem A. The in-vitro and in-vivo developmental potential of frozen and non-frozen biopsied 8-cell mouse embryos. Hum Reprod 1993; 8(9): 1481–6. Cohen J, Wells D, Munne S. Removal of 2 cells from cleavage stage embryos is likely to reduce the efficacy of chromosomal tests that are used to enhance implantation rates. Fertil Steril 2007; 87(3): 496–503. Van de Velde H, De Vos A, Sermon K, et al. Embryo implantation after biopsy of one or two cells from cleavage-stage embryos with a view to preimplantation genetic diagnosis. Prenat Diagn 2000; 20(13): 1030–7. Goossens V, De Rycke M, De Vos A, et al. Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis. Hum Reprod 2008; 23(3): 481–92. Thornhill A, Repping S. Quality control and quality assurance in preimplantation genetic diagnosis. In: Harper J. Preimplantation Genetic Diagnosis. London: John Wiley & Sons, 2008. Mastenbroek S, Twisk M, van Echten-Arends J, et al. In vitro fertilization with preimplantation genetic screening. N Engl J Med 2007; 357(1): 9–17. Cohen J, Grifo J. Multicentre trial of preimplantation genetic screening reported in the New England Journal of Medicine: an in-depth look at the findings. Reprod Biomed Online 2007; 15(4): 365–6. Rossant J. Postimplantation development of blastomeres isolated from 4- and 8-cell mouse eggs. J Embryol Exp Morphol 1976; 36: 283–90. Sutcliffe AG, D’Souza SW, Cadman J, et al. Outcome in children from cryopreserved embryos. Arch Dis Child 1995; 72(4): 290–3. Harper JC, Coonen E, Ramaekers FC, et al. Identification of the sex of human preimplantation embryos in two hours using an improved spreading method and fluorescent in situ hybridization (FISH) using directly labelled probes. Hum Reprod 1994; 9: 721–4. Munné S, Cohen J. Unsuitability of multinucleated human blastomeres for preimplantation genetic diagnosis. Hum Reprod 1993; 8: 1120–5. Kuo HC, Ogilvie CM, Handyside AH. Chromosomal mosaicism in cleavage-stage human embryos and

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the accuracy of single-cell genetic analysis. J Assist Reprod Genet 1998; 15: 276–80. Strom CM, Levin R, Strom S, et al. Neonatal outcome of preimplantation genetic diagnosis by polar body removal: the first 109 infants. Pediatrics 2000; 106(4): 650–3. Banerjee I, Shevlin M, Taranissi M, et al. Health of children conceived after preimplantation genetic diagnosis: a preliminary outcome study. Reprod Biomed Online 2008; 16(3): 376–81. Nekkebroeck J, Bonduelle M, Desmyttere S, Van den Broeck W, Ponjaert-Kristoffersen I. Mental and psychomotor development of 2-year-old children born after preimplantation genetic diagnosis/screening. Hum Reprod 2008; Feb 19 [Epub ahead of print]. Harper JC, Coonen E, Handyside AH, et al. Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos. Prenat Diagn 1995; 15(1): 41–9. Magli MC, Jones GM, Gras L, et al. Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in vitro. Hum Reprod 2000; 15(8): 1781–6. Bielanska M, Tan SL, Ao A. Chromosomal mosaicism throughout human preimplantation development in vitro: incidence, type, and relevance to embryo outcome. Hum Reprod 2002; 17(2): 413–19. Holding C, Bentley D, Roberts R, Bobrow M, Mathew C. Development and validation of laboratory procedures for preimplantation diagnosis of Duchenne muscular dystrophy. J Med Genet 1993; 30(11): 903–9. Handyside AH, Robinson MD, Simpson RJ, et al. Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease. Mol Hum Reprod 2004; 10(10): 767–72. Nijs M, Van Steirteghem A. Developmental potential of biopsied mouse blastocysts. J Exp Zool 1990; 256: 232–6. Dokras A, Sargent IL, Ross C, Gardner RL, Barlow DH. Trophectoderm biopsy in human blastocysts. Hum Reprod 1990; 5: 821–5. Dokras A, Sargent IL, Ross C, Gardner RL, Barlow DH. The human blastocyst: morphology and human chorionic gonadotrophin secretion in vitro. Hum Reprod 1991; 6: 1143–51. Muggleton-Harris AL, Glazier AM, Pickering SJ. Biopsy of the human blastocyst and polymerase chain reaction (PCR) amplification of the beta-globin gene and a dinucleotide repeat motif from 2–6 trophectoderm cells. Hum Reprod 1993; 8(12): 2197–205. Veiga A, Sandalinas M, Benkhalifa M, et al. Laser blastocyst biopsy for preimplantation diagnosis in the human. Zygote 1997; 5(4): 351–4. Papanikolaou EG, Kolibianakis EM, Tournaye H, et al. Live birth rates after transfer of equal number of blastocysts or cleavage-stage embryos in IVF. A systematic review and meta-analysis. Hum Reprod 2008; 23(1): 91–9. Kokkali G, Traeger-Synodinos J, Vrettou C, et al. Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation genetic

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diagnosis of beta-thalassaemia: a pilot study. Hum Reprod 2007; 22(5): 1443–9. Khalaf Y, El-Toukhy T, Coomarasamy A, et al. Selective single blastocyst transfer reduces the multiple pregnancy rate and increases pregnancy rates: a pre- and postintervention study. BJOG 2008; 115(3): 385–90. Kalousek D, Vekemans M. Confined placental mosaicism. J Med Genet 1996; 33(7): 529–33. James R, West J. A chimaeric animal model for confined placental mosaicism. Hum Genet 1994; 93(5): 603–4. Ruangvutilert P, Delhanty JD, Serhal P, et al. FISH analysis on day 5 post-insemination of human arrested and blastocyst stage embryos. Prenat Diagn 2000; 20(7): 552–60. Evsikov S, Verlinsky Y. Mosaicism in the inner cell mass of human blastocysts. Hum Reprod 1998; 13(11): 3151–5. Derhaag JG, Coonen E, Bras M, et al. Chromosomally abnormal cells are not selected for the extra-embryonic compartment of the human preimplantation embryo at the blastocyst stage. Hum Reprod 2003; 18(12): 2565–74. Baart EB, Martini E, van den Berg I, et al. Preimplantation genetic screening reveals a high incidence of aneuploidy and mosaicism in embryos from young women undergoing IVF. Hum Reprod 2006; 21(1): 223–33. Geber S, Winston RM, Handyside AH. Proliferation of blastomeres from biopsied cleavage stage human embryos in vitro: an alternative to blastocyst biopsy for preimplantation diagnosis. Hum Reprod 1995; 10: 1492–6. Geber S, Sampaio M. Blastomere development after embryo biopsy: a new model to predict embryo development and to select for transfer. Hum Reprod 1999; 14: 782–6. McArthur SJ, Leigh D, Marshall JT, de Boer KA, Jansen RP. Pregnancies and live births after trophectoderm biopsy and preimplantation genetic testing of human blastocysts. Fertil Steril 2005; 84(6): 1628–36. Kokkali G, Vrettou C, Traeger-Synodinos J, et al. Birth of a healthy infant following trophectoderm biopsy

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from blastocysts for PGD of beta- thalassaemia major. Hum Reprod 2005; 20(7): 1855–9. de Boer KA, Catt JW, Jansen RP, Leigh D, McArthur S. Moving to blastocyst biopsy for preimplantation genetic diagnosis and single embryo transfer at Sydney IVF. Fertil Steril 2004; 82(2): 295–8. Magli MC, Gianaroli L, Grieco N, et al. Cryopreservation of biopsied embryos at the blastocyst stage. Hum Reprod 2006; 21(10): 2656–60. Parriego M, Sole M, Aurell R, Barri PN, Veiga A. Birth after transfer of frozen-thawed vitrified biopsied blastocysts. J Assist Reprod Genet 2007; 24(4): 147–9. Joris H, Van den Abbeel E, Vos AD, Van Steirteghem A. Reduced survival after human embryo biopsy and subsequent cryopreservation. Hum Reprod 1999; 14: 2833–7. Magli MC, Gianaroli L, Fortini D, Ferraretti AP, Munné S. Impact of blastomere biopsy and cryopreservation techniques on human embryo viability. Hum Reprod 1999; 14: 770–3. Jericho H, Wilton L, Gook DA, Edgar DH. A modified cryopreservation method increases the survival of human biopsied cleavage stage embryos. Hum Reprod 2003; 18: 568–71. Stachecki JJ, Cohen J, Munné S. Cryopreservation of biopsied cleavage stage human embryos. Reprod Biomed Online 2005; 11(6): 711–15. Agca Y, Monson RL, Northey DL, et al. Normal calves from transfer of biopsied, sexed and vitrified IVP bovine embryos. Theriogenology 1998; 50(1): 129–45. Baranyai B, Bodo S, Dinnyes A, Gocza E. Vitrification of biopsied mouse embryos. Acta Vet Hung 2005; 53(1): 103–12. Isachenko V, Montag M, Isachenko E, van der Ven H. Vitrification of mouse pronuclear embryos after polar body biopsy without direct contact with liquid nitrogen. Fertil Steril 2005; 84(4): 1011–16. Edwards R, Hollands P. New advances in human embryology: implications of the preimplantation diagnosis of genetic disease. Hum Reprod 1988; 3(4): 549–56. Le Caignec C, Spits C, Sermon K, et al. Single-cell chromosomal imbalances detection by array CGH. Nucleic Acids Res 2006; 34(9): e68.

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15 Analysis of fertilization Lynette Scott

Introduction The efficiency of assisted reproductive technologies (ART) and embryo transfer (ET) in the human is low, with fewer than 30% of embryos that are transferred ever realizing full developmental potential, which should mean the delivery of a healthy infant.1 This has risen slightly over the years with better stimulation protocols and improved culture media, leading to better embryo development. However, the number of oocytes that are used to result in a delivery has still not altered; in other words, we have improved in our ability to grow and perhaps select advanced embryos but we have not improved in our ability to develop viable oocytes and/or select them at early stages. To overcome the low implantation potential, there has been a practice of replacing multiple embryos,2–6 depending on age group,7 in order to increase the likelihood of a pregnancy. This has led to an unacceptable level of highorder multiple pregnancies, which is being addressed at many levels worldwide. Some countries have mandated the number of embryos that can be replaced, in some instances limiting this number to one in certain age groups. Attempts are also being made in many centers to actively engage in elective single embryo transfer (eSET), but this practice is still not widespread. Although eSET will reduce the level of multiple pregnancies it can also reduce pregnancy rates, since there are data showing that the number of embryos replaced affects the pregnancy rate, especially when there are no high-quality embryos available.8 The use of eSET does not necessarily reduce the cost of delivering an infant through ART, although for target groups it should be the aim.9–11 What is required is a reliable way of selecting embryos that have the most potential for implantation, thus reducing the number of embryos needed without compromising the patient. Currently the only method of embryo selection is morphology, which operates under a fairly broad bell curve of success and is dependent on many factors. Other techniques are being tried but have not been fully clinically validated and at present are still expensive and in the experimental phases.

Initially, most in vitro fertilization (IVF) centers replaced embryos on the second day of culture, at the 2–4-cell stage. By allowing development for an additional day, permitting more critical assessment of the embryos after further cleavage divisions, Dawson et al2 demonstrated an increased implantation rate. This system has been widely adopted, with a concomitant increase in implantation rates. For both day-212–15 and day-32,4,5 embryo’s transfers, embryo selection is based on key morphologic features of cleaving embryos that have been previously correlated with increased implantation. However, even a good scoring system on day 3 will not give an accurate prediction of an embryo’s ability to implant.3,16 Two 8-cell embryos that look identical do not necessarily have the same implantation potential, as evidenced by the low implantation rates of transferred embryos. With the introduction of extended culture and blastocyst transfer, pregnancy and implantation rates have increased,17 which has enabled the selection of one or two blastocysts for transfer. This has been accomplished without reducing pregnancy rates but minimizing the incidence of high-order multiple pregnancies, a highly desired outcome. A major drawback is that only 40–50% of all fertilized oocytes placed in extended culture reach the blastocyst stage, and, of these, only 30–50% implant. Furthermore, blastocyst transfer has been shown to benefit implantation rates only in select groups of women,16,18 mainly those who are good-prognosis patients to begin with. This implies that even extended culture with blastocyst transfer is inefficient in a human IVF program. However, there is no way of knowing whether the in vitro culture system is selecting only for embryos that can develop in that particular system. Moreover, the strategy of allowing development in vitro as a means of embryo selection is not feasible in certain countries, since their state and government policies preclude the destruction of any fertilized embryos.19 In order to reduce the numbers of embryos replaced with constraints such as these, embryos at the first stages of development, or even oocytes, would need to be selected for transfer. If the early

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events of fertilization and the fertilized oocyte can be combined with later developmental competence, more accurate embryo selection should be feasible, allowing the use of fewer embryos or one embryo in embryo transfer.10,11,20,21 The morphology of human fertilized oocytes 22–24 has been used successfully as selection criteria for day-1 pronuclear transfers.23 The use of pronuclear scoring has also allowed the use of fewer embryos, but with increased implantation on days 2 and 3. 14,19,23,25 Pronuclear scoring was used in conjunction with day-3 morphology scoring for increased implantation,26,27 and has been correlated with blastocyst development. 14,20,26–29 The pronuclear score has also been linked with sperm source in intracytoplasmic sperm injection (ICSI) cases, and with implantation potential.30,31 Finally, there is growing evidence that pronuclear morphology correlates with the chromosomal status of the embryos, with aneuploidies being linked to certain abnormal pronuclear morphologies.32 Pronuclear scoring takes into account a number of aspects of the fertilized oocyte: namely, the position of the nuclei and the morphology of the nuclei in terms of the nucleolar precursor bodies (NPBs), which are located in the nucleus. The original Scott scoring system involved the nuclei, nucleoli, the cytoplasm, and progression to nuclear membrane breakdown, and was used prospectively to select embryos on day 1 for transfer.23 Tesarik and Greco24 reported a singleobservation grading system in which the nucleoli size, number, and distribution were utilized. Embryos were replaced on the third day of culture, at which point embryo morphology was used as the primary selection criterion. In a retrospective analysis of their data, they found a high correlation between implantation and the equality of nucleoli within each nucleus of the pronuclear embryos from which the resulting transferred cleaving embryos arose. Ludwig et al19 used a pronuclear grading system based on a combination of the Scott and Tesarik systems, and demonstrated no reduction in pregnancy rate using only two rather than three embryos on day 3 of culture. The embryos for transfer were selected according to pronuclear morphology. Screening embryos at the pronuclear stage enabled the group to reduce the risks of high-order multiple pregnancies without a reduction in pregnancy rates. The Scott system was revised, for ease of use,14,20 allowing a single observation to be performed and eliminating the scoring of entry into the first cleavage division. Basically, all pronuclear scoring systems take into account the same parameters: nucleus position and size and the morphology of the nuclei in terms of NPBs. The revised Scott and the Tesarik systems are equivalent, with different nomenclature. Further refinement of the scoring system is occurring to allow for more accurate assessment of the NPBs in the nuclei in terms of numbers, size, and location,

which is showing correlations with both embryo morphology and implantation and delivery.14

The oocyte The development of a mature oocyte within the follicle is a complex and coordinated series of events that ends with ovulation, when the oocyte surrounded by the cumulus cells enters the oviduct and awaits fertilization. Only a mature oocyte can be fertilized. The nuclear and cytoplasmic maturation of the oocyte and the growth and differentiation of the somatic cells in the follicle all play a crucial, and linked, role in the formation of a mature functional oocyte. The events of nuclear maturation include resumption of the first meiotic division and progression to metaphase II. Cytoplasmic maturation includes all events that prepare the oocyte for successful fertilization, including zona pellucida acquisition, cortical granule formation and the ability to release them and calcium, mitochondrial changes, protein synthesis involved in growth of the oocyte, and cytoskeletal changes. If all the events of somatic cell follicle development have not occurred systematically, aspects of the final stages of maturation and oocyte nutrition will not occur correctly.33 If all the events of nuclear and cytoplasmic maturation have not occurred synchronously, development will not be normal. Each of these events can proceed independently with the production of seemingly normal follicles and oocytes, which can be fertilized. However, the resulting embryos will be abnormal.34–36

Polar bodies It has been reported that the morphology of the first polar body is related to oocyte quality, which in turn will dictate the quality of the fertilized oocyte and embryo.37,38 The ovulated or retrieved oocyte has an intact polar body, and is arrested at metphase II of the second meiotic division. The first polar body is formed through an asynchronous division at completion of the first meiotic division. In the human oocyte this structure will begin to divide and disintegrate with time. This is a natural and controlled event that is complete by approximately 20 hours after extrusion,39 and is controlled by the c-mos viral oncogene and mitogen-activated protein (MAP) kinase.33,40–42 When ICSI is being performed, the first polar body can be easily visualized and assessed. This is not the case in normally fertilized oocytes in which polar body visualization can occur only after the events of fertilization. An increase in fertilization rates, embryo morphology scores,37 and implantation rates38 of embryos resulting from oocytes with polar bodies that were round or oval with no fragmentation or rough cell membranes has been shown. Since polar body

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formation is linked to spindle formation, meiosis, and the cell cycle, abnormal polar bodies may indicate abnormalities at the cellular and molecular levels. Polar body abnormalities include fragmentation (see Fig 15.4e), abnormal size, and cell membrane degeneration. The morphology of oocytes destined for conventional insemination cannot be scored as easily without disrupting the cumulus complex. It has been observed that small, cellular debris or inclusions in the perivitelline space, attached to the zona pellucida, are correlated with high estrogen levels. However, this was not related to any decrease in pregnancy rate. There have been no studies in which this phenomenon is correlated with either polar body morphology or with fertilized oocyte score.

Fertilization The ovulated or retrieved oocyte is activated when the sperm enters, either by normal fertilization or artificially with ICSI. Activation is a complex series of events that result in the release of the cortical granules, activation of membrane-bound adenosine triphosphatases (ATPases), resumption of meiosis, and finally formation of the male and female pronuclei with extrusion of the second polar body. The process of fertilization encompasses the entry of the sperm, activation, resumption of meiosis, and finally the first mitotic division resulting in a 2-cell embryo. During these phases, mitochondria are also activated and play a vital role in fertilization and subsequent embryo development.43 In human fertilization, the centriole, which is the microtubule-organizing center, is derived from the sperm.44,45 These structures are responsible for bringing the male and female pronuclei together. Within the nuclei are structures known as the nucleoli, which are the protein powerhouses of any cell. All mitotically active cells have nucleoli, which are located on the DNA where ribosomal genes are transcribed. There are generally between two and seven per human nucleus, with equal numbers in the two daughter cells in any mitotic division. Nucleoli develop on the chromosomes at the sites coding for ribosomal DNA (rDNA). These sites are referred to as the nucleolus organizing regions (NORs). There are only 5 NOR-bearing pairs of chromosomes,13, 14, 15, 21, and 22, which are also the heterochromatic chromosomes. Nucleoli consist of a dense fibrillar component (DFC), a fibrillar center (FC), and a granular component (GC). rDNA transcription requires the DF but not the FC component, and is restricted to foci on the DNA. The FCs act as structural centers for rDNA transcription and, in addition, store inactive transcription factors. The GC is a group of preribosomes.46,47 During mitosis the nucleoli fuse, with more being present at the beginning of the cell cycle (G1 phase) than at the S1 phase, where there are only 1–2 large nucleoli per nucleus.46 There is synchrony in the number and form of fusion of nucleoli in the daughter

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cells, and there is always synchrony in the daughter cells. Asynchronous fusion of nucleoli indicates aberrant chromosomal function.46 Asynchronous fusion is a method of distinguishing normal from abnormal cells in cervical cancer and many other forms of cancer cells. In these, the daughter cells have more than the expected numbers of nucleoli or abnormal or unequal sizes of nucleoli. Another aspect of abnormal nucleoli morphology is fragmentation due to aging, resulting in increased numbers of dense bodies in the cells.48 The nucleolus is also involved in cell cycle control, since many mitogenic and growth regulatory proteins are located in the nucleolus.49 Nucleoli are first seen in oocytes in antral follicles where they are well defined and synthesize rRNA. This synthesis is essential for meiotic competence and since at this stage the oocyte is growing exponentially.50,51 During oocyte maturation leading to ovulation, RNA synthesis decreases and the nucleoli become small and disocciate, leaving only the FC region attached to the chromatin, and it is the FC region that is termed the nucleolar precusor bodies or NPBs.52 At fertilization, rRNA synthesis resumes. This is accompanied by changes in the nucleoli, which reform and begin to grow. As more synthesis occurs, they begin to coalesce.53,54 Full development and activation of the nucleolus occurs through the first few mitotic cell cycles of the newly formed embryo.53–55 The nucleolus will be fully functional at the time of embryonic genome activation.46,56,57

Fertilized oocyte scoring Fertilized oocyte scoring involves careful analysis of the pronuclei and the NPBs within the nuclei in a single observation at 16–18 hours after fertilization. What is being observed at this time are the structural parts of the nucleoli on the chromatin, left after dissociation, or the FC regions/NPBs. Since there are only NORs on chromosomes 13, 14, 15, 21, and 22, what is being observed in NPB scoring is the coalecence of the chromatin in these chromosomes. If it is possible to follow the progression from pronuclei formation to the first cleavage division, more information can be obtained. However, within the limitations of a busy IVF laboratory, a single observation can aid in embryo selection for transfer and decisions of day of embryo transfer.

Nuclei The pronuclei are the first easily observable signs of fertilization. Both pronuclei should appear within the same time frame and be together (Fig 15.1a). Failure to have moved together by 16–18 hours postfertilization (Fig 15.1b) could indicate some disruption in aster and microtubule formation, which will lead to abnormal development.44,45 These embryos rarely progress well or form blastocysts.28,58

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c

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Fig 15.1 (a) Normal fertilization, even-sized nuclei correctly located in the oocyte with aligned nucleoli. (b) Nuclei not aligned by 18 hours after insemination; abnormal. (c) Nuclei of distinctly different sizes; abnormal. (d) Nuclei incorrectly positioned within the oocyte and small.

The pronuclei should be approximately the same size (Fig 15.1a). Fertilized oocytes that have pronuclei of very different sizes (Fig 15.1c) have an 87% incidence of chromosomal abnormalities.59,60 Likewise, fertilized oocytes that have very small nuclei or fragmented nuclei are generally abnormal, displaying poor and retarded development (Fig 15.1d). The position of the pronuclei within the fertilized oocyte is also important. As fertilization progresses, the microtubules in the aster pull the female pronucleus towards the male pronucleus. The male pronucleus moves to a central position within the oocyte. Thus, the pronuclei should be centrally placed in the oocyte or just into the hemisphere containing the first polar body at 16–18 hours after insemination or ICSI. Fertilized oocytes with nonaligned pronuclei, pronuclei in the hemisphere without the second polar body, and pronuclei of distinctly different sizes, fragmenting pronuclei, or very small pronuclei are probably abnormal. They should not be considered for transfer. Only fertilized oocytes with normal-sized pronuclei are used for the second phase of fertilized oocyte scoring.

Nucleolar precursor bodies The NPBs can be visualized within the nuclei on any inverted microscope with contrast optics (Hoffman or Nomarski). They can also be seen on high-power, good binocular microscopes that have the ability to tilt the mirror and throw shadows through the fertilized oocyte. This technique can be used for initial sorting, but contrast optics is recommended for final scoring. The size, number, and distribution of the nucleoli within the nuclei of fertilized oocytes, in a single observation at 16–18 hours after insemination/ICSI, form the central aspect of pronuclear scoring. There are currently two scoring systems in place which classify fertilized oocytes as Z1, Z2, Z3, or Z413 or pattern 0–520 depending on the size, number, and distribution of nucleoli within the nuclei (Fig 15.2 shows the Z system). The systems describe the same features, using different nomenclature. The number of nucleoli should ideally be between five and seven per nucleus (Fig 15.3a and b).14 Fertilized oocytes with many small pinpoint nucleoli are probably delayed in nuclear events and formation of the NORs

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Normal = equality between nuclei

Z1

0A

Z2

0B

Z3-1

Z3-3

Z3-2

Z3-4

5

1

4

2

Abnormal = any inequality

Figure 15.2

Nucleolar precursor body (NPB) pattern.

(Fig 15.4a). These fertilized oocytes are slow in development and result in suboptimal embryos with only 10–15% blastocyst development. The Z-score describes the number, size, and position of the nucleoli and the equality between the nuclei for these characteristics. Z1 zygotes have equal numbers of nucleoli between 3 and 7 that are aligned at the pronuclear junctions. Z2 zygotes have equality in size and number between the nuclei, but the nucleoli have not yet aligned at the pronuclear junction. Z3 zygotes are characterized by inequality between the nuclei: unequal-sized nucleoli, unequal numbers of nucleoli, or unequal alignment at the pronuclear junction. Z4 zygotes are grossly abnormal and present with unequal-sized nuclei, nuclei that have not aligned, small, and misplaced nuclei. Fertilized oocytes with unequal numbers of nucleoli in the two nuclei also have reduced developmental potential (Fig 15.4b–f). Those with unequal numbers or unequal sizes of nucleoli are probably displaying asynchrony between male and female pronuclei development. Since the nucleoli progress from small centers, coalesce, and align at the pronuclear junction, any inequality between them could result in abnormal development.14,33,41,58 The alignment or polarized distribution of chromatin, and therefore of the NPBs, at the pronuclear junction is a desired feature (Figs 15.1a, 15.2, and 15.3a–c).61–64 This

is related to the metabolic status of the embryo and the ability of the nuclei to fuse and form the unique embryonic genome.61,63,64 There is a pH gradient between the two sets of NPBs which is lower than elsewhere in the fertilized oocyte and which is an important factor in the event of nuclei fusion. This alignment is also seen in mouse fertilized oocytes, where it is speculated that this feature is indicative of intact DNA, normal metabolic characteristics, and appropriate assembly of microtubules.65 Failure to align or the inequality of nuclei could alter this pH gradient, leading to abnormal development when the male and female genomes combine. Therefore, the desired fertilized oocyte is one that has equal numbers of even-sized NPBs with between five and about seven per pronucleus that are beginning to align or have aligned at the pronuclear junction (Z1, Z2, or pattern 0A, 0B) (Figs 15.1a and 15.3a–c). These have been shown to give optimal development and implantation at the 1-cell,19 cleaving day-3 cell stage and blastocyst stage.14,20,23–25,28,58,66 Those in which there is alignment at the pronuclear junction are designated Z1/pattern 0A (Fig 15.2), and those whose nucleoli are still scattered are designated Z2/pattern 0B (Figs 15.2 and 15.3d–f). Fertilized oocytes with unequal numbers and/or unequal sizes of nucleoli are designated Z3/pattern 1, 4, and 5 (Fig 15.4b–f). These fertilized oocytes have been

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Fig 15.3 Z1 and Z2 zygotes. (a–d) Z1: equal numbers of equal-sized nucleoli aligned at the pronuclear junction. (e and f ) Z2: equal numbers of equal-sized nucleoli still scattered in the nuclei.

shown to result in lower-grade day-3 embryos, lower in vitro blastocyst formation, and lower implantation rates when using day-5 transfers.13,14,60 Inequality in the state of coalescence of nucleoli in the two nuclei and progression to the S phase will lead to gross abnormalities in development. This is directly reflected in the decreased development recorded for these fertilized oocytes. Another aspect that is now evident is that the ratio of NPBs per nucleus is highly correlated with developmental competence.14 Fertilized oocytes displaying between five and seven NPBs per nucleus that are symmetrically aligned have a higher developmental potential than when there are more or less or when the ratio varies from 1 significantly (Fig 15.5). A second level of assessment of the fertilized oocytes that can be used is the appearance of the cytoplasm. The

presence of a halo23 is associated with the development of high-quality embryos on day 3 and day 5 (Figs 15.1a and 15.3a–c).58 The halo has not been shown to be associated with any specific fertilized oocyte morphology, but has been consistently linked to cohorts of fertilized oocytes that developed to good-quality blastocysts.58 The clearing of the cytoplasm on the fertilized oocyte periphery has been termed cytoplasmic streaming.22 Mouse67 and hamster68 1-cell fertilized embryos have differential mitochondrial distribution, which is related to the cell cycle. In the mouse fertilized oocytes the mitochondria migrate to the periphery of the cell, whereas in the hamster fertilized oocytes the mitochondria migrate and aggregate in the center of the cell, around the pronuclei. The pattern of cytoplasmic streaming or the halo effect in human fertilized oocytes

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Fig 15.4 Z3 zygotes. (a) Many small pinpoint nucleoli. (b–e) Inequality in size or number or alignment of nucleoli between the two nuclei. (f) many small scattered pinpoint nucleoli.

Figure 15.5

NPB ratio.

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would seem to mimic that seen in hamster fertilized oocytes, with an aggregation in the center of the cell.22,23 It would seem reasonable to assume that the mitochondria are aggregating at the site of highest metabolic activity, the pronuclei. Fertilized oocytes in which this movement is not so pronounced or does not occur could be metabolically compromised, leading to delayed and poor development. However, in a prospective trial the presence of a halo did not affect delivery14 in high-prognosis patients. It could, however, be of benefit in lower-prognosis or older patients.29

40 Percentage of total zygotes

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30

1166

20

10 129 0 Z1

Z2

Z3

Z4

Fig 15.6 Distribution of Z-scores for 4318 zygotes.

Application of fertilized oocyte scoring Percentage of total embryos

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50

0 Z4 Z3 Z2 Z1 Grade 1: 8 cells, <10% fragmentation, good cell-to-cell con Grade 2: 8 cells, 10−20% fragmentation, lacking good cellto-cell contact Grade 3: 6−8 cells, 20% fragmentation, uneven blastomere Grade 4: < 8 cells with fragmentation or uneven blastomere or at least one multinucleate blastomere Grade 5: < 6 cells or grossly fragmented or > half blastome multinucleate

Fig 15.7 The distribution of day-3 embryo morphologies of the four Z-scores for a large cohort of embryos over a 12-month period.

80 Percentage blastocyst development

Fertilized oocyte scoring involves grading fertilized oocytes at 16–18 hours postinsemination, in a one-time scoring, and designating them as Z1–420 or pattern 0–5,24 as depicted in Fig 15.2. Fertilized oocytes of a like score can be cultured in groups or separately, according to laboratory protocol. The spread of pronuclear scores is not affected by age, infertility type, route of sperm entry, and numbers of oocytes retrieved, or medium used for insemination. However, there is a trend for high-scoring or low-scoring cohorts of embryos. Some women will have all Z1/0A and Z2/0B, whereas others will have predominantly Z3/pattern 1–5. Overall, less than 10% of fertilized oocytes are Z4 (unequal nuclei). The Z4’s are generally grossly abnormal, but can develop into embryos with good morphology and into high-grade blastocysts.58 However, since Z4 fertilized oocytes are considered abnormal, and have been shown to have a high degree of aneuploidy,60,69 they should not be used for transfer or cryopreservation. Fig 15.6 shows the spread of pronuclear scores for a large cohort of fertilized oocytes over an 18-month period in a group of patients ranging in age from 22 to 43 years old, with varying infertility. Overall, no one type is predominant. Once fertilized oocytes are scored and sorted, they can be followed to day 2, 3, or 5 when a secondary scoring system, based on key morphologic features, can be used. The use of high-grade day-3 embryos resulting from abnormal fertilized oocytes does not result in implantation.70 It can be argued that embryos that result from abnormal fertilized oocytes are those in a cohort of day-3 embryo transfers that do not implant. Fig 15.7 shows the day-3 embryo grading of a large cohort of pronuclear-scored embryos. This demonstrates that, although the majority of high-grade embryos, with little fragmentation and adequate cell number, are from Z1 and Z2 fertilized oocytes, there is a wide spread of morphologies for all Z-scores. This was further shown in a larger cohort of embryos, where the rate of development and the fragmentation patterns were taken into account.14 The concept of pronuclear scoring followed by traditional day-3 or day-5 scoring is to select against embryos that have little continued developmental potential but which can

Day 5 60

Day 5 + 6

40

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0 Z1

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Z4

Fig 15.8 The percentage of blastocyst development after 96 hours of culture (day 5) and 120 hours of culture (day 5 + 6) for each of the Z-scores over a 2-year time period for all infertility and age presentations.

cleave to at least the 8-cell or blastocyst stage. Using a gated scoring system will aid in embryo selection and reduce the numbers of embryos used in transfer. Fig 15.8 shows the relationship between fertilized oocyte morphology and development to the blastocyst stage on day 5 of culture and for total blastocyst

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development (day 5 + 6) (see also Scott58). Again, although the majority of high-grade blastocysts on day 5 originate from Z1/pattern 0A fertilized oocytes, there are still a number of blastocysts that are morphologically high grade arising from lower-scoring fertilized oocytes. When these blastocysts are transferred, they have little implantation potential.70 When a continuous grading system is used for embryo selection, based initially and primarily on fertilized oocyte scores, the implantation and pregnancy rates for both day-3 and day-5 embryo transfers can be significantly increased.14,19,20,24–28 This is very important for limiting the number of embryos used to achieve high pregnancy rates. By applying fertilized oocyte scoring in conjunction with day-3 or day-5 morphology, only one or two embryos per transfer need to be used, thus limiting the potential for highorder multiple pregnancies, a highly desired outcome in human IVF–ET. Thus, the application of scoring fertilized embryos at the 1-cell stage can be used in a number of ways. Primarily, it can be used as the first line of embryo selection for transfer, whether on day 1, 3, or 5. The embryo morphology on day 3 or day 5 could be used as the secondary system. This can also be applied to selecting embryos for cryopreservation. It could be argued from the day-5 transfer data that embryos originating from fertilized oocytes with poor pronuclear score should not be frozen, since they have very little implantation potential.70 Fertilized oocyte scoring can also be used to select patients who would benefit from a day-3 vs day-5 transfer, based on how many high-grade fertilized oocytes they have. For countries where there are strict laws regarding the culture and freezing of cleaving embryos, the application of fertilized oocyte scoring could also reduce the numbers of embryos they both freeze and use for embryo transfer.

References 1. Edwards R, Beard H. Blastocyst stage transfer: pitfalls and benefits. Hum Reprod 1999; 14: 1–6. 2. Dawson KJ, Conaghan J, Ostera GR, Winston RML, Hardy K. Delaying transfer to the third day postinsemination, to select non-arrested embryos, increases development to the fetal heart stage. Hum Reprod 1995; 10: 177–82. 3. Milki AA, Hinckley MD, Gebhardt J, et al. Accuracy of day 3 criteria for selecting the best embryos. Fertil Steril 2002; 77: 1191–5. 4. Puissant F, Van Rysselberge M, Deweze J, Leroy F. Embryo score as a prognostic tool in IVF treatment. Hum Reprod 1987; 2: 705–8. 5. Steer C.V, Mills CL, Tan SL, Campbell S, Edwards RG. The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in-vitro fertilization and embryo transfer programme. Hum Reprod 1992; 7: 117–19.

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Analysis of fertilization 57. Hyttel P, Viuff D, Laurincik J, et al. Risk of in-vitro production of cattle and swine embryos: aberrations in chromosome numbers, ribosomal RNA gene activation and perinatal physiology. Hum Reprod 2000; 15(Suppl 5): 87–97. 58. Scott L. Pronuclear scoring as a predictor of embryo development. Reprod Biomed Online 2003; 6: 57–70. 59. Munné S, Cohen J. Chromosome abnormalities in human embryos. Hum Reprod Update 1998; 4: 842–55. 60. Sadowy S, Tomkin G, Munné S. Impaired development of zygotes with uneven pronuclear size. Zygote 1998; 63: 137–41. 61. Van Blerkom J. Occurrence and developmental consequences of aberrant cellular organization in meiotically mature human oocytes after exogenous ovarian hyperstimulation. J Electron Microsc Tech 1990; 16: 324–46. 62. Van Blerkom J, Davis P, Merriman J, Sinclair J. Nuclear and cytoplasmic dynamics of sperm penetration, pronuclear formation and microtubule organization during fertilization and early preimplantation development in the human. Hum Reprod Update 1995; 1: 429–61. 63. Van Blerkom J, Henry G. Oocyte dysmorphism and aneuploidy in meiotically-mature human oocytes after ovulation stimulation. Hum Reprod 1992; 7: 379–90.

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64. Van Blerkom J, Runner MN. Mitochondrial reorganisation during resumption of arrested meiosis in the mouse oocyte. Am J Anat 1984; 171: 335–55. 65. Dozortsev D, Coleman A, Nagy P. Nucleoli in pronuclei-stage mouse embryo are represented by major satellite DNA of interconnecting chromosomes. Fertil Steril 2000; 73: 366–71. 66. De Placido G, Wilding M, Strina I, et al. High outcome predictability after IVF using a combined score for zygote and embryo morphology and growth rate. Hum Reprod 2002; 17: 2402–9. 67. Muggleton-Harris AL, Brown JJG. Cytoplasmic factors influence mitochondrial reorganization and resumption of cleavage during culture of early mouse embryos. Hum Reprod 1998; 3: 1020–8. 68. Barnett DK, Kimura J, Bavister BD. Translocation of active mitochondria during hamster preimplantation embryo development studied by confocal laser scanning microscopy. Dev Dyn 1996; 205: 64–72. 69. Sandalinas M, Sadowy S, Alikani M, et al. Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage. Hum Reprod 2001; 16: 1954–8. 70. Scott L. Embryological strategies for overcoming recurrent assisted reproductive technology treatment failure. Hum Fertil (Camb) 2002; 5: 206–14.

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16 Culture systems for the human embryo David K Gardner, Michelle Lane

Introduction Upon first sight, embryo culture appears a rather simple procedure. In reality, it is far from it, requiring proactive quality control and quality assurance programs, together with a high level of training for embryologists. Furthermore, a sufficient number of incubation chambers are required to maintain a stable environment for development in vitro. Therefore, embryo culture is far more involved than simply using the appropriate culture media formulations. In order to optimize embryo development in vitro and maintain the viability of the conceptus, it is essential to consider the embryo culture system in its entirety. The embryo culture system consists of the media, gas phase, type of medium overlay, the culture vessel, the incubation chamber, ambient air quality, and the embryologists themselves. The concept of an embryo culture system highlights the interactions that exist not only between the embryo and its physical surroundings but also between all parameters within the laboratory (Fig 16.1). Only by taking such a holistic approach can one optimize embryo development in vitro. It is also important to appreciate that it is not possible to make a good embryo from poor-quality gametes. Rather, the role of the laboratory is to maintain the inherent viability of the oocyte and sperm from which the embryo is derived. Ultimately therefore, the in vitro fertilization (IVF) laboratory is dependent on the quality of the ovarian stimulation decided by the physician, as well as patient factors, and hence emphasizes the need for a broader perspective of patient management as well as laboratory management. Adequate communication pathways should exist between physicians and scientists to ensure all variables are monitored and that action plans are put in place should changes need to be implemented.

The human embryo in culture Serendipitously, the human embryo exhibits a considerable degree of plasticity, enabling it to develop under a wide variety of culture conditions. Indeed it would be fair to say that the human preimplantation embryo is the most resilient of all mammalian species

studied to date. However, this should be perceived as a testament of the ability of the human embryo to adapt to its surroundings and not our ability to culture it. Undoubtedly, having to adapt to suboptimal collection and/or culture conditions comes at the cost of impaired viability and potentially compromised pregnancy outcomes.1,2 Therefore, it is important to focus on the generation of healthy embryos, as it is evident that embryo development in culture, even to the blastocyst stage per se, does not necessarily equate to the development of a viable embryo.3 Viability is best defined as the ability of the embryo to implant successfully and give rise to a normal healthy term baby. Subsequently, implantation rate (fetal heart rate, as opposed to fetal sac) and, ideally, live birth rates, should always be reported and considered, as they help to reflect the true efficacy of a given IVF system. Today, clinics are not only faced with a multitude of embryo culture media to choose from, but also with the decision of whether to transfer at the cleavage stage or the blastocyst. There remains some confusion about optimal culture conditions and at which stage of embryo development to transfer to the uterus. However, with the publication of evidence-based Cochrane4 and meta-analysis,5 demonstrating a clear increase in pregnancy and implantation rates and reduced pregnancy loss following blastocyst culture in sequential media, and with morphological selection being more predictive at the blastocyst stage, there is an increasing awareness and need for laboratories to be able to support extended culture.3 Therefore, the aim of this chapter is to discuss the types of media and culture systems currently available, to describe how they can be implemented in a clinical setting, and to assess which parameters are important when considering extended culture.

Impact of single embryo transfer on the laboratory It is evident that with the development of enhanced culture systems and better methods for embryo selection (see Chapter 17) and cryopreservation (Chapters 20 and 21), the move to single embryo transfer (SET)

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Fig 16.1 The relationship between patient stimulation, the laboratory, and transfer outcome in human in vitro fertilization (IVF). This figure serves to illustrate the complex and interdependent nature of human IVF treatment. For example, the stimulation regimen used not only impacts on oocyte quality (and hence embryo physiology and viability174 but can also affect subsequent endometrial receptivity.122,175–177 Furthermore, the health and dietary status of the patient can have a profound effect on the subsequent developmental capacity of the oocyte and embryo.64,178 The dietary status of patients attending IVF is typically not considered as a compounding variable, but growing data would indicate otherwise. In the schematic, the laboratory has been broken down into its core components, only one of which is the culture system. The culture system has in turn been broken down to its components, only one of which is the culture media. Therefore, it would appear rather simplistic to assume that by changing only one part of the culture system (i.e. culture media), that one is going to mimic the results of any given laboratory or clinic. One of the biggest impacts on the success of a laboratory and culture system is the level of quality control (QC) and quality assurance (QA) in place. For example, one should never assume that anything coming into the laboratory that has not been pretested with a relevant bioassay (e.g. mouse embryo assay), is safe merely because a previous lot has performed satisfactorily. Only a small percentage of the contact supplies and tissue culture-ware used in IVF comes suitably tested. Therefore it is essential to assume that everything entering the IVF laboratory without a suitable pretest is embryotoxic until proven otherwise. In our program the 1-cell mouse embryo assay (MEA) is employed to prescreen every lot of tissue culture-ware that enters the program, i.e. plastics that are approved for tissue culture. Around 25% of all such material fails the 1-cell MEA (in a simple medium lacking protein after the first 24 h).109 Therefore, if one does not perform QC to this level, one in four of all contact supplies used clinically will be sub-optimal. In reality many programs cannot allocate the resources required for this level of QC and when embryo quality is compromised in the laboratory it is the media that are frequently held responsible, when in fact the laboratory-ware is more often the culprit. Modified from Gardner and Lane88 with permission.

for a significant number of patients is now a practical reality. Indeed, in several countries this is now mandated. Far from being exceptional, SET (or single blastocyst transfer [SBT]) is becoming the primary standard of care for the majority of patients that seek IVF treatment.6 (Chapter 51) One of the impacts of single

embryo transfers is the increased reliance on a successful frozen embryo program. Therefore, an important consideration in assessing the efficacy of any culture system is for its ability to produce high-quality embryos that can survive cryopreservation by either freezing and thawing or by vitrification followed by

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warming. This has significant implications for cumulative pregnancy rates per retrieval.

Dynamics of embryo and maternal physiology Before attempting to culture any cell type, be it embryonic or somatic, it is important to consider the physiology of the cell in order to establish its nutrient requirements. The mammalian embryo presents an intriguing problem in that it undergoes significant changes in its physiology, molecular regulation, and metabolism during the preimplantation period. The preimplantation human embryo is, therefore, a highly dynamic entity with its needs changing as development proceeds. Indeed, it goes from being one of the most quiescent tissues in the body (the oocyte), to being amongst the most metabolically active (the blastocyst) within just 4 days.1 Interestingly, the pronucleate oocyte, like the Metaphase stage II (MII) oocyte from which it was derived, exhibits relatively low levels of oxygen consumption and has a preference for carboxylic acids, such as pyruvate, as its primary energy source.7,8 Little glucose is consumed by the early embryo, but it is still utilized.9 In particular the balance of mitochondrial and cytoplasmic metabolism is critical at these early stages of development to maintain adequate levels of ATP production.10 However, despite the low levels of biosynthetic activity at these early stages of development, there is an increasing awareness of a significant amount of remodeling of the nucleus. For example, there are major changes in methylation and acetylation levels, with many of the processes involved still to be elucidated.11–13 Nevertheless, what is critical is that many key developmental events, such as activation of the egg and regulation of acetylation, are regulated by proteins whose activity is dependent on metabolic regulation.14–16 Therefore, maintenance of metabolic homeostasis at these early stages is paramount for the maintenance of viability. Consequently this continues to be a major focus of the culture system in the laboratory.1 As development proceeds and energy demands increase with cell multiplication, transcription following activation of the embryonic genome, and an increase in protein synthesis, there is a concomitant increase in energy requirement and in glucose utilization. By the blastocyst stage, the embryo exhibits high oxygen utilization and an ability to readily utilize glucose, along with other energy sources. Table 16.1 highlights some of the differences between the preand post-compacted embryo. In many ways the physiology of the cells of the embryo prior to compaction, and hence before the formation of a transporting epithelium, can be likened to unicellular organisms.17 This in part explains why those amino acids in the so-called nonessential group are beneficial to the cleavage stage embryo (see below). Significantly, the nutrients available within the human female reproductive tract mirror the changing

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Table 16.1 Differences in embryo physiology pre- and post-compaction Pre-compaction

Post-compaction

Low biosynthetic activity

High biosynthetic activity High QO2 Glucose preferred nutrient

Low QO2 Pyruvate preferred nutrient Nonessential amino acids Maternal genome Individual cells One cell type

Nonessential + essential amino acids Embryonic genome Transporting epithelium Two distinct cell types: ICM and trophectoderm

QO2, oxygen consumption; ICM, inner cell mass.

nutrient preference of the embryo. At the time when the embryo resides in the oviduct, the fluid within is characterized by relatively high concentrations of pyruvate (0.32 mmol/l) and lactate (10.5 mmol/l), and a relatively low concentration of glucose (0.5 mmol/l).18 In contrast, uterine fluid is characterized by relatively low levels of pyruvate (0.1 mmol/l) and lactate (5.87 mmol/l), and a higher concentration of glucose (3.15 mmol/l).

Susceptibility of the preimplantation embryo to stress There is an increasing understanding in mammalian embryology that the early embryo is highly adaptive to its environment. The embryo appears to have the ability to continue development, even to the blastocyst stage, at the cost of normal cellular processes and checkpoints that may be essential for viability. Therefore, as a result, many embryos can appear to be morphologically normal, while at a cellular level they are actually highly perturbed and unlikely to be viable.2,17 Therefore, one of the key focuses of the embryology laboratory should be to ensure its collection and culture system are able to maintain normal development at a cellular level. Although the human embryo has a plasticity to adapt to its environment, as already highlighted, this is at a cost of cellular regulation either through metabolic adaptations or adaptations at a molecular level. Therefore, the laboratory should seek to employ systems that reduce these adaptations and thereby maintain viability.

Cleavage stages versus post-compaction embryo and stress As a result of its ‘primitive’ physiology, the precompaction stage embryo is highly susceptible to stress compared with the post-compaction stage embryo. A stress applied in vitro at the two pronuclei (2PN) to the 8-cell stage can have devastating effects

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Increasing sensitivity and susceptibility

Primordial Antral IVM IVF Zygote 8-cell Blastocyst stage follicle follicle or S/O

Impact of external factors

Fig 16.2 Sensitivity and susceptibility of germ cells and embryos to external factors. IVM, in vitro maturation; S/O, super ovulation.

on normal cellular physiology and viability of the subsequent blastocyst and fetus (Fig 16.2).17,19–21 At these early stages of development prior to activation of the embryonic genome, the embryo does not possess the capacity at a molecular level to respond to a stress. In somatic cells, when a cell finds itself in a hostile environment it can activate a cascade of molecular signaling pathways to engage systems to maintain normal development. However, the precompaction stage embryo has a limited capacity for gene transcription22 and, therefore, the human embryo prior to the 8-cell stage is highly vulnerable to any perturbed environment. At these early stages of embryo development prior to compaction, there is limited capacity to maintain normal cellular functions such as regulation of intracellular pH, alleviation of oxidative stress, and ionic homeostasis.2,17 Therefore, a stress applied at these early stages of development can result in major disruptions to subsequent viability. In contrast, the application of the same stress postcompaction and post-embryonic genome activation typically has limited negative impact on subsequent developmental competence.17,20,21 A concept that has emerged from the animal literature is that the manifestation of a stress can be masked at the level of morphological assessment and may only therefore become evident downstream compared to the timing of the stress itself. It has been shown that the detrimental effects of a stress applied at the early stage of development during handling and culture of the oocyte and 2PN may not be evident until the blastocyst stage. Even then, the effects may only be at a subcellular level, with the embryo having reduced metabolic capacity and high levels of apoptosis, which ultimately result in a reduction in pregnancy rates.19–21 Therefore, the conditions employed for the collection and culture of the human cleavage stage embryo will directly affect the ability of the embryo to implant and form a viable pregnancy, independent

of morphological assessments within the laboratory. The inability of the analysis of morphology alone to distinguish viable and nonviable embryos highlights a major limitation in the field and confirms the need for more diagnostic parameters of normal development (see Chapter 17).

Composition of culture media There are several extensive treatise on the composition of embryo culture media,23–27 and it is beyond the scope of this chapter to discuss in detail the role of individual medium components. However, two key components, amino acids and macromolecules, will be discussed due to their significant impact on cycle outcome. Understanding their effects on embryo physiology should help clinics make a more informed decision regarding their choice of culture media.

Amino acids It is certainly the case that the human embryo can grow in the absence of amino acids. The real questions are: how well do they develop in their absence and how viable are the resultant embryos? There are several reasons for the inclusion of amino acids in embryo culture media. Oviduct and uterine fluids contain significant levels of free amino acids,28–33 while both oocytes and embryos possess specific transport systems for amino acids34 to maintain an endogenous pool.35 Amino acids are readily taken up and metabolized by the embryo.36,37 Table 16.2 lists the roles amino acids can fulfill during the pre- and peri-implantation period of mammalian embryo development. Oviduct and uterine fluids are characterized by high concentrations of the amino acids alanine, aspartate, glutamate, glycine, serine and taurine.28–33 With the exception of taurine, the amino acids at high concentrations in oviduct fluid bear a striking homology to those amino acids present in Eagle’s nonessential amino acids.38 Studies on the embryos of several mammalian species, such as mouse,39–42 hamster,43,44 sheep,45,46 cow,47,48 and human,49,50 have all demonstrated that the inclusion of amino acids in the culture medium enhances embryo development to the blastocyst stage. More significantly, it has been demonstrated that the preimplantation embryo exhibits a switch in amino acid requirements as development proceeds. Up to the 8-cell stage nonessential amino acids and glutamine increase cleavage rates;48,51,52 i.e. those amino acids present at the highest levels in oviduct fluid stimulate the cleavage-stage embryo. However, after compaction, nonessential amino acids and glutamine increase blastocoel formation and hatching, while the essential amino acids stimulate cleavage rates and increase development of the inner cell mass (ICM) in the blastocyst.19,51 Most importantly, amino acids have been reported to increase viability of cultured embryos from several species after transfer to recipients23,46,51 as

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Table 16.2 Functions of amino acids during preimplantation mammalian embryo development

Role Biosynthetic precursors Energy source Regulators of energy metabolism Osmolytes Buffers of pHi Antioxidants Chelators Signaling Regulation of differentiation

Reference 165 166 1,10 167 53 168 169 170,171 51,172

well as increasing embryo development in culture. In the mouse, equivalent implantation rates to in vivo developed blastocysts were achieved when pronucleate oocytes were cultured with nonessential amino acids to the 8-cell stage followed by culture with all 20 amino acids from the 8-cell stage to the blastocyst.51 The terms nonessential and essential have little meaning in terms of embryo development and differentiation; rather, they reflect the requirements of certain somatic cells in vitro.38 More appropriate terminology would reflect the ability of the nonessential group to stimulate early cleavage (cleavage amino acids or CAA), while the essential group stimulate the development of the ICM (ICMAA). The reasons for this switch undoubtedly stem from the nature of the nonessential amino acids; they act as good intracellular buffers of pH due to their zwitterionic nature,43 and they are able to chelate toxins. As discussed, prior to compaction the blastomeres of the mammalian embryo appear to behave like unicellular organisms and use exogenous amino acids to help regulate their homeostasis. In contrast, post-compaction and the generation of a transporting epithelium, the embryo is able to regulate its internal environment and is not as dependent on the nonessential amino acids to regulate intracellular function.53 As evidence of the significance of amino acids, it has been shown that even a transient exposure (about 5 minutes) of mouse zygotes to medium lacking amino acids impairs subsequent developmental potential.54 During this 5-minute period in a simple medium the zygote loses its entire endogenous pool of amino acids, which takes several hours of transport to replenish after returning the embryo to medium with amino acids. This, therefore, has implications for the collection of oocytes, and more importantly the manipulation of denuded oocytes during intracytoplasmic sperm injection (ICSI), where plausibly the inclusion of amino acids in the holding medium will decrease or prevent intracellular stress (see below). Similarly, the work of Ho et al.55 on gene expression in mouse embryos goes some way to confirm this

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hypothesis, in that gene expression in mouse embryos cultured in the presence of amino acids was comparable to that of embryos developed in vivo. In contrast, mouse embryos cultured in the absence of amino acids, i.e. in a medium based on a simple salt solution, exhibited aberrant gene expression and altered imprinting of the H19 gene.56

Cautionary tale Even though the formulations of embryo culture media have improved significantly over the years, and for the most part have become more physiological in their basis, there is nothing physiological about a polystyrene culture dish. Therefore, one has to be careful about in vitro artifacts induced by a static environment. A good example of this is the production of ammonium by both embryo metabolism of amino acids and by the spontaneous breakdown of amino acids in the culture medium once incubated at 37oC (Fig 16.3).39 Although amino acids are used by embryos, it is their spontaneous breakdown that results in the vast majority of ammonium produced in the medium. Ammonium build-up in culture medium can not only have negative effects on embryo development and differentiation in culture,39,45,57 but can affect subsequent fetal growth rates and normality at a concentration of around 300 µmol/l.19,58 Furthermore, it has been shown that ammonium affects embryo metabolism, pHi regulation, and gene expression.59 As amino acids are such important regulators of embryo development, it is essential to alleviate this in vitro problem. The immediate answer is to renew the culture medium, thereby bringing the ammonium concentration under control. A second solution is to replace the most labile amino acid, glutamine, with a dipeptide form such as alanyl-glutamine. This dipeptide is just as effective as glutamine and has the advantage of not breaking down at 37oC. Therefore, media containing this stable form of glutamine do not produce significant levels of ammonium. Although there is some debate as to the level of concern one should place on ammonium toxicity in culture medium,60,61 there is growing data to support the appearance of ammonium in the culture medium over time39,57,62,63 and its toxicity to embryos, including the human.57 Furthermore, exposure of gametes and embryos to increasing concentrations of ammonium in vivo is not consistent with maintained embryo viability.64–66 It is, therefore, the authors’ humble opinions that one should err on the side of caution, take the data from animal and human in vitro and in vivo studies, and take the appropriate action.

Macromolecules Most culture media for the human embryo contain serum albumin as the protein source. The use of whole serum can no longer be condoned due its documented detrimental effects on embryos.46,67–70

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Fig 16.3 Production of ammonium into the culture medium (lacking embryos) by the spontaneous breakdown of amino acids in culture media. Solid circles, KSOMAA; open circles, G1/G2. The media were placed in the incubator at 4 p.m. the day before culture for equilibration purposes. The line at time zero represents when embryos would be placed into culture (although these measurements were taken in the absence of embryos). Medium KSOMAA contains 1 mmol/l glutamine and therefore releases significant levels of ammonium into the culture medium. Media G1/G2 do not contain glutamine, but rather the stable dipeptide form, alanyl-glutamine, and therefore these media do not release significant levels of ammonium. At a concentration of just 75 µmol/l ammonium can induce a 24-h developmental delay in mouse fetal development by day 15 and induces the neural tube defect exencephaly in 20% of all fetuses.53,61 It is therefore evident that dangerously high levels of ammonium are produced by media containing glutamine. From88 with permission.

Although serum albumin is a relatively pure fraction, it is still contaminated with fatty acids and other small molecules. The latter includes an embryotrophic factor, citrate, which stimulates cleavage and growth in rabbit morulae and blastocysts.71 Not only are there significant differences between sources of serum albumin,72,73 but also between batches from the same source.72,74 Therefore, when using serum albumin or any albumin preparation, it is essential that each batch is screened for its ability to adequately support embryo development in the mouse prior to clinical use. Recombinant human serum albumin has recently become available, which should eliminate the problems inherent with using blood-derived products, and lead to the standardization of media formulations. Recombinant human albumin has now been shown to be as effective as blood-derived albumin in supporting fertilization75 and embryo development, and its

efficacy has been proven in a prospective randomized trial.76 Significantly, embryos cultured in the presence of recombinant albumin exhibit an increased tolerance to cryopreservation.77 A further macromolecule present in the female reproductive tract is hyaluronan, which in the mouse uterus increases at the time of implantation.78 Hyaluronan is a high molecular mass polysaccharide that can be obtained endotoxin and prion free from a yeast fermentation procedure. It has been demonstrated that not only can hyaluronan improve mouse and bovine embryo culture systems,79,80 but also its use for embryo transfer results in a significant increase in embryo implantation.79,81,82 In the largest prospective trial to date, which enrolled 1282 cycles of IVF, it was determined that the use of hyaluronanenriched medium was associated with significant increases in clinical pregnancy rates and implantation rates, both for day 3 and day 5 embryo transfers. The beneficial effect was most evident in women who were >35 years of age, in women who had only poorquality embryos available for transfer, and in women who had previous implantation failures.82 However, another highly significant effect of the inclusion of hyaluronan in the culture medium is its beneficial effects on cryosurvivability of cultured embryos from a number of species, including the human, mouse, sheep, and cow.77,83,84 As IVF programs are moving to transfer fewer embryos, there is an increasing need to be able to cryopreserve supernumerary embryos. Therefore, increased cumulative pregnancy outcome is an important factor in deciding which culture system to use in the laboratory. In Fig 16.4 the effects of culture medium composition on the cryosurvival and subsequent implantation of human embryos is shown. Embryos were cultured in media with or without hyaluronan prior to slow freezing at the cleavage stage. Both survival and viability were higher if the embryos had been cultured in the presence of hyaluronan.

Monoculture or sequential media? It was established in the 1960s that it was feasible to culture the 1-cell mouse embryo to the blastocyst stage in a medium lacking amino acids. In the intervening decades, it has become apparent that amino acids have a significant role to play during embryo development (discussed above), and that the medium needs to be renewed at least every 48 h to ensure minimal accumulation of embryotoxic ammonium. Subsequently, all culture systems have become, by default, dynamic.85 From a practical point of view, therefore, the amount of work and embryo manipulations required are the same whether one is working with sequential media or a monophasic system (i.e. one medium formulation for the entire preimplantation period). However, the two approaches to embryo culture do have some fundamental differences. Specifically,

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the literature using an evidence-based Cochrane review4 has determined that the benefit of blastocyst culture over cleavage stage culture is only evident in a sequential culture system, thereby indicating that there is a compromise in developmental competence when embryos are grown beyond the cleavage stage in a single medium. This finding for human IVF cycles mirrors the literature on animal embryos that has established developmental outcomes more similar to in vivo results after culture in sequential-type culture media compared with a monoculture system.88

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20 10 0 Culture medium without hyaluronan

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Fig 16.4 Effect of culture medium on the subsequent cryosurvival of cleavage-stage human embryos. Embryos were cultured either in medium without (n = 1235) or with elevated hyaluronan (n = 1351). Solid bars represent survival rate, assessed as ≥50% of blastomeres being intact. Open bars represent implantation rates, as assessed by fetal heart at 8-week scan. ∗ Significantly different, p <0.05.

monoculture is based on the principle of letting the embryo choose what it wants during development. In contrast, sequential media were developed to accommodate the dynamics of embryo nutrition and to mirror the environment of the female reproductive tract (in which the embryo is exposed to a gradient of nutrients as it passes along the oviduct into the uterus).18,32,33 The significance of these nutrient gradients to the embryo in culture warrants further research, as existing data on the mouse indicates that such gradients in vitro do impact embryo viability following transfer. For example, when the mouse zygote is cultured to the 8-cell stage and then transferred, embryo viability is highest after exposure of the embryo to a high lactate concentration (>20 mmol/l D/L-lactate), while when the embryo is cultured postcompaction to the blastocyst stage, viability is highest after exposure to lower levels of lactate (<5 mmol/l 86 D/L-lactate). These data support the hypothesis that the physiology of the developing conceptus is temporally regulated by concentration of nutrients available.87 Sequential media are, therefore, better able to meet the changing needs of the embryo compared to a single culture medium, which must make a compromise between supporting the pre- or post-compaction stage embryo. Sequential media have now proven themselves to be highly effective in clinical settings. An assessment of

How far behind embryo development in vivo is development in vitro? Historically, embryos cultured in vitro lag behind their in vivo-developed counterparts.89,90 However, with the development of sequential media based on the premise of meeting the changing requirements of the embryo and minimizing trauma, in vivo rates of embryo development can now be attained in vitro in the mouse.1,91 The one proviso is that each laboratory must have sufficient quality systems in place to ensure the optimum operation of a given culture system. Such advances in culture systems represent a significant development for the laboratory, for there now exists a means of producing blastocysts at the same time and with the same cell number and allocation to the ICM as embryos developed in the female tract.85,88 Using sequential media in a highly controlled environment, as detailed throughout this book, it is possible to attain high rates of human embryo development to the blastocyst stage. Using an oocyte donor model to evaluate the efficacy of culture approaches, where the age of the oocyte donor is typically under 30 years, it is possible not only to obtain a blastocyst formation rate of 65% but also a resultant viability (as determined by fetal heart beat following transfer) of >65% (Table 16.3). As such, oocyte donors represent as close to a human ‘gold standard’ as one can have in an infertility clinic. With this in mind, ensuring one can attain blastocyst development of >50% and implantation rates of >50% when using donated oocytes is a good potential starting point for introducing blastocyst culture clinically.

Culture systems Several key components of the culture system are reviewed here, none of which should be considered in isolation as all directly impact upon media performance.

Incubation chamber Whatever incubation chamber is chosen, a key to successful embryo culture is to minimize perturbations in the atmosphere around the embryo. The two key perturbations to avoid are pH and temperature changes.

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Table 16.3 Viability of human embryos conceived in vitro using an oocyte donor model Mean blastocyst development (%) Mean number of blastocysts transferred Mean age of recipient Fetal heart (per blastocyst transferred) (%) Clinical pregnancy rate (per retrieval) (%) Twins (%)

65.1 2.05 40.3 68.0 85.2 59.9

All pronucleate oocytes were cultured for 48 hours in medium G1 at 5% O2, 6% CO2, and 89% N2. On day 3 of development, embryos were washed and transferred into medium G2 under the same gaseous environment. Embryos were cultured in groups of 4 in 50 µl drops of medium under Ovoil (Vitrolife AB, Sweden) in 60-mm Falcon Primaria dishes. All embryos were transferred on day 5 of development. n = 950 patients.173

have limited ability to maintain temperature or pH in the event of an emergency. What is evident is that it is imperative to have sufficient numbers of incubator chambers to match the caseload. This is especially true when performing extended culture. For around 800 retrievals, it is advisable to have between 16 and 20 incubator chambers (present in the laboratory as double stacks). The top chamber of each stack can be for media equilibration, while the bottom chamber can be used for inseminations and embryo culture, thereby minimizing the amount of access to incubators containing embryos. This means that one double stack of chambers is used for just 2–3 patients per week.

pH and carbon dioxide

This means that ideally the environment in which the embryo is placed is not disturbed during the culture period. Practically, this is difficult to achieve in a busy clinical laboratory. The use of an individual incubation chamber, such as a modular incubator chamber or glass desiccator, which can be purged with the appropriate gas mix, can alleviate such concerns. Using such incubator chambers, each patient’s embryos can be completely isolated within an incubator, the gas phase and, for the most part, temperature being unaffected when the incubator door is opened. We like to consider such chambers as ‘a womb with a view.’ However, a downside of this approach is that only three modular chambers can be placed in one incubator, thereby necessitating the acquisition of sufficient incubators. An alternative to the use of modular chambers is the use of inner doors within an incubator to significantly reduce fluctuations in the gaseous environment upon opening the incubator door. Several incubator manufacturers make incubators with inner doors. Incubators with infrared (IR) as opposed to thermocouple (TC) CO2 sensors are quicker at regulating the internal environment of the chamber; they are less sensitive to environmental factors and, subsequently, better able to maintain a constant CO2 level in the incubator. Therefore, incubators equipped with IR sensors will provide a more stable environment for embryo development. With regard to temperature changes, incubators with an air jacket are less susceptible to large temperature fluctuations than those with a water jacket. Again, the use of inner doors will aid in minimizing environmental fluctuations within the chamber. An alternative to classic incubators are the mini incubators with constant flow chambers, which allow for direct heat transfer by contact between the chamber and culture vessel. Such chambers also allow for a direct flow of premixed gas and therefore minimize changes in pH. However, such systems come at high running costs (using premixed gas cylinders), and

When discussing pH it is worth considering that the actual pH of the surrounding medium (pHo, typically 7.4) is different from that inside the embryo (pHi, 7.2).92–94 Specific media components, such as lactic and amino acids, directly affect and buffer pHi, respectively. Of the two isomers of lactate, D- and L-, only the L-form is biologically active. However, both the D- and L- forms decrease pHi of the embryo.94 Therefore, it is advisable to use only the L-isomer of lactate and not a medium containing both the D- and L- forms. While high concentrations of lactate in the culture medium can drive pHi down,94 amino acids increase the intracellular buffering capacity and help maintain the pHi at around 7.2.53 As the embryo has to maintain pHi against a gradient when incubated at pH 7.4, it would seem prudent to culture embryos at lower pHo. The pH of a CO2/bicarbonate-buffered medium is not easy to quantitate. A pH electrode can be used, but one must be quick and the same technician must take all readings to ensure consistency. An alternative approach is to take samples of medium and measure the pH with a blood-gas analyzer. A final method necessitates the presence of phenol red in the culture medium and the use of Sorenson’s phosphate buffer standards. This method allows visual inspection of a medium’s pH with a tube in the incubator, and is accurate to 0.2 pH units.8,26 When using bicarbonate-buffered media, the concentration of CO2 has a direct impact on medium pH.26 Although most media work over a wide range of pH (7.2–7.4), it is preferable to ensure that pH does not go over 7.4. Therefore it is advisable to use a CO2 concentration of between 6 and 7% to yield a medium pH of around 7.3. The amount of CO2 in the incubation chamber can be calibrated with a Fyrite, although such an approach is only accurate to ± 1%. A more suitable method is to use a hand-held infrared metering system (Vaisala) that can be calibrated and is accurate to around 0.2%. When using a CO2/bicarbonate-buffered medium it is essential to minimize the amount of time the culture dish is out of a CO2 environment to prevent

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increases in pH. To facilitate this, modified pediatric isolettes designed to maintain temperature, humidity, and CO2 concentration can be used. However, should it not be feasible to use an isolette, then the media used can be buffered with either 20–23 mmol/l 4(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES)95 or 4-morpholinopropane sulfonic acid (MOPS)96 together with 2–5 mmol/l bicarbonate. Such buffering systems do not require a CO2 environment. An oil overlay also reduces the speed of CO2 loss and the associated increase in pH.

Oxygen The fact that both human and F1 mouse embryos can grow at atmospheric oxygen concentration (~20%) has lead to some confusion regarding the optimal concentration for embryo culture. The concentration of oxygen in the lumen of the rabbit oviduct is reported to be 2–6%,97,98 whereas the oxygen concentration in the oviduct of the hamster, rabbit, and rhesus monkey is ~8%.99 Interestingly, the oxygen concentration in the uterus is significantly lower than in the oviduct, ranging from 5% in the hamster and rabbit to 1.5% in the rhesus monkey.99 Significantly, it has been demonstrated that optimum embryo development of other mammalian species occurs at an oxygen concentration below 10%.72,100,101 Furthermore, it has been documented that mouse embryos cultured to the blastocyst stage in the presence of 20% oxygen have altered gene expression and perturbed proteome compared with embryos developed in vivo.17,102 In contrast, culture in 5% oxygen had significantly less effect on both embryonic gene expression and proteome. It is our experience that human embryos cultured in a low oxygen environment (5%) produce blastocysts with significantly more cells than those embryos cultured in a high oxygen environment (20%). Considering the physiology of the reproductive tract and the beneficial effects of using a reduced oxygen concentration, as determined in controlled studies, it is advisable to culture embryos at low oxygen concentrations. This can easily be achieved with the modern trigas incubators or by using a premixed cylinder to purge a modular chamber.

Incubation vessel and the embryo:volume ratio Culture of embryos in drops of culture medium under an oil overlay is the preferred and effective method of culturing embryos. Within the lumen of the female reproductive tract the developing embryo is exposed to microliter volumes of fluid.103 In contrast, the embryo grown in vitro is subject to relatively large volumes of medium of up to 1 ml. Consequently, any autocrine factor(s) produced by the developing embryo will be diluted and may therefore become ineffectual. It has been demonstrated in the mouse

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that cleavage rate and blastocyst formation increase when embryos are grown in groups (up to 10) or reduced volumes (around 20 µl).104–106 Of greatest significance is the observation that decreasing the incubation volume significantly increases embryo viability106 due to an increase in ICM development. Similar results have been obtained with sheep45 and cow embryos.107 It is therefore apparent that the preimplantation mammalian embryo produces a factor(s) capable of stimulating development of both itself and surrounding embryos. Furthermore, embryos of one species can be used to promote development and differentiation of another.108 In order to culture in such reduced volumes (of 20– 50 µl) an oil overlay is required. Although the use of an oil overlay is time-consuming, it prevents the evaporation of media, thereby reducing the harmful effects of increases in osmolality, and reduces changes in pH caused by a loss of CO2 from the medium when culture dishes are taken out of the incubator for embryo examination. If oil is to be used, then light paraffin oil is recommended. Such an overlay also serves another purpose in being able to trap a number of volatile organic compounds. The benefits of using drops of medium under oil would obviously be negated should the oil be embryotoxic. Therefore, care must be taken in selecting and storing oil, which if done incorrectly will lead to it becoming toxic. Oil should be stored in the dark and in glass. It should not be stored for extended periods in the incubator. Oil should never be aliquoted into tissue culture flasks as these are styrene-based and oils are able to leach styrene from such containers at very high rates. Always use a batch of oil prescreened with an appropriate mouse embryo bioassay before clinical use. Oil toxicity may not necessarily show up by simply culturing mouse embryos to the blastocyst stage. Rather, one should also look for signs of necrosis, which is most evident at the blastocyst stage, and perform cell counts on the blastocysts developed.

Medium storage Commercially available culture media have several labile components and it is therefore important to know how to handle and store such solutions. Two of the most labile components are amino acids and vitamins. Glutamine is the most labile amino acid and produces the highest levels of ammonium of any amino acid. The significance of this is that ammonium impairs embryo development both in vitro and subsequent development in utero after transfer. It is therefore essential that when using culture media containing amino acids that they are placed in the incubator for the minimum time required for equilibration and they should certainly never be stored in the incubator. Fortunately, glutamine can be replaced with alanylglutamine, a dipeptide that is stable at 37oC. Vitamins are light-sensitive and therefore care should be taken to

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minimize exposure to light by storing the culture media in dark bottles or wrapping them in foil.

Quality control Establishing an appropriate quality control system for the IVF laboratory is a prerequisite in the establishment of a successful laboratory (Chapter 2). The types of bioassays conducted for this have been the focus of much discussion.109 In reality there is no perfect model for the human, save for the very patients we treat. Therefore, it is important to understand the limitations of the assays performed and to use data obtained from bioassays in an appropriate fashion. Quality control should not be limited to the culture media used, but should include all contact supplies and gases used in an IVF procedure. The bioassay we favor is the culture of pronucleate mouse oocytes in protein-free media. There has been a lot of conflicting data regarding the use of the mouse embryo bioassay, but by adjusting conditions one can not only increase the sensitivity of the assay but can also attempt to quantitate quality with it. First, the stage at which the embryo is cultured from has an impact on development: embryos collected at the pronucleate stage do not tend to fair as well in culture as those collected at the 2-cell stage. Secondly, the strain of mice is important: embryos from hybrid parents have a decided advantage in culture, and do not represent the diverse genetic background one is dealing with in an infertility clinic; therefore, a randombred strain of mice provides greater genetic diversity. Thirdly, the embryo cultures should be performed in the absence of protein, as protein has the ability to mask the effects of any potential toxins present. Reports that mouse embryos can develop in culture in medium prepared using tap water110,111 should be interpreted carefully after taking into account the strain of mouse, types of media used, and the supplementation of medium with protein. Silverman et al110 used Ham’s F-10. This medium contains amino acids, which can chelate any possible toxins present in the tap water, e.g. heavy metals. George et al111 included high levels of bovine serum albumin (BSA) in their zygote cultures to the blastocyst. Albumins can chelate potential embryotoxins and thereby mask the effect of any present in the culture medium.112,113 Furthermore, all studies used blastocyst development as the sole criterion for assessing embryo development. Blastocyst development is a relatively poor indicator of embryo quality and does not accurately reflect developmental potential.51 Therefore, rates of development should be determined by scoring the embryos at specific times during culture. Key times to examine the embryos include the morning of day 3 to determine the extent of compaction, the afternoon of day 4 to determine the degree of blastocyst formation, and the morning of day 5 to assess the initiation of hatching. Finally, the embryos that form blastocysts in a given time, typically on the morning of day 5, should have

their cell numbers determined, as blastocyst cell number is a good indicator of subsequent development potential. When components of certain culture media can affect the development of the ICM directly, such as essential amino acids, a differential nuclear stain should be performed in order to determine the extent of ICM development. Using such an approach it is possible to identify potential problems in culture media before they are used clinically. In our experience around 25% of all contact supplies fail such pre-screening.109 Although some of the contact supplies that fail the bioassay are not outright lethal, they do compromise embryo development. If undetected this would result in reduced clinical pregnancy rates. Therefore, this can help to explain periodic changes in clinical pregnancy rates and therefore emphasizes the significance of an ongoing quality control program.

What day should embryo transfer be performed? For the past three decades the majority of embryos conceived through IVF have been transferred between days 1 and 3 at either the pronucleate or cleavage stages. The reason for this stems primarily from the inability of past culture systems to support the development of viable blastocysts at acceptable rates. However, with the advent of sequential culture media3 it is feasible to perform day 5 blastocyst transfers as a matter of routine in an IVF clinic.114,115 This now facilitates an answer to the question; on which day of embryo development should embryos be transferred? Before answering this question, the potential advantages and disadvantages of blastocyst culture and transfer are considered.

Blastocyst transfer: advantages and disadvantages The potential advantages of blastocyst culture and transfer have been well documented116–119 and include: 1. Synchronizing embryonic stage with the female tract: this is important as the levels of nutrients within the fallopian tube and uterus do differ, and therefore the premature transfer of the cleavagestage embryo to the uterus could result in metabolic stress.1 Furthermore, the uterine environment during a stimulated cycle can not be considered normal. Certainly it is known from animal studies that the hyperstimulated female tract is a less than optimal environment for the developing embryo, resulting in impaired embryo and fetal development.120–122 Therefore, it would seem prudent to shorten the length of time an embryo is exposed to such an environment before implantation. 2. When embryos are selected for transfer at the 2- to 8cell stage the embryonic genome has only just begun to be transcribed,22,123 and therefore it is not possible to identify from within a given cohort the embryos

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3.

4.

5.

6.

with the highest developmental potential. Only by culturing embryos past the maternal/embryonic genome transition and up to the blastocyst does it become possible to identify those embryos with limited or no developmental potential. Relatively recently, there has been a move to assess pronucleate stage oocytes in order to select embryos for transfer,124 with the report that implantation rates can be increased by the assessment of pronuclear morphology. Similarly, Gerris et al125 have used a scoring system for day 3 to increase implantation rates. However, assessment of the embryos at either the pronuclear or cleavage stages can at best be considered as an assessment of the oocyte. The quality of the oocyte is important, as the quality of the developing embryo is ultimately dependent on the quality of the gametes from which it is derived, but it provides limited information regarding true embryo developmental potential. Not all fertilized oocytes are normal and, therefore, a percentage always exists that is not destined to establish a pregnancy or go to term. Factors contributing to embryonic attrition include an insufficiency of stored oocyte coded gene products, and a failure to activate the embryonic genome.126 The culmination of this is that many abnormal embryos arrest during development in vitro. So by culturing embryos to the blastocyst stage, one has already selected against those embryos with little if any developmental potential. Sandalinas and colleagues127 have confirmed that some chromosomally abnormal human embryos can reach the blastocyst stage in vitro. However, even though aneuploid embryos form blastocysts at lower rates than their euploid counterparts, this means that blastocyst culture can not be used as the sole means of identifying chromosomally abnormal embryos. Uterine contractions have been negatively correlated with embryo transfer outcome, possibly by the expulsion of embryos from the uterine cavity.128 Uterine junctional zone contractions have been quantitated and found to be strongest on the day of oocyte retrieval.129 All patients exhibited such contractions on days 2 and 3 after retrieval, but contractility decreased and was barely evident on day 4. It is therefore feasible that the transfer of blastocysts on day 5 is, by default, associated with reduced uterine contractions and therefore there is less chance for embryonic expulsion and loss.130 Cryopreservation of embryos at the blastocyst stage appears to be more successful than at earlier stages.131 Trophectoderm biopsy and analysis may prove to be more reliable than current procedures performed on cleavage-stage embryos.

The potential disadvantage of extended embryo culture in a program where only blastocyst culture and transfer is offered is the possibility that a patient will not have a blastocyst for transfer. Certainly there has

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been an increase in the percentage of patients who do not have an embryo transfer from 2.9% on day 3 to 6.7% on day 5 in one clinic,115 and from 1.3% on day 3 to 2.8% on day 5 in another.114 Interestingly, in spite of the increase in patients not having an embryo transfer, there was a significant increase in pregnancy rate per retrieval with blastocyst culture, due to a significant increase in implantation rates. In support of the move to blastocyst transfer, as opposed to the transfer of embryos at the cleavage stage, 17 prospective randomized trials on blastocyst transfer following the use of sequential media have been published (Table 16.4).132–148 Eight have reported a significant increase in implantation/pregnancy rates when embryos were transferred at the blastocyst stage on day 5 rather than at the cleavage stage. Eight of the trials reported no difference in implantation rate with respect to day of transfer, while only one clinic reported a lower implantation rate when day 5 transfer was used. Interestingly, of the trials listed in Table 16.4, there is incomplete information published on the culture system used. Although the media types are listed, there is limited information available on the other components of the culture system used. Furthermore, neither the number and type of incubators in each laboratory nor the types of air handling systems are documented. It is important to encourage clinics to report such parameters in order to be able to assess the merits and impacts of such variables on IVF outcome. In a meta-analysis of prospective trials, in which equal numbers of embryos were transferred, it was concluded that: The best available evidence suggests that the probability of live birth after fresh IVF is significantly higher after blastocyst-stage embryo transfer as compared to cleavagestage embryo transfer when equal number of embryos are transferred …5 Additionally, in the most recent Cochrane report on blastocyst transfer it was concluded that: … there is a significant difference in pregnancy and live birth rates in favour of blastocyst transfer with good prognosis patients…. There is emerging data to suggest that in selected patients, blastocyst culture may be applicable for single embryo transfer.4 In support of such analyses, from a model previously developed to determine which patients should have single embryo transfer, it was determined that pregnancy outcome was more favorable with day 5 than day 3 transfer.149 As well as the published prospective randomized trials, there are retrospective studies that have concluded that day 5 transfer exhibits significant benefits for human assisted reproductive technologies (ART) in both nonselected and specific patient populations.114,115,150

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Table 16.4 Outcome of prospective randomized trials on embryo transfer at the cleavage and blastocyst stages when sequential media have been used for embryo culture Cleavage stage No. of embryos transferred

Blastocyst

Implantation Pregnancy No. of embryos rate (%) rate (%) transferred

Implantation Pregnancy rate (%) rate (%)

Author

Patient population

Gardner et al (1998)132

≥10 follicles of >12 mm on day of hCG

3.7

37.0

66

2.2

55.4∗∗

71

Coskun et al (2000)133

≥4 2PN

2.3

21

39

2.2

24

39

Karaki et al (2002)134

≥5 2PN

3.5

13

26

2.0

26∗∗

29

Levron et al (2002)135

<38 years old and >5 2PN

3.1

38.7∗∗

45.5

2.3

20.2

18.6

Utsonomiya et al (2002)136

All

2.9

11.7

26.3

3.0

9.2

24.9

Rienzi et al (2002)137

<38 years old and ≥8 2PN by ICSI

2

35

58

2

38

62

Van der Auwera et al (2002)138

All (day 2 transfers)

1.86

29

32

1.87

46∗

44

Frattarelli et al (2003)139

<35 years old, no previous IVF and ≥10 follicles of ≥14 mm on day of hCG

2.96

26.1

43.5

2.04

43.4∗

69.2

Emiliani et al (2003)140

<39 (day 2 transfers)

2.1

29

49

1.9

30

44

Magreiter et al (2003)141

Mean age of 32.1 years old, with a mean of 7 pronucleate oocytes (days 1–5 transfers)

Bungum et al (2003)142

<40 years old, FSH <12 IU/l, >2 8-cell embryo with <20% fragmentation

2.0

43.9

61

1.96

36.7

51

Pantos et al (2004)143

<40 years old, <4 previous failures, days 2 and 3

4.0

15.7 and 16

46.9 and 48.1

3.39

15.6

37

Levitas et al (2004)144

<37 years old and 3 failed IVF attempts

3.4

6.0

12.9

1.9

21.2∗∗

21.7

Hreinsson et al (2004)145

>5 follicles, days 2 and 3

1.8

20.9

36.7

1.9

21.1

32.5

Kolibianakis et al (2004)146

<43 years old

1.9

24.5

33.1

1.8

24.5

33.2

Papanikolaou et al (2005)147

<37 years old, >3 embryos with >5 cells and <20% fragmentation

2.0

20.6

27.4

1.97

37.0∗∗

51.3∗∗

Papanikolaou et al (2006)148

<36 years old, 1st or 2nd cycle, FSH <13

1

23.3

21.6

1

33.1∗

33.1∗

50∗ on days 4 and 5

20 on day 1 and 30.4 on days 2 and 3

hcg, human chorionic gonadotropin; 2PN, 2 pronuclei; ICSI, intracytoplasmic sperm injection; FSH, follicle-stimulating hormone; IVF, in vitro fertilization. Significantly different from cleavage stage: ∗, p <0.05; ∗∗, p <0.01.

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80 70 60

60.9

60.9 56.0 47.4

Percent

For patients having oocyte donation, blastocyst culture and transfer is the most effective course of treatment.151 Oocytes from donors generally represent a more viable cohort of gametes, as they tend to come from young fertile women. Embryos derived from oocyte donors tend to reach the blastocyst stage at a higher frequency than those from IVF patients, and be of higher quality. It is possible to attain an implantation rate of >65% when transferring blastocysts to recipients whose mean age is over 40 years old (Table 16.3).151 Such data not only reflect the competency of modern embryo culture systems but also emphasize the need to move to single embryo transfers, especially when performing day 5 transfers.152

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50 40 30 20

Toward single embryo transfer Several reviews have discussed the development of scoring systems used in clinical IVF and their significance in identifying the most viable embryo(s) for transfer153–155 (see also Chapter 17). Certainly with newer types of embryo culture media, implantation rates are increasing whether embryos are transferred at the cleavage stage or blastocyst. It is envisaged that for a significant number of patients, blastocyst culture and transfer will be the most effective means of being able to transfer a single embryo while maintaining high pregnancy rates, as it is evident that blastocyst score is highly predictive of implantation potential. A prospective randomized trial of one versus two blastocysts transferred in patients with 10 or more follicles has been performed. The data in Fig 16.5 indicate that it is possible to transfer a single blastocyst and obtain an ongoing pregnancy rate of 60%.152 Subsequent trials of single blastocyst transfer versus cleavage-stage embryo transfer have confirmed the higher implantation rate of the later stage embryo. It has also been established that fetal loss is significantly less following blastocyst transfer.156

Cumulative pregnancy rates per retrieval: the significance of cryopreservation The introduction of blastocyst culture was met with much speculation, as not all laboratories were able to cryopreserve blastocysts that were not transferred. However, with the development of more suitable cryopreservation procedures, it is now possible to obtain implantation and ongoing pregnancy rates of greater than 30 and 60%, respectively, using frozen–thawed blastocysts.131 Consequently, the ability of a given culture system to support embryo cryosurvival is of great significance. Media containing hyaluronan appear to confer great advantage in this regard.

Future developments in embryo culture systems An area not discussed in this text has been the role of growth factors in regulating embryo development in

10 0 Implantation

Ongoing pregnancy

Twins

Fig 16.5 In vitro fertilization (IVF) outcome following the transfer of either one or two blastocysts. Dark bars represent the transfer of a single blastocyst (group I), open bars represent the transfer of two blastocysts (group II). Implantation and pregnancy rates were not statistically different between the two groups of patients. There were no twins in group I in contrast to 47.4% twins in group II. The biochemical pregnancy rate was equivalent between the two groups (group I, 12.5%; group II, 5%). From Gardner et al152 with permission.

culture and subsequent fetal development. Although numerous growth factors in isolation have been shown to modulate embryo development in culture,157,158 we currently do not know their optimal concentration for clinical use, nor which groups of growth factors should be present together in the medium. However, growing research on the effects of such factors at the physiological, genomic, and proteomic levels could lead to their introduction into clinical IVF over the next few years. As discussed previously, there is nothing physiological about the physical conditions in which embryos are cultured. Rather than a static drop of medium, the future may engage perfusion culture systems, enabling the embryo to be exposed to a flux of nutrients and factors (Figs 16.6, 16.7).23,159,160 This latter approach has the advantage of being able to expose embryos to numerous gradients and fresh media throughout development. Furthermore, samples of medium can be taken and analyzed for carbohydrates,161 amino acids,162 and other factors related to implantation potential post transfer163 (Chapter 17). Research in this area is growing, and the application of novel elastomers, together with soft lithography, is starting to produce a new generations of chips capable of moving submicroliter volumes accurately for both culture and analysis.159,160,164

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Medium introduced: changing metabolite pool, introduction of stagespecific factors, etc.

Channel or microchamber created in biologically compatible, gas permeable and transparent material, such as PDMS

Medium expelled: Removal of toxins such as ammonium

Embryos cultured individually or in groups in volumes ranging from nanoliters to microliters

Fig 16.6 Schematic of an embryo perfusion culture system. Culture media are continuously passed over the embryo(s). The composition of the culture media can be changed according to the specific requirements of each stage of embryonic development. Toxins such as ammonium are not able to build up and impair embryo development, while more labile components of the culture system are not denatured. PDMS, polydimethylsiloxane. Modified from Gardner.23

a

V

b

S

c

Fig 16.7 Culture of preimplantation mouse embryos in a ‘Lab-on-a-chip’ microfluidic channel. The chip is constructed using polydimethylsiloxane (PDMS) and soft lithography.164 (a) A 5 µl channel for embryo culture. Embryo entry into the channel is regulated by a pneumatic valve (V), and medium can leave the channel through a sampling port (S). (b) Day 4 mouse embryos cultured in a 5 µl drops of medium G1 under paraffin oil. (c) Sibling day 4 mouse embryos to (b) but cultured in a 5 µl channel in the chip shown in (a). There was no difference in blastocyst development of cell numbers between the two treatments over several replicates.179

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Conclusions The culture system in the clinical laboratory is one part of the overall treatment cycle. Good oocytes, derived from appropriate stimulation regimens, are able to give rise to good embryos. However, it is not currently feasible to obtain good embryos from poor oocytes. Furthermore, the embryo transfer technique and subsequent luteal support administered have an impact on cycle outcome (see Chapters 43 and 49). Significant improvements in culture media formulations have certainly provided better conditions for the human embryo to develop. With this, it has become possible to culture to the blastocyst stage. Increasing evidence supports the move to day 5 transfers for a significant number of patients attending an infertility program, especially those undergoing oocyte donation, or who are <36 years old and in their first cycle. However, there are certain exceptions to this, such as clinics in countries like Germany, who are only able to culture as many pronucleate oocytes as they will transfer, and this can be no more than three. In such cases, people may be deterred from trying blastocyst culture, as certainly there would be an increase in the number of patients not having an embryo transfer. It is evident that culture conditions affect the ability of embryos to survive cryopreservation. As we move closer to the day when single embryo/blastocyst transfer is considered the standard of care, it becomes more important than ever to ensure embryos are given the best chance of being cryopreserved for transfer at a later date, thereby increasing the efficiency of each oocyte retrieval procedure. In this chapter we have outlined how human embryos can be successfully cultured. As with all aspects of biomedicine, not everyone will agree with our position on everything. Irrespective of opinion, one thing is clear: the systems outlined do work very effectively, as reflected in the implantation and pregnancy rates that can be attained, especially in an oocyte donation model. Together with the information elsewhere in this book, our patients are in better hands today than they have ever been.

Appendix Embryo culture In order to perform oocyte isolation, preparation for ICSI, or embryo manipulation outside a CO2 incubator there are two distinct approaches: one can use media that have a second buffer system in them, such as MOPS or HEPES (both of which will keep the pH of the medium relatively constant in air), or one can employ a pediatric isolette-type system. The latter maintains both temperature and CO2, and negates the use of buffer systems other than bicarbonate. Both

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approaches can be made to work effectively, and the choice is one of preference and cost. Embryo culture should be performed in a reduced O2 environment (typically 5%, but the optimum value below 10% is yet to be determined) and 5–7% CO2, depending on the media system chosen and altitude. This multigas environment can be created using either a trigas incubator or a modular incubator chamber/desiccator and a cylinder of special gas mix. It is advisable to minimize the number of observations made during embryo development, and to minimize the number of cases per incubator. It is also essential that all contact supplies, media, oil, etc., are prescreened with a suitable test.109

Pronucleate oocytes to Day 3 culture Embryo manipulation (following fertilization assessment) Once the cumulus is removed (see Chapter 7), all manipulations should be performed using a glass capillary style pipette or a displacement pipette. Fine control can be attained with both approaches. It is important to use a pipette with the appropriate size tip (day 1–3; around 200 µm). Using the appropriate size tip minimizes the volumes of culture medium moved with each embryo, which typically should be less than 1 µl. Such volume manipulation is a prerequisite for successful culture.

Setting up culture dishes (day 1 to day 3) At around 4 p.m. on the day of oocyte retrieval, label pretested 60-mm dishes with the patient’s name. Using a single-wrapped tip, first rinse the tip once, then place 6 × 25 µl drops of phase 1 medium, for example G1, into the plate. Four drops should be at the 3, 6, 9, and 12 o’clock positions (for embryo culture); the fifth and sixth drops should be in the middle of the dish (wash drops). Immediately cover drops with 9 ml of prescreened paraffin oil. Prepare no more than two plates at one time. Using a new tip for each drop, first rinse the tip, and then add a further 25 µl of medium to each original drop. Immediately place the dish in the incubator. Gently remove the lid of the dish and set at an angle on the side of the plate. Dishes must gas in the incubator for a minimum of 4 h (this is the minimal measured time for the media to reach the correct pH under oil). For each patient, set up a wash dish at the same time as the culture dishes. Place 1 ml of phase 1 medium into the center of an organ well dish. Place 2 ml of medium into the outer well. Place immediately into the incubator. If working outside an isolette, use HEPES/MOPS buffered medium with amino acids. This should not be placed in a CO2 incubator, but rather warmed on a heated stage.

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Morning of day 1 Culture in phase 1 medium Following removal of the cumulus cells, embryos are transferred to the organ well dish and washed in the center well drop of medium in the culture dish. Washing entails picking up the embryo 2–3 times and moving it around within the well. Embryos should then be washed in the two center drops in the culture dish and up to four embryos placed in each drop of the

culture medium. Four is the maximum number of embryos that can be cultured in each drop due to their nutrient requirements. More than four embryos may result in a significant depletion of the nutrient pool by the embryos. This will result in no more than 16 embryos per dish. Return the dish to the incubator immediately. It is advisable to culture embryos in at least groups of two. Therefore, for example, for a patient with six embryos it is best to culture in two groups of three and not four and two or five and one.

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Fig 16.8 Human embryos on the morning of day 5 (4 days of culture from the pronucleate stage). Embryos were cultured from the pronucleate oocyte until midday on day 3 in medium G1.3. Cleavage-stage embryos were then washed in medium G2.3 and cultured in G2.3 for a further 48 h. a b c d e f g

0 – morula or lesser stage 1 – early blastocyst 2 – blastocyst 3 – full blastocyst 4 – expanded blastocyst 5 – hatching blastocyst 6 – hatched blastocyst

no blastocoel cavity seen blastocoel less than half the volume of the embryo blastocoel ≥ half of the volume of the embryo blastocoel completely fills the embryo zona thinning and overall increase in size trophectoderm has started to herniate through the zona blastocyst has completely escaped from the zona

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On day 3, embryos can be transferred to the uterus in an appropriate hyaluronan-enriched transfer medium.

Day 3 embryos to the blastocyst Setting up culture dishes (day 3 to day 5) On day 3 before 8:30 a.m. label a 60-mm dish with the patient’s name. Using a single-wrapped tip, first rinse the tip once, then place 6 × 25 µl drops of phase 2 medium, for example G2, into the plate. Immediately cover with 9 ml of oil. Never prepare more than two plates at one time. Using a new tip for each drop, rinse the tip and then add a further 25 µl of medium to each original drop. Immediately place the dish in the incubator. Gently remove the lid and set on the side of the plate. For each patient, set up one wash dish per 10 embryos. Place 1 ml of phase 2 medium into the center of an organ well dish. Place 2 ml of medium into the outer well. Place immediately into the incubator. Dishes must gas in the incubator for a minimum of 4 h. If working outside an isolette, use HEPES/MOPS buffered medium with amino acids. This should not be placed in a CO2 incubator, but rather warmed on a heated stage. For each patient, set up one sorting dish before 8:30 a.m. Place 1 ml of phase 2 medium into the center of an organ well dish. Place 2 ml of medium into the outer well. Place immediately into the incubator. If working outside an isolette, use HEPES/MOPS buffered medium with amino acids. This should not be placed in a CO2 incubator, but rather warmed on a heated stage.

Culture in phase 2 medium Moving embryos from phase 1 to phase 2 media should occur between 9 a.m. and midday. Wash embryos in the organ well dish. Washing entails picking up the embryo 2–3 times and moving it around within the well. Transfer embryos to the sorting dish and group like embryos together. Rinse through the wash drops of medium and again place up to four embryos in each drop of phase 2 medium. This will result in no more than 16 embryos per dish. Return the dish to the low O2 incubator immediately. If working outside an isolette, use HEPES/MOPS buffered medium with amino acids in the sorting dish. This should not be placed in a CO2 incubator, but rather warmed on a heated stage. On the morning of day 5, embryos should be scored (Fig 16.8; see Gardner and Schoolcraft180 and Chapter 17) and the one or two top scoring embryos selected for transfer. Transfers should be performed in a medium enriched with hyaluronan. Any blastocysts not transferred can be cryopreserved. Should an embryo not have formed a blastocyst by day 5, then it should be cultured in a fresh drop of the phase 2 medium for 24 h and assessed on day 6. Embryos at different stages of development are shown in Fig 16.8. For more details on blastocyst grading, see Chapter 17 and Gardner et al.181

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Culture systems for the human embryo 118. Olivennes F, Hazout A, Lelaidier C, et al. Four indications for embryo transfer at the blastocyst stage. Hum Reprod 1994; 9: 2367−73. 119. Scholtes MC, Zeilmaker GH. A prospective, randomized study of embryo transfer results after 3 or 5 days of embryo culture in in vitro fertilization. Fertil Steril 1996; 65: 1245−8. 120. Ertzeid G, Storeng R. Adverse effects of gonadotrophin treatment on pre- and postimplantation development in mice. J Reprod Fertil 1992; 96: 649−55. 121. Ertzeid G, Storeng R, Lyberg T. Treatment with gonadotropins impaired implantation and fetal development in mice. J Assist Reprod Genet 1993; 10: 286−91. 122. Van der Auwera I, Pijnenborg R, Koninckx PR. The influence of in-vitro culture versus stimulated and untreated oviductal environment on mouse embryo development and implantation. Hum Reprod 1999; 14: 2570−4. 123. Taylor DM, Ray PF, Ao A, et al. Paternal transcripts for glucose-6-phosphate dehydrogenase and adenosine deaminase are first detectable in the human preimplantation embryo at the three- to four-cell stage. Mol Reprod Dev 1997; 48: 442−8. 124. Scott LA, Smith S. The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum Reprod 1998; 13: 1003−13. 125. Gerris J, De Neubourg D, Mangelschots K, et al. Prevention of twin pregnancy after in-vitro fertilization or intracytoplasmic sperm injection based on strict embryo criteria: a prospective randomized clinical trial. Hum Reprod 1999; 14: 2581−7. 126. Tesarik J. Developmental failure during the preimplanation period of human embryogenesis. In: Van Blerkom J, ed. The Biological Basis of Early Human Reproductive Failure. New York: Oxford University Press, 1994: 327−44. 127. Sandalinas M, Sadowy S, Alikani M, et al. Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage. Hum Reprod 2001; 16: 1954−8. 128. Fanchin R, Righini C, Olivennes F, et al. Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization. Hum Reprod 1998; 13: 1968−74. 129. Lesny P, Killick SR, Tetlow RL, et al. Uterine junctional zone contractions during assisted reproduction cycles. Hum Reprod Update 1998; 4: 440−5. 130. Fanchin R, Ayoubi JM, Righini C, et al. Uterine contractility decreases at the time of blastocyst transfers. Hum Reprod 2001; 16: 1115−19. 131. Veeck LL. Does the developmental stage at freeze impact on clinical results post-thaw? Reprod Biomed Online 2003; 6: 367−74. 132. Gardner DK, Schoolcraft WB, Wagley L, et al. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 1998; 13: 3434−40. 133. Coskun S, Hollanders J, Al-Hassan S, et al. Day 5 versus day 3 embryo transfer: a controlled randomized trial. Hum Reprod 2000; 15: 1947−52. 134. Karaki RZ, Samarraie SS, Younis NA, et al. Blastocyst culture and transfer: a step toward improved in vitro fertilization outcome. Fertil Steril 2002; 77: 114−18.

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135. Levron J, Shulman A, Bider D, et al. A prospective randomized study comparing day 3 with blastocyststage embryo transfer. Fertil Steril 2002; 77: 1300−1. 136. Utsunomiya T, Naitou T, Nagaki M. A prospective trial of blastocyst culture and transfer. Hum Reprod 2002; 17: 1846−51. 137. Rienzi L, Ubaldi F, Iacobelli M, et al. Day 3 embryo transfer with combined evaluation at the pronuclear and cleavage stages compares favourably with day 5 blastocyst transfer. Hum Reprod 2002; 17: 1852−5. 138. Van der Auwera I, Debrock S, Spiessens C, et al. A prospective randomized study: day 2 versus day 5 embryo transfer. Hum Reprod 2002; 17: 1507−12. 139. Frattarelli JL, Leondires MP, McKeeby JL, et al. Blastocyst transfer decreases multiple pregnancy rates in in vitro fertilization cycles: a randomized controlled trial. Fertil Steril 2003; 79: 228−30. 140. Emiliani S, Delbaere A, Vannin AS, et al. Similar delivery rates in a selected group of patients, for day 2 and day 5 embryos both cultured in sequential medium: a randomized study. Hum Reprod 2003; 18: 2145−50. 141. Margreiter M, Weghofer A, Kogosowski A, et al. A prospective randomized multicenter study to evaluate the best day for embryo transfer: does the outcome justify prolonged embryo culture? J Assist Reprod Genet 2003; 20: 91−4. 142. Bungum M, Bungum L, Humaidan P, et al. Day 3 versus day 5 embryo transfer: a prospective randomized study. Reprod Biomed Online 2003; 7: 98−104. 143. Pantos K, Makrakis E, Stavrou D, et al. Comparison of embryo transfer on day 2, day 3, and day 6: a prospective randomized study. Fertil Steril 2004; 81: 454−5. 144. Levitas E, Lunenfeld E, Har-Vardi I, et al. Blastocyst-stage embryo transfer in patients who failed to conceive in three or more day 2-3 embryo transfer cycles: a prospective, randomized study. Fertil Steril 2004; 81: 567−71. 145. Hreinsson J, Rosenlund B, Fridstrom M, et al. Embryo transfer is equally effective at cleavage stage and blastocyst stage: a randomized prospective study. Eur J Obstet Gynecol Reprod Biol 2004; 117: 194−200. 146. Kolibianakis EM, Zikopoulos K, Verpoest W, et al. Should we advise patients undergoing IVF to start a cycle leading to a day 3 or a day 5 transfer? Hum Reprod 2004; 19: 2550−4. 147. Papanikolaou EG, D’Haeseleer E, Verheyen G, et al. Live birth rate is significantly higher after blastocyst transfer than after cleavage-stage embryo transfer when at least four embryos are available on day 3 of embryo culture. A randomized prospective study. Hum Reprod 2005; 20: 3198−203. 148. Papanikolaou EG, Camus M, Kolibianakis EM, et al. In vitro fertilization with single blastocyst-stage versus single cleavage-stage embryos. N Eng J Med 2006; 354: 1139−46. 149. Hunault CC, Eijkemans MJ, Pieters MH, et al. A prediction model for selecting patients undergoing in vitro fertilization for elective single embryo transfer. Fertil Steril 2002; 77: 725−32. 150. Balaban B, Urman B, Alatas C, et al. Blastocyststage transfer of poor-quality cleavage-stage embryos results in higher implantation rates. Fertil Steril 2001; 75: 514−18.

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151. Schoolcraft WB, Gardner DK. Blastocyst culture and transfer increases the efficiency of oocyte donation. Fertil Steril 2000; 74: 482−6. 152. Gardner DK, Surrey E, Minjarez D, et al. Single blastocyst transfer: a prospective randomized trial. Fertil Steril 2004; 81: 551–5. 153. Cummins JM, Breen TM, Harrison KL, et al. A formula for scoring human embryo growth rates in in vitro fertilization: its value in predicting pregnancy and in comparison with visual estimates of embryo quality. J In Vitro Fert Embryo Transf 1986; 3: 284–95. 154. Steer CV, Mills CL, Tan SL, et al. The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in-vitro fertilization and embryo transfer programme. Hum Reprod 1992; 7: 117–19. 155. Scott L. The biological basis of non-invasive strategies for selection of human oocytes and embryos. Hum Reprod Update 2003; 9: 237–49. 156. Papanikolaou EG, Camus M, Fatemi HM, et al. Early pregnancy loss is significantly higher after day 3 single embryo transfer than after day 5 single blastocyst transfer in GnRH antagonist stimulated IVF cycles. Reprod Biomed Online 2006; 12: 60–5. 157. Kane MT, Morgan PM, Coonan C. Peptide growth factors and preimplantation development. Hum Reprod Update 1997; 3: 137–57. 158. Hardy K, Spanos S. Growth factor expression and function in the human and mouse preimplantation embryo. J Endocrinol 2002; 172: 221–36. 159. Suh RS, Phadke N, Ohl DA, et al. Rethinking gamete/embryo isolation and culture with microfluidics. Hum Reprod Update 2003; 9: 451–61. 160. Wheeler MB, Walters EM, Beebe DJ. Toward culture of single gametes: the development of microfluidic platforms for assisted reproduction. Theriogenology 2007; 68(Suppl 1): S178–89. 161. Lane M, Gardner DK. Selection of viable mouse blastocysts prior to transfer using a metabolic criterion. Hum Reprod 1996; 11: 1975–8. 162. Brison DR, Houghton FD, Falconer D, et al. Identification of viable embryos in IVF by noninvasive measurement of amino acid turnover. Hum Reprod 2004; 19: 2319–24. 163. Gardner DK, Sakkas D. Assessment of embryo viability: the ability to select a single embryo for transfer – a review. Placenta 2003; 24(Suppl B): S5–12. 164. Urbanski JP, Thies W, Rhodes C, et al. Digital microfluidics using soft lithography. Lab Chip 2006; 6: 96–104. 165. Crosby IM, Gandolfi F, Moor RM. Control of protein synthesis during early cleavage of sheep embryos. J Reprod Fertil 1988; 82: 769–75. 166. Rieger D, Loskutoff NM, Betteridge KJ. Developmentally related changes in the uptake and metabolism of glucose, glutamine and pyruvate by cattle embryos produced in vitro. Reprod Fertil Dev 1992; 4: 547–57.

167. Van Winkle LJ, Haghighat N, Campione AL. Glycine protects preimplantation mouse conceptuses from a detrimental effect on development of the inorganic ions in oviductal fluid. J Exp Zool 1990; 253: 215–19. 168. Liu Z, Foote RH. Development of bovine embryos in KSOM with added superoxide dismutase and taurine and with five and twenty percent O2. Biol Reprod 1995; 53: 786–90. 169. Lindenbaum A. A survey of naturally occurring chelating ligands. Adv Exp Med Biol 1973; 40: 67–77. 170. Wu G, Morris SM Jr. Arginine metabolism: nitric oxide and beyond. Biochem J 1998; 336(Pt 1): 1–17. 171. Martin PM, Sutherland AE, Van Winkle LJ. Amino acid transport regulates blastocyst implantation. Biol Reprod 2003; 69: 1101–8. 172. Martin PM, Sutherland AE. Exogenous amino acids regulate trophectoderm differentiation in the mouse blastocyst through an mTOR-dependent pathway. Dev Biol 2001; 240: 182–93. 173. Gardner DK. Dissection of culture media for embryos: the most important and less important components and characteristics. Reprod Fertil Dev 2008; 20: 9–18. 174. Hardy K, Robinson FM, Paraschos T, et al. Normal development and metabolic activity of preimplantation embryos in vitro from patients with polycystic ovaries. Hum Reprod 1995; 10: 2125–35. 175. Simon C, Garcia Velasco JJ, Valbuena D, et al. Increasing uterine receptivity by decreasing estradiol levels during the preimplantation period in high responders with the use of a follicle-stimulating hormone step-down regimen. Fertil Steril 1998; 70: 234–9. 176. Ertzeid G, Storeng R. The impact of ovarian stimulation on implantation and fetal development in mice. Hum Reprod 2001; 16: 221–5. 177. Kelley RL, Kind KL, Lane M, et al. Recombinant human follicle-stimulating hormone alters maternal ovarian hormone concentrations and the uterus and perturbs fetal development in mice. Am J Physiol Endocrinol Metab 2006; 291: E761–70. 178. Kwong WY, Wild AE, Roberts P, et al. Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension. Development 2000; 127: 4195–202. 179. Potter DL, Johnson MJ, Gardner DK, et al. Culture of mammalian embryos in a microfluidic chip. 2007; unpublished observations. 180. Gardner DK, Schoolcraft WB. In vitro culture of human blastocyst. In: Jansen R, Mortimer D, eds. Towards Reproductive Certainty: Fertility and Genetics Beyond 1999. Carnforth, UK: Parthenon Publishing, 1999: 378–88. 181. Gardner DK, Stevens J, Sheehan CB, Schoolcraft WB. Analysis of blastocyst morphology. In: Elder KC, Cohen J, eds. Human Preimplantation Embryo Selection. London: Informa Healthcare, 2007: 79–87.

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17 Evaluation of embryo quality: new strategies to facilitate single embryo transfer Denny Sakkas, David K Gardner

Introduction Utilization of assisted reproductive technologies (ART) continues to increase annually, with over 125 000 treatment cycles being initiated in the United States alone in 2004. This trend is driven by the steady improvement in ART delivery rates, improving access to care in many areas, and the relative ineffectiveness of other treatment options. At the current time, more than 1% of all children born in the United States and Europe are from ART-related conceptions. The high success rates established through in vitro fertilization (IVF) are attained, in many cases, only through the simultaneous transfer of multiple embryos. In 2004 in the United States, a mean number of 2.8 embryos per patient were transferred, leading to a 27.7% delivery rate per initiated IVF cycle and an overall multiple birth rate of 33.5%. In oocyte donation cycles, where the embryos should possess the highest reproductive potential, the transfer of more than one embryo led to a multiple gestation rate of 42%. The risks related to multiple gestations are well known and include preterm delivery, low birth weight, and a dramatic increase in the relative risk for cerebral palsy (reviewed by Adashi et al1). These complications lead to a higher incidence of medical, perinatal, and neonatal complications and a 10-fold increase in healthcare costs compared to a singleton delivery.2 Decreasing the prevalence of multiple gestations while maintaining or improving overall pregnancy rates remains the most significant contemporary goal of infertility research. In a number of countries, including Norway, Sweden, Denmark, Belgium, England, Italy, Germany, and Australia, the dangers associated with multiple pregnancies have been allayed by legal restrictions on the number of embryos that can be transferred in a single IVF cycle. For example, in most Scandinavian countries and Belgium the government has set a legal limit of single embryo transfer (i.e. only one embryo to be transferred per cycle) for specific patient groups, while many other European countries have restricted the number of transferred embryos to a maximum of

two. In other parts of the world, where no legal restrictions exist, the onus is on the individual clinic (as well as the patient) to decrease the number of embryos transferred so that an acceptable balance can be achieved between the risks associated with multiple gestations and ‘acceptable’ pregnancy rates. The current indications are that (in the future) clinics in the United States, and other countries currently lacking legislation, will be compelled via legal, financial, and/or moral obligation to restrict the number of embryos transferred in order to minimize the risk of multiple gestations. A major issue in limiting the number of embryos transferred is the apparent inability to accurately estimate the reproductive potential of individual embryos within a cohort of embryos using the existing selection techniques, which largely encompasses morphological evaluation. Faced with the scenario that we, the worldwide IVF community, will in the future have to select only one or two embryos for transfer, we will be forced to make certain choices. The first choice may be to rely on less aggressive stimulation protocols, hence generating a lower number of eggs at collection. Paradoxically, the generation of a smaller number of oocytes could lead to a greater percentage of viable embryos within a given cohort.3,4 The second choice is to improve the selection process for defining the quality of individual embryos so that the ones we choose for transfer are more likely to implant. This chapter will discuss new strategies in selection criteria that will help us achieve this second choice.

Morphology as an assessment tool Morphological assessment has been a stalwart in the armory of the embryologist for selecting which embryo(s) to replace. Since the early years of IVF it was noted that embryos cleaving faster and those of better morphological appearance were more likely to lead to a pregnancy.5,6 Morphological assessment systems have evolved over the past decade and, in addition to the classical parameters of cell number and

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Textbook of Assisted Reproductive Technologies Ideal features shared by pronucleate oocytes that have high viability: β

(i) the number of nucleolar precursor bodies (NPBs) in both pronuclei never differed by more than three (ii) the NPBs are always polarized or nonpolarized in both pronuclei but never polarized in one pronucleus and not in the other (iii) the angle β from the axis of the pronuclei and the furthest polar body is less than 50°

18-19h post insemination/injection

fragmentation, numerous other characteristics have been examined, including pronuclear morphology, early cleavage to the 2-cell stage, top quality embryos on successive days, and various forms of sequential assessment of embryos [see reviews in references 7–9]. In addition, the ability to culture and assess blastocyst-stage embryos has also significantly improved the ability to select embryos on the basis of morphology.10

The pronucleate oocyte The many transformations that take place during the fertilization process make this a dynamic stage to assess. The oocyte contains the majority of the developmental materials, maternal mRNA, for ensuing that the embryo reaches the 4- to 8-cell stage. In human embryos, embryonic genome activation has been shown to occur between the 4- and 8-cell stage.11 The quality of the oocyte, therefore, plays the lead role for determining embryo development and subsequent viability. A number of studies have postulated that embryo quality can be predicted at the pronucleate oocyte stage. Separate studies by Tesarik and Greco12 and Scott et al13 concentrated on the predictive value of the nucleoli. Tesarik and Greco12 postulated that the normal and abnormal morphology of the pronuclei were related to the developmental fate of human embryos. They retrospectively assessed the number and distribution of nucleolar precursor bodies (NPBs) in each pronucleus of fertilized oocytes that led to embryos that implanted. The characteristics of these concepti were then compared to those that led to failures in implantation. The features that were shared by zygotes that had the 100% implantation success were (1) the number of NPBs in both pronuclei never differed by more than 3 and (2) the NPBs were always polarized or nonpolarized in both pronuclei but never polarized in one pronucleus and not in the other. Pronucleate oocytes not showing the above criteria were more likely to develop into preimplantation embryos that had poor morphology and/or experienced cleavage arrest. The presence of at least one embryo

Fig 17.1 Ideal features shared by pronucleate oocytes that have high viability as described by Tesarik and Greco,12 Garello et al,17 and Scott and Smith.18

that had shown the above criteria at the pronuclear stage in those transferred led to a pregnancy rate of 22/44 (50%), compared to only 2/23 (9%) when none were present. A further criterion of pronucleate oocytes that may affect embryo morphology is the orientation of pronuclei relative to the polar bodies. Oocyte polarity is clearly evident in nonmammalian species. In mammals, the animal pole of the oocyte may be estimated by the location of the first polar body, whereas after fertilization, the second polar body marks the embryonic pole.14 In human oocytes a differential distribution of various factors within the oocyte has been described and anomalies in the distribution of these factors, in particular the side of the oocyte believed to contain the animal pole, are thought to affect embryo development and possibly fetal growth.15,16 Following on from this hypothesis Garello et al17 examined pronuclear orientation, polar body placement, and embryo quality to ascertain if a link existed between a plausible polarity of oocytes at the pronuclear stage and further development. The most interesting observation involved the calculation of angle β (Fig 17.1), which represented the angle between a line drawn through the axis of the pronuclei and the position of the furthest polar body. They found that as the angle β increased, there was a concurrent decrease in the morphological quality of preimplantation-stage human embryos. They postulated that the misalignment of the polar body might be linked to cytoplasmic turbulence, hence disturbing the delicate polarity of the zygote. A further study by Scott and Smith18 devised an embryo score on day 1 on the basis of alignment of pronuclei and nucleoli, the appearance of the cytoplasm, nuclear membrane breakdown, and cleavage to the 2-cell stage. Patients who had an overall high embryo score (>15) had a pregnancy and implantation rate of 34/48 (71%) and 49/175 (28%), respectively, compared to only 4/49 (8%) and 4/178 (2%) in the low embryo score group. The use of pronuclear scoring has been reviewed by Scott.19

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Pregnancy (%)

40

30

20

10

0 0 (159)

1 (42)

2 (78)

No of early 2-cell embryos transferred

Cleavage-stage embryos The most widely used criteria for selecting the best embryos for transfer have been based on cell number and morphology.5 A vast number of variations on the theme have been published. Some of the key studies presented by Gerris et al20 and Van Royen et al21 employed strict embryo criteria to select single embryos for transfer. The necessary characteristics of their ‘top’-quality embryos were established by retrospectively examining embryos that had a very high implantation potential.21 These top-quality embryos had the following characteristics: four or five blastomeres on day 2 and at least seven blastomeres on day 3 after fertilization, absence of multinucleated blastomeres, and <20% of fragments on day 2 and day 3 after fertilization. When these criteria were utilized in a prospective randomized clinical trial comparing single and double embryo transfers, it was found that in 26 single embryo transfers where a top-quality embryo was available, an implantation rate of 42.3% and ongoing pregnancy rate of 38.5% was obtained. In 27 double embryo transfers an implantation rate of 48.1% and ongoing pregnancy rate of 74% was obtained. A larger study analyzing the outcome of 370 consecutive single top-quality embryo transfers in patients <38 years old for pregnancy showed that the pregnancy rate after single top-quality embryo transfer was 51.9%.22 Recently, the same group of authors have provided further evidence of the importance of multinucleation as part of the equation in selecting top-quality embryos.23 The majority of studies that have used and reported embryo selection criteria on the basis of cell number and morphology, do so by stating that embryos were selected on day 2 or day 3. As discussed by Bavister,24 one of the most critical factors in determining selection criteria is to ascertain strict time points to compare the embryos. A 4-cell embryo scored in the morning of day 2 is definitely not the same as one that was scored as a 4-cell in the afternoon. Sakkas and

Fig 17.2 The percentage of clinical pregnancies (light columns) and implantation rate (dark columns) in relation to whether patients had 0, 1, or 2 early-cleavage embryos transferred. The numbers in parentheses indicate the number of cycles per group.

colleagues have therefore used cleavage to the 2-cell stage at 25 h post insemination or microinjection as the critical timepoint for selecting embryos.25–27 In a larger series of patients, it was found that 45% of patients undergoing IVF or intracytoplasmic sperm injection (ICSI) have early cleaving 2-cell embryos. Patients who have early-cleaving 2-cell stage embryos allocated for transfer on day 2 or 3 have significantly higher implantation and pregnancy rates.27 Furthermore, nearly 50% of the patients who have two early-cleaving 2-cell embryos transferred achieve a clinical pregnancy (Fig 17.2). The most impressive data investigating the usefulness of earlycleaving 2-cell embryos is that supported by single embryo transfer.28,29 In one of these studies Salumets et al28 showed that when transferring single embryos a significantly higher clinical pregnancy rate was observed after transfer of early-cleaving (50%) rather than non-early-cleaving (26.4%) embryos. The embryos that cleave early to the 2-cell stage have also been reported to have a significantly higher blastocyst formation rate.30,31 It is also interesting to note that, in the embryo scoring system described by Scott and Smith,18 embryos that had already cleaved to the 2-cell stage by 25–26 h postinsemination were assigned an additional score of 10. This score is a sizable part when the high-quality embryos were judged to be those scoring >15. Recently a large data set has been reported examining the sequential growth of 4042 embryos individually cultured from day 1 to day 5/6.31 Pronuclear morphology on day 1, and early cleavage, cell number, and fragmentation rate on day 2 were evaluated for each zygote. Interestingly, early cleavage and cell number on day 2 were the most powerful parameters to predict the development of a good-morphology blastocyst at day 5. Overall, the study found that parameters of early development were not helpful in predicting implantation ability and that transfer of a good-morphology blastocyst was still associated with high implantation and live birth rates.

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Development to the blastocyst stage The commercial availability of sequential culture media systems has led to the routine use of blastocyst culture in many IVF clinics. The type of blastocyst obtained is, however, of critical importance. As with the scoring of embryos during the cleavage stages, time and morphology play an important part in selecting the best blastocyst. The scoring assessment for blastocysts devised by Gardner and Schoolcraft32 is based on the expansion state of the blastocyst and on the consistency of the inner cell mass and trophectoderm cells (Fig 17.3). Examples of high-quality blastocysts are shown in Fig 17.4. Using such a grading system, it was determined that when two high-scoring blastocysts (>3AA) – i.e. expanded blastocoel with compacted inner cell mass and cohesive trophectoderm epithelium – are transferred, the clinical pregnancy and implantation rates of >80% and 69%, respectively, can be attained.33 When two blastocysts not achieving these scores (<3AA) are transferred, the clinical pregnancy and implantation rates are significantly lower, at 50% and 33%,34 respectively. Although reduced from the values obtained with top-scoring blastocysts, it is evident that early blastocysts on day 5 still have high developmental potential. The time of blastocyst formation is also crucial. When cases were compared where only day 5 and 6 frozen blastocysts were transferred, compared with those frozen on or after day 7 and transferred, the pregnancy rates were 7/18 (38.9%) and 1/16 (6.2%), respectively.35 In these cases, expanded blastocysts with a definable inner cell mass and trophectoderm were frozen. These results show that even though blastocysts can be obtained, a crucial factor is when they form blastocysts. When taking this into account, the best blastocysts would be those that develop by day 5. Selecting the fastest blastocysts had been proposed to create a bias in sex selection, as Menezo et al36 reported that blastocysts transferred after development in coculture gave rise to the birth of more male offspring. Milki et al37 also reported that combined data from the literature show a male-to-female ratio of 57.3%/42.7% in blastocyst transfer compared with 51.2%/48.8% in day 3 embryo transfer (p = 0.001). Extensive use of sequential culture media appears to indicate that the skewing of the sex ratio following blastocyst transfer is not a major concern.38 Although day 5 blastocyst culture remains as one of the strongest indicators of all morphological parameters,39 the reality is that most practicing IVF centers still choose to perform the majority of transfers at an earlier date.

A strategy for selecting the best embryo by morphology The above selection criteria have all shown that they generate some benefit in identifying individual embryos that have a high viability.

How do we implement a strategy for selecting a single embryo when we have many to choose from? A multiple-step scoring system that encompasses all the above criteria would allow us to reach this goal. The use of sequential scoring systems has been shown to be beneficial by a number of authors.21,30,40 Here we propose the following plan for sequential embryo assessment: 18–19 h postinsemination/ICSI: (Fig 17.1) The pronuclei are examined for: • symmetry • the presence of even numbers of NPBs • the positioning of the polar bodies. 25–26 h postinsemination/ICSI: (Fig 17.5) • embryos that have already cleaved to the 2-cell stage • zygotes that have progressed to nuclear membrane breakdown. 42–44 h postinsemination/ICSI: (Fig 17.5) • number of blastomeres should be greater or equal to four • fragmentation of less than 20% • no multinucleated blastomeres. 66–68 h postinsemination/ICSI: (Fig 17.5) • number of blastomeres should be greater or equal to eight • fragmentation of less than 20% • no multinucleated blastomeres. 106–108 h postinsemination/ICSI: (Figs 17.3, 17.4) • the blastocoelic cavity should be full • inner cell mass should be numerous and tightly packed • trophectodermal cells should be numerous and cohesive. Which of the above criteria would be the most important? To select the best embryos we could envisage a fluid selection process that would mark embryos as they develop. The above criteria would therefore be seen as ideal hurdles of development. At every step an embryo would be given a positive mark when it reached the ideal criteria of a certain stage. It would, however, be possible that an embryo may not pass one step, but would pass the hurdle at a following step. The embryo or embryos attaining the best criteria at each step would therefore be the ones that would be selected for transfer. For example, if we are attempting to transfer a single embryo to a patient, the following scenario could be envisaged. An embryo may not pass any of the earlier hurdles but still form a high-grade blastocyst on

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Early Blastocyst – blastocoel being less than half the volume of the embryo

2

Blastocyst – blastocoel being greater than half the volume of the embryo

Full Blastocyst – blastocoel completely fills embryo

3

4

Expanded Blastocyst – blastocoel volume is now larger than that of early embryo and zona is thinner

ICM grading

A Tightly packed and many cells

B Loosely grouped and several cells

Very few cells

Trophectoderm grading

A Many cells forming cohesive epithelium

B Few cells forming loose epithelium

C Very few large cells

Fig 17.3

The blastocyst grading system. ICM, inner cell mass. Modified from Gardner and Schoolcraft.32

C

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b

c

Fig 17.4 Day 5 human blastocysts. Using the grading system reported by Gardner and Schoolcraft,32 blastocysts in (a) and (b) would both score 4AA, whereas the embryo in (c) would only score 4CA owing to the apparent absence of an inner cell mass, in spite of the development of an excellent trophectoderm.

25–26 h postinsemination/injection – embryo should be at the 2-cell stage with equal blastomeres and no fragmentation

42–44 h postinsemination/injection – embryo should have 4 or more blastomeres and less than 20% fragmentation

66–68 h postinsemination/injection – embryo should have 8 or more blastomeres and less than 20% fragmentation

Fig 17.5 Ideal features of embryos scored at 25–26 h, 42–44 h, and 66–68 h postinsemination/ICSI. For greater details on the scoring criteria, see Sakkas et al,26 Shoukir et al,25 and Van Royen et al.21

day 5. If this were the most successful of the cohort of embryos, then this would be the one selected. If, however, six blastocysts were observed on day 5, all of equally high grade, then the blastocyst that had achieved the most positive scores at each of the previous hurdles could be transferred. Furthermore, patients who have low numbers of embryos, and will have transfer on day 2 or 3, could be assessed using the initial criteria and the embryo that passed the initial hurdles would be selected. A proposed schedule of embryo selection is given in Fig 17.6. It is important to

note that to date the strongest criteria of selection appear to be the selection of a high-quality blastocyst on day 5 of development.31,33 The practical issues for performing such a selection process would be that embryos would need to be cultured in individual drops. This may remove any necessary benefits of culturing embryos in groups.41–43 A further practical issue is that embryos will need to be observed more often; however, using drop culture systems under oil will reduce CO2 loss. The extra observations, if performed under a controlled heated and gassed climate, should not be detrimental to the embryo. Recently imaging incubators have become available to facilitate this (Sanyo). In prolonged culture, pronuclear assessment, change over into new media on day 3, and assessment of the blastocyst is already performed. The extra assessment periods would be the check of earlycleaving 2-cell embryos and assessment of embryos on day 2. Optional observations could include that of the polar body placement, as described by Garello et al.17 This assessment criteria involves photography, followed by calculation from the photograph, which would involve a further manipulation of the zygote once the polar body displacement has been calculated. A further observation would be to determine the degree of blastulation the afternoon of day 4, which reflects the speed at which a given embryo is developing. So it is evident that with improved culture conditions, together with suitable grading systems, it is possible to dramatically increase implantation rates and therefore decrease the number of embryos transferred. However, this approach raises two issues: first, if the laboratory in question is not performing blastocyst transfer, then it cannot rely on advanced grading systems; and secondly, morphology can only tell us so much about the physiological status of the embryo. The rest of this chapter is therefore devoted to the application of novel tests of embryonic function. It is assumed that such tests must be noninvasive for their adoption in clinical use. Therefore, methods that can be considered as semi-invasive, i.e. those that involve embryo biopsy prior to cell analysis, are not considered here.

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Amino acids

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Fig 17.7 Noninvasive analysis of human embryo nutrient consumption and metabolite/factor production. Individual embryos are incubated in 1–10 µl volumes of defined medium. Serial or endpoint samples of medium can then be removed for analysis. An indirect measurement of metabolic pathways, i.e. glycolysis and transamination, can be obtained by measuring specific nutrients in combination, such as glucose uptake and lactate production, or amino acid turnover with ammonium production. SHLA-G, soluble human leukocyte antigen G; LDH, lactate dehydrogenase; PAF, Platelet-activating factor; HOXA10, homeobox A10 gene.

Non-invasive assessment of embryo culture media The inherent ease for the laboratory to assess various morphological markers makes it the preferred assessment technique to transfer embryos. Even with the adoption of more complex forms of assessment it will still remain as one of the tools we have in our armory for assessment. However, a number of quantitative techniques are now being optimized that can monitor the uptake of specific nutrients by the embryo from the surrounding medium and detect the secretion of specific metabolites and factors into the medium (Fig 17.7). To measure such changes in culture media, the noninvasive assessment tools must fulfill a number of criteria so that they can be applicable in IVF clinics. The problems for clinical IVF have always been: 1. 2. 3.

The ability to measure the change without harming the embryo. The ability to measure the change quickly. The ability to measure the change consistently and accurately.

Historically, it is accepted that there is a relationship between metabolic parameters and embryo viability. In 1980, Renard et al44 observed that day 10 cattle blastocysts which had an elevated glucose uptake developed better, both in culture and in vivo after transfer, than those blastocysts with a lower glucose uptake. Subsequently, in 1987, using a relatively new technique of non-invasive microfluorescence, Gardner and Leese45 measured glucose uptake by individual day 4 mouse blastocysts prior to transfer to recipient females. Those embryos that went to term had a significantly higher glucose uptake in culture than those embryos that failed to develop after transfer.

This work was then built on by Lane and Gardner,46 who showed that glycolytic rate of mouse blastocysts could be used to select embryos for transfer prospectively. Morphologically identical mouse blastocysts with equivalent diameters were identified using metabolic criteria, as ‘viable’ prior to transfer and had a fetal development of 80%. In contrast, those embryos that exhibited an abnormal metabolic profile (compared to in vivo developed controls), developed at a rate of only 6%. Clearly, such data provide dramatic evidence that metabolic function is linked to embryo viability (Figs 17.8, 17.9). Only a few studies have been performed on nutrient uptake and the subsequent viability of the human embryo. In a retrospective analysis, Conaghan et al47 observed an inverse relationship between pyruvate uptake by 2- to 8-cell embryos and subsequent pregnancy. In a study by Jones et al48 on human morulae and blastocysts of different degrees of expansion, no conclusive data were generated on nutrient consumption or utilization to predict pregnancy outcome.48 Unfortunately, in both the above studies the medium used to assess embryo metabolism was a simple one, lacking lactate, amino acids, and vitamins. Under such culture conditions, the resultant stress on the embryos could have been detrimental, and therefore it is questionable whether any meaningful data could have been obtained. In contrast, Van den Bergh et al49 showed that in patients who conceived following blastocyst transfer, their embryos had an elevated glucose uptake and a higher oxidative rate than to those blastocysts which failed to establish a pregnancy (Fig 17.10). Significantly, in the work of Van den Bergh et al,49 a complete medium was used for the metabolic assessment, thereby alleviating the culture-induced metabolic stress. Furthermore, two studies have determined the relationship between embryo nutrition and subsequent

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development in vitro.50,51 Gardner et al50 determined that glucose consumption on day 4 by human embryos was twice as high in those embryos that went on to

0 Pregnant

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Fig 17.10 Glycolytic flux of human blastocysts. There is a significant difference in the flux between patients who conceived and those who did not; **, p <0.01. Data from Van den Berg et al.49

form blastocysts. Furthermore, it was determined that blastocyst quality affected glucose uptake. Poor-quality blastocysts consumed significantly less glucose than top-scoring embryos. In studies on amino acid turnover by human embryos, Houghton et al51 determined that alanine release into the surrounding

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medium on day 2 and day 3 was highest in those embryos that did not form blastocysts. Therefore, assessing metabolic activity and metabolic normality may prove to be a feasible way to determine human embryonic ‘health’. To this end, Brison et al,52 using high-performance liquid chromotography, reported changes in concentration of amino acids in the spent medium of human zygotes cultured for 24 h in an embryo culture medium containing a mixture of amino acids. They found that asparagine, glycine, and leucine were all significantly associated with clinical pregnancy and live birth. Other techniques have also been reported to measure metabolic parameters in culture media; however, they have yet to be tested in a clinical IVF setting. These techniques include the self-referencing electrophysiological technique, which is a noninvasive measurement of the physiology of individual cells and monitors the movement of ions and molecules between the cell and the surrounding media.53,54 One technique that is of the above mode is that which noninvasively measures oxygen consumption of developing embryos. Interestingly, although this technology has been shown to correlate with bovine blastocyst development, it was less successful in predicting mouse embryo development.55,56 The technology has yet to be assessed in a clinical IVF setting, however. A number of studies have also investigated the assessment of secreted factors in the embryo culture media (Fig 17.7) and correlated them with better embryo development and pregnancy rates. One such factor is soluble human leukocyte antigen G (sHLAG),57,58 which is believed to protect the developing embryo from destruction by the maternal immune response. Soluble HLA-G has been found in media surrounding the early embryo and a number of papers have also reported that its presence correlates with the improved pregnancy potential of an embryo.59–61 Recently, some studies have raised some serious concerns regarding the use of HLA-G production as a marker of further developmental potential,62–64 and

prospective clinical trials are needed to further evaluate this parameter. Included, in the studies examining secretion of factors in the media by embryos are numerous papers examining the secretion of platelet-activating factor (PAF). The clinical utility of PAF in an IVF setting has also yet to be stringently examined (see review by O’Neill65). Another indirect assay of soluble markers that may be present in embryo culture media was that described by Sakkas et al,66 where it was determined that cell-free media from human embryos cultured to the blastocyst stage contained a soluble molecule that induced HOXA10 (homeobox A10 gene) expression in an endometrial epithelial cell line (Ishikawa). Finally, a more direct analysis of protein markers in embryo culture media has been shown by Katz-Jaffe et al,67 using proteomic-based technology. They found differential protein expression profiles between early and

expanded blastocysts, as well as between developing blastocysts and degenerate embryos.

Metabolomics A new and emerging technology that may allow us in the future to measure factors in embryo culture media is metabolomics. The complete array of small-molecule metabolites that are found within a biological system constitutes the metabolome and reflects the functional phenotype.68 Metabolomics is the systematic study of this dynamic inventory of metabolites, as small molecular biomarkers representing the functional phenotype in a biological system. Using various forms of spectral and analytical approaches, metabolomics attempts to determine metabolites associated with physiological and pathological states.69 Metabolic studies of embryos are beginning to indicate that embryos that result in pregnancy are different in their metabolomic profile compared with embryos that do not lead to pregnancies.70 Investigation of the metabolome of embryos, as detected in the culture media they grow in, using targeted spectroscopic analysis and bioinformatics, may therefore divulge these differences. The Seli et al70 study established that these differences are detectable in the culture media using both Raman and nearinfrared (NIR) spectroscopy. Briefly, a total of 69 day 3 spent embryo culture media samples from 30 patients with known outcome (0 or 100% sustained implantation rates) were evaluated using Raman and/or NIR spectroscopy. A statistical formula was used to assign a relative ‘embryo viability score’ – equating to embryo reproductive potential – and it was found that this score correlated to positive or negative implantation outcomes. Both Raman and NIR spectroscopic analysis of the spent culture media of embryos with proven reproductive potential demonstrated significantly higher viability indices than those that failed to implant (Fig 17.11). Interestingly, when human embryos of similar morphology are examined using the same NIR spectral profile, their viability scores vary remarkably in relation to morphology, indicating that the metabolome of embryos that look similar differ significantly (Fig. 17.12). This observation is in agreement with the studies of Katz-Jaffe et al,67,71 who revealed that the proteome of individual human blastocysts of the same grade differed between embryos, again indicating that embryo morphology is not completely linked to its physiology. In a concurrent metabolomic study to that above, the individual idiosyncrasies of the spectral profiles of embryos that did and did not lead to pregnancy were used to create a statistical formula (genetic algorithm). The genetic algorithm established was subsequently used to predict the likelihood of pregnancy from blinded embryo culture media samples. When the model developed using NIR was used to test a subgroup of 16 day 3 embryo samples collected at a different center and cultured using a different type of

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Fig 17.11 Metabolomic analysis, resulting in viability indices calculated using Raman and near-infrared (NIR) spectroscopy, of culture media for embryos that implanted and led to delivery, and those that did not implant. For Raman, mean values were calculated for culture media of embryos that implanted and led to delivery (n = 15), and those that did not implant (n = 21). For NIR, mean values were calculated for culture media of embryos that implanted and led to delivery (n = 16), and those that did not implant (n = 17). Adapted from Seli et al.70

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Fig 17.12 Distribution of the viability score of individual day 3 embryos calculated using Raman spectroscopy within embryos sharing the same morphology; grade A embryos are excellent, grade B are good, grade C are poor, and grade D are very poor morphology. Data courtesy of Molecular Biometrics LLC.

commercial media, by an observer blinded to pregnancy outcome, viability indices of embryos with proven reproductive potential were significantly higher than embryos that failed to implant.72 A larger analysis of single embryo transfer cycles has also been undertaken whereby an NIR spectral analysis of frozen day 2 and day 3 embryo culture media samples was performed blinded to outcome. Individual metabolic profiles were established from 7 µl of the samples, with each measurement taking less than 1 minute. Statistical analysis performed on the metabolic profiles established a viability score (as generated above) that was significantly different (p <0.001) between the

0.00 (156) (108) A

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Fig 17.13 Single embryo transfer (SET) implantation rates of day 2 and 3 embryos, comparing embryos transferred with either a grade A or B morphology and a Raman spectroscopy viability score of greater (black column) or less than 0.3 (gray column). The number of SETs performed are in parentheses. Data courtesy of Molecular Biometrics LLC.

pregnant and nonpregnant patients. A cut-off value for predicting pregnancy was taken at >0.3. When this cut-off was used to examine embryos of excellent and good morphology that underwent single embryo transfer, a significant difference was found in the establishment of pregnancy (Fig 17.13). Although this technology awaits to be further proven, it does fit the three key criteria necessary to be used routinely in a clinical IVF setting.

Summary Analysis of embryo morphology and the development of suitable grading systems have assisted in the selection of human embryos for transfer. However, it is envisaged that in the near future selection will also be significantly aided by the noninvasive analysis of embryo physiology and function, using techniques such as metabolomics or proteomics. The addition of this technology will be of immense value in helping both clinicians and embryologists to more confidently select the most viable embryos within a cohort, helping the move to single embryo transfers.

References 1. Adashi EY, Barri PN, Berkowitz R, et al. Infertility therapy-associated multiple pregnancies (births): an ongoing epidemic. Reprod Biomed Online 2003; 7: 515–42.

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2. Ledger WL, Anumba D, Marlow N, Thomas CM, Wilson EC. The costs to the NHS of multiple births after IVF treatment in the UK. BJOG 2006; 113: 21–5. 3. Inge GB, Brinsden PR, Elder KT. Oocyte number per live birth in IVF: were Steptoe and Edwards less wasteful? Hum Reprod 2005; 20: 588–92. 4. Patrizio P, Sakkas D. From oocyte to baby: a clinical evaluation of the biological efficiency of in vitro fertilization. Fertil Steril 2008; [Epub ahead of print]. 5. Cummins J, Breen T, Harrison K, et al. A formula for scoring human embryo growth rates in in vitro fertilization: its value in predicting pregnancy and in comparison with visual estimates of embryo quality. J In Vitro Fert Embryo Transf 1986; 3: 284–95. 6. Edwards R, Fishel S, Cohen J. Factors influencing the success of in vitro fertilization for alleviating human infertility. J In Vitro Fert Embryo Transf 1984; 1: 3–23. 7. De Neubourg D, Gerris J. Single embryo transfer – state of the art. Reprod Biomed Online 2003; 7: 615–22. 8. Gardner DK, Sakkas D. Assessment of embryo viability: the ability to select a single embryo for transfer – a review. Placenta 2003; 24 (Suppl B): 55–12. 9. Sakkas D, Gardner DK. Noninvasive methods to assess embryo quality. Curr Opin Obstet Gynecol 2005; 17: 283–8. 10. Gardner DK, Surrey E, Minjarez D, et al. Single blastocyst transfer: a prospective randomized trial. Fertil Steril 2004; 81: 551–5. 11. Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 1988; 332: 459–61. 12. Tesarik J, Greco E. The probability of abnormal preimplantation development can be predicted by a single static observation on pronuclear stage morphology. Hum Reprod 1999; 14: 318–23. 13. Scott L, Alvero R, Leondires M, Miller B. The morphology of human pronuclear embryos is positively related to blastocyst development and implantation. Hum Reprod 2000; 15: 2394–403. 14. Gardner R. The early blastocyst is bilaterally symmetrical and its axis of symmetry is aligned with the animal–vegetal axis of the zygote in mouse. Development 1997; 124: 289–301. 15. Antczak M, Van Blerkom J. Oocyte influences on early development: the regulatory proteins leptin and STAT3 are polarized in mouse and human oocytes and differentially distributed within the cells of the preimplantation stage embryo. Mol Hum Reprod 1997; 3: 1067–86. 16. Antczak M, Van Blerkom J. Temporal and spatial aspects of fragmentation in early human embryos: possible effects on developmental competence and association with the differential elimination of regulatory proteins from polarized domains. Hum Reprod 1999; 14: 429–47. 17. Garello C, Baker H, Rai J, et al. Pronuclear orientation, polar body placement, and embryo quality after intracytoplasmic sperm injection and in-vitro fertilization: further evidence for polarity in human oocytes? Hum Reprod 1999; 14: 2588–95. 18. Scott LA, Smith S. The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum Reprod 1998; 13: 1003–13.

19. Scott L. Pronuclear scoring as a predictor of embryo development. Reprod Biomed Online 2003; 6: 201–14. 20. Gerris J, De Neubourg D, Mangelschots K, et al. Prevention of twin pregnancy after in-vitro fertilization or intracytoplasmic sperm injection based on strict embryo criteria: a prospective randomized clinical trial. Hum Reprod 1999; 14: 2581–7. 21. Van Royen E, Mangelschots K, De Neubourg D, et al. Characterization of a top quality embryo, a step towards single-embryo transfer. Hum Reprod 1999; 14: 2345–9. 22. De Neubourg D, Gerris J, Mangelschots K, et al. Single top quality embryo transfer as a model for prediction of early pregnancy outcome. Hum Reprod 2004; 19: 1476–9. 23. Van Royen E, Mangelschots K, Vercruyssen M, et al. Multinucleation in cleavage stage embryos. Hum Reprod 2003; 18: 1062–9. 24. Bavister B. Culture of preimplantation embryos: facts and artefacts. Hum Reprod Update 1995; 1: 91–148. 25. Shoukir Y, Campana A, Farley T, Sakkas D. Early cleavage of in-vitro fertilized human embryos to the 2-cell stage: a novel indicator of embryo quality and viability. Hum Reprod 1997; 12: 1531–6. 26. Sakkas D, Shoukir Y, Chardonnens D, Bianchi PG, Campana A. Early cleavage of human embryos to the two-cell stage after intracytoplasmic sperm injection as an indicator of embryo viability. Hum Reprod 1998; 13: 182–7. 27. Sakkas D, Percival G, D’Arcy Y, Sharif K, Afnan M. Assessment of early cleaving in vitro fertilized human embryos at the 2-cell stage before transfer improves embryo selection. Fertil Steril 2001; 76: 1150–6. 28. Salumets A, Hyden-Granskog C, Makinen S, et al. Early cleavage predicts the viability of human embryos in elective single embryo transfer procedures. Hum Reprod 2003; 18: 821–5. 29. Van Montfoort AP, Dumoulin JC, Kester AD, Evers JL. Early cleavage is a valuable addition to existing embryo selection parameters: a study using single embryo transfers. Hum Reprod 2004; 19: 2103–8. 30. Neuber E, Rinaudo P, Trimarchi JR, Sakkas D. Sequential assessment of individually cultured human embryos as an indicator of subsequent good quality blastocyst development. Hum Reprod 2003; 18: 1307–12. 31. Guerif F, Le Gouge A, Giraudeau B, et al. Limited value of morphological assessment at days 1 and 2 to predict blastocyst development potential: a prospective study based on 4042 embryos. Hum Reprod 2007; 22: 1973–81. 32. Gardner DK, Schoolcraft WB. In vitro culture of human blastocysts. In: Jansen R, Mortimer D, eds. Towards Reproductive Certainty: Infertility and Genetics Beyond. Carnforth: Parthenon Press, 1999: 378–88. 33. Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 2000; 73: 1155–8. 34. Gardner DK, Stevens J, Sheehan CB, Schoolcraft WB. Morphological assessment of the human blastocyst. In: Elder KT, Cohen J, eds. Analysis of the Human Embryo. London: Taylor & Francis, 2007: 79–87.

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52. Brison DR, Houghton FD, Falconer D, et al. Identification of viable embryos in IVF by non-invasive measurement of amino acid turnover. Hum Reprod 2004; 19: 2319–24. 53. Trimarchi JR, Liu L, Porterfield DM, Smith PJ, Keefe DL. A non-invasive method for measuring preimplantation embryo physiology. Zygote 2000; 8: 15–24. 54. Trimarchi JR, Liu L, Smith PJ, Keefe DL. Noninvasive measurement of potassium efflux as an early indicator of cell death in mouse embryos. Biol Reprod 2000; 63: 851–7. 55. Ottosen LD, Hindkjaer J, Lindenberg S, Ingerslev HJ. Murine pre-embryo oxygen consumption and developmental competence. J Assist Reprod Genet 2007; 24: 359–65. 56. Lopes AS, Larsen LH, Ramsing N, et al. Respiration rates of individual bovine in vitro-produced embryos measured with a novel, non-invasive and highly sensitive microsensor system. Reproduction 2005; 130: 669–79. 57. Kovats S, Main EK, Librach C, et al. A class I antigen, HLA-G, expressed in human trophoblasts. Science 1990; 248: 220–3. 58. Jurisicova A, Casper RF, MacLusky NJ, Mills GB, Librach CL. HLA-G expression during preimplantation human embryo development. Proc Natl Acad Sci U S A 1996; 93: 161–5. 59. Noci I, Fuzzi B, Rizzo R, et al. Embryonic soluble HLA-G as a marker of developmental potential in embryos. Hum Reprod 2005; 20: 138–46. 60. Sher G, Keskintepe L, Nouriani M, Roussev R, Batzofin J. Expression of sHLA-G in supernatants of individually cultured 46-h embryos: a potentially valuable indicator of ‘embryo competency’ and IVF outcome. Reprod Biomed Online 2004; 9: 74–8. 61. Yie SM, Balakier H, Motamedi G, Librach CL. Secretion of human leukocyte antigen-G by human embryos is associated with a higher in vitro fertilization pregnancy rate. Fertil Steril 2005; 83: 30–6. 62. Menezo Y, Elder K, Viville S. Soluble HLA-G release by the human embryo: an interesting artefact? Reprod Biomed Online. 2006; 13: 763–4. 63. Sageshima N, Shobu T, Awai K, et al. Soluble HLA-G is absent from human embryo cultures: a reassessment of sHLA-G detection methods. J Reprod Immunol 2007; 75: 11–22. 64. Sargent I, Swales A, Ledee N, et al. sHLA-G production by human IVF embryos: can it be measured reliably? J Reprod Immunol 2007; 75: 128–32. 65. O’Neill C. The role of PAF in embryo physiology. Hum Reprod Update 2005; 11: 215–28. 66. Sakkas D, Lu C, Zulfikaroglu E, Neuber E, Taylor HS. A soluble molecule secreted by human blastocysts modulates regulation of H0XA10 expression in an epithelial endometrial cell line. Fertil Steril 2003; 80: 1169–74. 67. Katz-Jaffe MG, Gardner DK, Schoolcraft WB. Proteomic analysis of individual human embryos to identify novel biomarkers of development and viability. Fertil Steril 2006; 85: 101–7. 68. Oliver SG, Winson MK, Kell DB, Baganz F. Systematic functional analysis of the yeast genome. Trends Biotechnol 1998; 16: 373–8. 69. Ellis DI, Goodacre R. Metabolic fingerprinting in disease diagnosis: biomedical applications of infrared and Raman spectroscopy. Analyst 2006; 131: 875–85.

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70. Seli E, Sakkas D, Scott R, et al. Noninvasive metabolomic profiling of embryo culture media using Raman and near-infrared spectroscopy correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril 2007; 88: 1350–7. 71. Katz-Jaffe MG, Schoolcraft WB, Gardner DK. Analysis of protein expression (secretome) by

human and mouse preimplantation embryos. Fertil Steril 2006; 86: 678–85. 72. Scott R, Seli E, Miller K, et al. Non-invasive metabolomic profiling of human embryo culture media using Raman spectroscopy predicts embryonic reproductive potential: a prospective blinded pilot study. Fertil Steril 2008; Epub ahead of print.

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18 The human oocyte: controlled rate cooling Andrea Borini, Giovanni Coticchio

Introduction As early as 1940, attempts were made to accomplish the cryogenic preservation of gametes and embryos, with the aim of expanding the potential of reproductive technologies in the animal and human fields. The initial experiences, initially limited to preservation of semen for practical reasons, were rapidly extended to embryos and oocytes, as soon as appropriate culture systems became available. From its very beginning, low-temperature storage has found its niche in the methodology of controlled rate cooling. Cells may be cooled from physiological to liquid nitrogen temperature, conditions under which preservation can be protracted safely and virtually indefinitely, controlling the formation of extracellular ice and ideally avoiding intracellular ice formation (IIF), which is the source of cell injury. In 1983 for the first time, live pregnancies were generated after transfer of cryopreserved human embryos.1 Since then, embryo cryopreservation has profoundly influenced the practice of clinical in vitro fertilization (IVF), contributing to a reduction in the incidence of multiple pregnancies, an increase in the overall efficiency of IVF treatment, and a decrease in the need for repeated cycles of ovarian stimulation. In principle, the same benefits could be gained from the cryopreservation of unfertilized oocytes, circumventing the well-known ethical and legal constraints of embryo storage. Not surprisingly, the possibility to store oocytes has always attracted the interest of IVF specialists. However, the pace of progress in oocyte cryopreservation has been slow. Compared with preimplantation embryos, the mature oocyte is less amenable to freezing as a consequence of its unicellular nature, unique membrane permeability, and subcellular attributes. For many years, poor post–thaw survival rates have represented an endemic problem that has prevented oocyte freezing from being adopted as an established form of treatment. This has meant embryo freezing has been perceived as the virtually exclusive option for the preservation of the reproductive potential generated at each cycle of ovarian stimulation. Low survival rates have also nurtured the widespread preconception that oocytes cannot be stored safely and that

oocyte cryopreservation as a treatment strategy is inapplicable. In reality, newly developed cryopreservation protocols can now produce survival rates comparable to the ones normally obtained with embryo freezing. In the last several years, these advances have stimulated a renewed interest that is conveying oocyte cryopreservation from the realm of academic interest to that of a routine clinical practice. Meanwhile, vitrification techniques have been developed, and are promising to bring a revolution in human oocyte storage. Unfortunately, many of the studies published on this matter have been episodic, not properly designed, and insufficient in terms of sample size. Assessment of the safety and clinical efficiency of oocyte cryopreservation is intrinsically difficult. The overall clinical outcome is largely determined by well-recognized factors, such as oocyte quality, post–thaw survival, and subcellular effects of cryopreservation conditions. However, other elements, e.g. oocyte and embryo selection criteria, reproducibility, adherence to cryopreservation protocols, and strategy of use of the stored material, can make particularly arduous the interpretation of the clinical significance of oocyte cryopreservation.

Essential principles of controlled rate cooling During their return journey to and from physiological temperatures to cryogenic storage in liquid nitrogen (at −196°C), cells are exposed to a number of conditions that often cause damage at the subcellular level or, in extreme cases, overt cell death. In fact, if carried out in the absence of appropriate measures, cooling to storage temperature is accompanied by formation of ice in the intracellular as well as the extracellular compartments. Extensive IIF is incompatible with the preservation of cell viability, causing widespread physical/structural damage. In order to succeed in cryostorage, it is therefore crucial to implement some form of cryoprotection to prevent IIF and other undesired effects that derive directly or indirectly from ice formation. Cryoprotection may be achieved through the use of cryoprotective agents (CPAs), chemicals that interfere

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Figure 18.1 Mean ± SD normalized volume of human oocytes during exposure to 1.5 mol/l propanediol (PrOH)22 (left) or ethylene glycol (EG)72 (right) at 25°C. The magnitude and kinetics of volume changes are dictated by the relative oolemma permeability to water and cryoprotective agent (CPA). Low permeability to CPA, and, consequently, more pronounced volume reduction, may cause major transient perturbations of the original spherical shape (right).

with the water–ice transition and interact with biomolecules acting as ‘water replacement.’ Some CPAs, classified as permeating or intracellular, are oligohydroxy compounds of relative low toxicity which can enter the cell across the plasmalemma. Other CPAs are polyhydroxylic and are unable to diffuse into the intracellular compartment, being unable to cross the plasmalemma. The action of the diverse CPAs is rather multifactorial and only partly understood at present. Before the cell is cooled to subzero temperatures, relatively low but significant concentrations of CPAs (1.0–1.5 mol/l) are utilized to dehydrate the intracellular environment and prevent IIF. Generally, this initial phase of dehydration involves an exposure to a single permeating CPA – e.g. propanediol (PrOH), dimethylsulfoxide (Me2SO), or ethylene glycol (EG). The presence of CPA in the extracellular compartment creates an osmotic gradient which draws water out of the cell. Because the permeability to water of the plasmalemma is higher than its permeability to intracellular CPAs, the net efflux of water is not initially counterbalanced by an equivalent influx of CPA. This generates a rapid reduction in the cell volume, followed by a slower recovery (Fig 18.1). A second phase of dehydration occurs when the cell is subsequently exposed to a mixture of the intracellular

CPA used in the initial step and an extracellular CPA, the latter being generally sucrose or another oligosaccharide. These conditions re-establish an osmotic disequilibrium and drive a further phase of dehydration on a kinetic basis. Afterwards, the cell is cooled down to subzero temperatures slowly (normally to a rate of −2°C/min) to avoid thermal shock. Once the sample temperature has been lowered to −6 to −8°C (just below the equilibrium freezing point of the mixture), ice nucleation is deliberately induced by touching the storage device (straws or vials) with precooled forceps, and a period of time (minutes) permitted to allow dissipation of latent heat of ice nucleation. Thereby, extracellular water is slowly converted into ice and, subsequently, the sample is subjected to a low cooling rate (−0.3°C/min). During the transition of water into ice, the solutes present in the freezing mixture selectively segregate into the nonfrozen fraction. If not controlled properly, the increase in solute concentration (dictated kinetically by cooling rate) may affect cell viability; having a high salt content a destabilizing effect on membranes and biomolecules. However, in practice the novel osmotic gradient caused by such an increase in solute concentration also draws further water out of the cell, reducing the risk of IIF. Therefore, the rate of

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cooling during the extracellular water–ice transition responds to a fine compromise between the need for sufficient dehydration on the one hand, and the limitation of the detrimental effects of increase in extracellular solute concentration on the other. At temperatures of between −30 and −40°C, almost all extracellular water has been converted into ice, whereas almost all freezable water has been extracted from the cell, and the mixture achieves a ‘glassy matrix’ of ice and highly viscous components. Under these conditions, the sample temperature can be quickly lowered to the liquid nitrogen temperature, essentially avoiding significant IIF. After storage, rewarming is performed in a fashion which depends on cooling conditions. If a sample has been plunged in liquid nitrogen from a relatively high temperature (−20°C to −30°C), it is possible that minute intracellular ice crystal nuclei have formed as a consequence of incomplete dehydration during conversion of extracellular water into ice. Under such conditions, rewarming should be fast to prevent growth of these intracellular ice crystals to a size that may cause harm to the cell organelles and organization. On the contrary, if samples are moved to liquid nitrogen from a lower plunge temperature, rewarming is compatible with slow warming rates, because it is likely that the more extended phase of dehydration during freezing has coincided with a complete extraction of freezable water from the intracellular environment. Once samples are thawed, cell rehydration is usually obtained by exposure to decreasing concentrations of the intracellular CPA present in the freezing mixture. Rehydration mixtures may also contain fixed or decreasing amounts of the extracellular CPA, such as sugars which act as osmotic buffers, in order to limit the net flux of water towards the intracellular compartment during the dilution of the intracellular CPA. Once rehydration and CPA dilution is completed, the cell is placed in standard culture conditions.

Oocyte selection Embryo cryopreservation benefits from selection criteria applicable to the fresh material, which can increase the perceived performance of the procedure. In fact, embryos displaying cleavage delay, pronounced fragmentation (>30% of the total embryo mass), or other anomalies (e.g. multinucleation) are not cryopreserved because their post–thaw survival or implantation ability is known to be compromised.2 In the case of oocyte cryopreservation, the identification of criteria for the selection of material more suitable for freezing appears rather more problematic, leaving the definition of oocyte quality an unresolved matter per se.3 During the few hours following retrieval, mature oocytes are in a static phase. Assessment criteria based on the normalcy and pace of cleavage, which are critical for embryo selection, are not relevant. On the other hand, the hypothesis that morphological characteristics, apart from gross dismorphisms

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and certain peculiarities such as a large first polar body,4 can predict the oocyte developmental potential remains controversial. After removal of cumulus cells, oocytes should be assessed for their meiotic status. Despite the fact that germinal vesicle (GV)-stage oocytes survive with high rates after thawing,5 their storage appears of little benefit. Rather paradoxically, cumulus cells are in fact especially vulnerable to freezing conditions and their loss would make the subsequent process of acquisition of developmental competence in vitro unattainable.6 It is also disputable that GV-stage oocytes may be cryopreserved after maturation in vitro, for the reason that a variety of culture conditions can easily compromise oocyte quality, irrespective of the ability to resume meiosis and extrude the first polar body. For example, inadequacies in the maturation medium may cause an increase in the incidence of metaphase II (MII) spindle abnormalities.7 Oocytes which do not display the first polar body and are usually referred to as ‘metaphase I’ also represent an improbable source of freezable material. In fact, their meiotic status is not readily assessable, as it is impossible using conventional light microscopy to discriminate among the diverse phases between germinal vesicle breakdown (GVBD) and first polar body extrusion. The inability to determine the precise stage of ‘metaphase I’ oocytes would presumably have notable implications on the freezing outcome. This is suggested by studies in the rat which have shown that during oocyte maturation the oolemma water permeability decreases, an event that may influence the oocyte osmotic response to CPA addition and dilution and ultimately affect post– thaw viability.8 In the last several years, polarized light microscopy (Polscope) has been developed to a stage that is compatible with the noninvasive visualization of the two birefringent structures of the mammalian oocyte, the meiotic spindle and the zona pellucida. Spindle presence is obviously essential for chromatid segregation during the second meiotic division, but it is also suspected to have an influence on fertilization and early cleavage. This makes the Polscope a potential tool for oocyte selection, considering that not all oocytes showing an extruded polar body and classified as mature possess a proper spindle. Nevertheless, several studies conducted using the Polscope have not demonstrated conclusively that spindle presence and location is an absolute requirement for fertilization and the generation of high-quality embryos.3,4 Polarized light analysis is therefore of limited significance for oocyte selection. Nevertheless, spindle visualization may have some relevance to oocyte assessment before freezing. In sporadic cases in which oocytes appear meiotically mature, fibers of microtubules extend from the oocyte cortex to the adjacent polar body, the first meiotic division having not been completed.4 These oocytes should be left in culture for a few hours before proceeding with their storage, to prevent

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Table 18.1

Oocyte cryopreservation outcome generated in three different studies adopting the same freezing protocol14

Patients Thaws Oocytes: Thawed Survived (%) Microinjected 2PN Embryos: Cleaved Mean transferred Pregnancies (% per ET) Implantations (%) Implantation efficiency per thawed oocyte

Borini et al, 200611

Levi Setti et al, 200612

De Santis et al, 200713

146 201

120 159

66 68

927 687 (74) 589 448 (76)

1087 760 (69) 687 464 (67)

396 282 (71.2) 194 156 (80.4)

404 (90) 2.1 ± 0.8 18 (9.7) 21 (5.2) 2.6%

413 (89)

142 (91)

18 (12.4) 19 (5.7) 1.9%

6 (9.5) 7 (5.7) 2.6%

2PN, two pronuclei; ET, embryo transfer.

possible loss of chromosome or elements of the segregation apparatus as an effect of polar body post–thaw degeneration.

Reproducibility One of the theoretical advantages of controlled rate freezing is represented by a high degree of reproducibility. Irrespective of the absolute efficiency of a given method, it should be possible to reproduce comparable inter-operator and inter-laboratory rates of success, provided strict adherence to protocols is assured. A possible source of variability may reside in the fact that, until recently, freezing–thawing solutions specifically designed for cryopreservation were not commercially available. Home-made solution preparation requires careful preliminary assessment of basic constituents. Chemicals may contain traces of toxic compounds; such is the case with certain batches of PrOH that have been found to be contaminated by formaldehyde.9 Buffer solutions may include diverse pH buffers (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid [HEPES] and phosphate-buffered saline [PBS], whose performance and relative toxicity may differ depending on certain temperature ranges. Pyruvate, essential to oocyte metabolism, may not be included in a basic buffer formulation and its use as a separate supplement may cause problems of chemical stability. Quality and quantity of macromolecular supplement may impact significantly on the oocyte’s survival and function.10 Exchanges of water and CPAs between the extra- and intracellular environments, which account for important effects such as dehydration/rehydration, osmotic stress, and chemical toxicity, require several minutes for their completion. Therefore, diversions of a few seconds from the prescribed time of exposure to a given dehydration/rehydration mixture, which are always possible for a number of practical reasons, should not

have measurable effects on the survival and quality of the frozen–thawed material. Nonetheless, oolemma permeability to water and CPA is temperature-dependent. Higher temperatures facilitate the exchange of water and CPA across the plasmalemma, limiting the possible impact of osmotic stress, but at the same time may increase the always present risk of chemical toxicity derived from CPA exposure. Lower suprazero temperatures reduce CPA toxicity, but may generate damage to the cell membrane and cytoskeleton. Therefore, the temperature at which dehydration/rehydration solutions are utilized should be precisely regulated. Evaporation of these solutions should be controlled to avoid changes in concentration, but the use of a covering layer of mineral oil is not recommended, because traces of oil may interfere with loading and freezing in the storage vessel. Care is advised in the choice and use of storage vessels. Plastic straws are almost universally employed for the cryopreservation of oocytes and embryos. It should be noted, though, that differences in size, volume, and wall thickness between various straw types may influence the transmission of heat and potentially affect the freezing–thawing process. For the same reason, the volume of the freezing mixture loaded into straws should be standardized. By definition, in controlled rate cooling methods, temperature changes are finely controlled. This is achieved through automated devices (cryofreezers) which provide printable records of temperature transitions. Cooling rates governing the water–ice transition are slow (−0.3°C/min) and protracted for over 70 minutes, conditions which are believed to ensure good consistency. A valuable example of protocol reproducibility is provided by the comparison of three studies11–13 in which the same method,14 involving freezing with an increased sucrose concentration (0.3 mol/l), was adopted (Table 18.1). Post-thaw survival, fertilization,

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cleavage, and implantation rates were rather similar, despite the fact that the three studies, performed with relatively large number of patients, were conducted independently. In contrast with such an evidence, a different set of data15 suggests that the 0.3 mol/l sucrose protocol can generate a considerably higher implantation rate (11%). The source of this inconsistency is not known. It should be considered that the study of Chen et al15 included a rather smaller number of patients, and the supplementation of dehydration/ rehydration solutions with maternal serum in place of serum albumin. It is difficult to conclude whether such a protocol modification may have influenced the viability of survived oocytes, but it is a fact that experiments carried out with mouse oocytes showed that the addition of serum in the dehydration/rehydration solutions as an alternative to albumin,16 or to an inert macromolecule such as polyvinyl alcohol,10 dramatically improves post-thaw survival. Whether serum is also beneficial to the overall oocyte viability remains to be established.

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Table 18.2 Schematic description of a slow cooling rate protocol involving the use of 0.2 and 0.3 mol/l sucrose for (A) dehydration and (D) rehydration, respectively. Solutions are prepared in PBS- or HEPES-buffered media and supplemented with 10 mg/ml of human serum albumin A

Dehydration

Solution

Time

Temperature

1.5 mol/l PrOH 1.5 mol/l PrOH, 0.2 mol/l sucrose

10 min 5 min

24°C 24°C

B

Controlled rate cooling

Ramp

Thermal interval

1 2 3 4 5

+20 to −8°C, −2.0°C/min −8°C, hold for 10 min. Seed at about 30% of ramp −8°C to −30°C, −0.3°C/min −30°C to −150°C, −50.0°C/min −150°C, hold for 10 min, then plunge into LN2

C

Thawing

Time

Survival and insemination of frozen–thawed oocytes

30 sec 40 sec

Poor survival rates17–20 have long been the limiting factor that has prevented widespread experimentation and adoption of oocyte storage. This constraint is primarily ascribable to inadequate pre-freeze exposure to the extracellular CPA. In fact, the method initially adopted for freezing oocytes21 involved a concentration of sucrose (0.1 mol/l), which a posteriori was recognized to cause only a marginal dehydration effect,22 presumably insufficient to prevent IIF. A few years ago, the demonstration that by increasing to 0.3 mol/l the sucrose concentration in the freezing solution it was possible to improve dramatically the survival rate up to 80%14 marked a breakthrough. Survival rates in excess of 70% are currently the norm.11–13 More recently, more light has been shed on the possibility to modulate sucrose concentration in freezing–thawing solutions as a means of optimizing dehydration/ rehydration conditions. In osmotic response experiments,23 it has been observed that after around 3 minutes of exposure to 1.5 mol/l PrOH in the presence of 0.3 mol/l sucrose, oocyte volume decreases rapidly, reaching values below the 30% threshold excursion, which may be detrimental to cell viability. In the presence of a sucrose concentration of 0.2 mol/l, the 30% volume change is not reached until after 10 minutes of exposure. Therefore, by finely tuning concentration and time of exposure to 0.2 mol/l sucrose, sustained dehydration may be achieved more moderately and presumably less traumatically. These concepts have been applied to test an alternative protocol,24 based on a freezing solution containing 0.2 mol/l sucrose and rehydration solutions including 0.3 mol/l sucrose (Table 18.2). The principle of using a medium-strength

D

Temperature 24°C 30°C, water bath

Rehydration

Solution

Time

Temperature

1.0 mol/l PrOH, 0.3 mol/l sucrose 0.5 mol/l PrOH, 0.3 mol/l sucrose 0.3 mol/l sucrose Buffer Buffer

5 min 5 min 10 min 10 min 10 min

24°C 24°C 24°C 24°C 37°C

ProH, propanediol; LN2, liquid nitrogen.

sucrose concentration at freezing and a higher sucrose concentration at thawing is not novel, having been implemented successfully for improving the survival rate and implantation ability of biopsied embryos, which are not amenable to freezing.25 This protocol has shown the ability to produce survival rates (75.9%) equivalent to those derived from the 0.3 mol/l method, but with the potential advantage of a better control of osmotic stress. An alternative route has also been attempted with the aim of improving post-thaw survival rates. Considering the possibility that the increase in solute concentration that occurs during ice formation may be source of biochemical toxicity, it has been proposed that replacement of sodium, the most abundant solute in freezing solutions, with the less toxic cation choline may reduce the detrimental effects of high solute concentration on oocyte viability. Experiments in the mouse have fully confirmed the hypothesis,16 but tests conducted in the human appear less conclusive. In one case choline-based freezing media have generated a survival rate of 90%,26 while in others the

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proportion of survived oocytes ranged between 59% and 63%.27,28 In addition, it should be noted that these attempts involved other protocol modifications, such as increased sucrose concentrations in the freezing solutions, or alternative modes to perform the dehydration/rehydration steps. Therefore, it appears difficult to conclude that sodium-depleted freezing solutions can improve the survival of human oocytes. Cryopreservation bears potentially important implications for the way in which fertilization may be achieved, because cryptic damage at the subcellular level may be not easily ascertained. In particular, it is commonly believed that, irrespective of the method adopted, cryopreservation inevitably causes hardening of the zona pellucida and consequent inhibition of sperm penetration, leading to decreased rates of fertilization via standard IVF. This notion probably has its roots in early studies carried out in mouse oocytes, where it was shown that the rate of fertilization was dramatically reduced in frozen oocytes (48.8%) when compared with unfrozen controls (87.8%) unless the zona was subjected to drilling with an acidified solution (88.0%).29 The hypothesis of a modification in the zona pellucida is consistent with the recent finding that freezing and thawing with PrOH induces extensive loss of specific cortical granules (CG) staining from the cortex of human oocytes.30 Electron microscopy analysis has confirmed that the population of subcortical CG undergoes a decrease in human oocytes frozen with PrOH.31 The magnitude of this phenomenon appears to be influenced by the type of protocol. In fact, a low concentration (0.1 mol/l) of the extracellular CPA sucrose in the freezing solution is associated with a more conspicuous loss of CG in comparison to a higher concentration (0.3 mol/l) of the same CPA. As early as 1997,32 intracytoplasmic sperm injection (ICSI) was adopted as the method for achieving fertilization in frozen–thawed oocytes. In effect, in general, ICSI has been able to guarantee rates of fertilization in the range of 70–75%,11–13,24 but in other cases it has not proven itself to be decisive. For example, the fertilization rate of oocytes stored via the conventional Lassalle protocol21 appears compromised even after microinjection.19 This is also the case of oocytes cryopreserved with a method based on the replacement of sodium with choline, in which the rate of fertilization may remain below 56%,28 showing that cryopreservation can cause sublethal damage associated with limited fertilization ability, irrespective of possible alterations to the zona. A recent study has challenged the credence that cryopreserved oocytes should be necessarily microinjected, describing fertilization rates of about 80% after standard IVF insemination.33 The very small numbers of oocytes tested in this study (16–43) should suggest caution. Nevertheless, the possibility of achieving high fertilization rates (75–80%) in frozen oocytes without resorting to microinjection emerges also from the experiences of Chen34 and AlHasani et al,35 who pioneered oocyte storage well

before the introduction of ICSI. The hypothesis that, under appropriate conditions, in frozen oocytes fertilization may be obtained with standard IVF is supported also by studies in the mouse, in which after oocyte cryopreservation ICSI is not applied and high fertilization rates are routinely attained with standard IVF.10,36 Therefore, the current practice of using ICSI for the insemination of frozen oocytes is likely to reflect inadequacies of current protocols or an insufficiency of the data available rather than an objective and well-documented necessity.

Timing of freezing and insemination During the routine IVF procedure, following retrieval, oocytes are cultured for a few hours and inseminated at about 40 h from human chorionic gonadotropin (hCG) administration. This is believed to respond to the necessity of attaining maturation of the nuclear and cytoplasmic compartments of the oocyte, a process which is largely accomplished in vivo in the follicular environment but that may require to be completed in vitro. Oocyte freezing creates a discontinuity in the oocyte life during the critical period encompassed between retrieval and insemination. In different laboratories, oocytes are frozen at different times (1 to 6–7 h) following recovery, depending on arbitrary guidelines, workload on the day, or other reasons. Polarized light analysis may offer a guidance concerning the choice of an appropriate time of freezing. When spindle detection is conducted at least 38 h following hCG administration, spindle visualization occurs with higher frequency compared to oocytes, in which the assessment is conducted at an earlier time (81.5% vs 61.6%). 37 This may be explained with the fact that oocytes with an extruded polar body may be at a meiotic stage that precedes MII, typically telophase I or prometaphase II. Therefore, Polscope evaluation may be included in the cryopreservation procedure to ensure that oocytes of the appropriate meiotic phase are frozen, setting for each IVF program a suitable time interval between hCG induction and/or egg retrieval and freezing. This should increase the consistency of freezing conditions and thereby improve the standardization of the methodology. Likewise the freezing time: there is no clear consensus of the time during which oocytes should be left in culture undisturbed to allow recovery after thawing. In fact, the concept of post-thaw recovery itself is rather vague, although it generally refers to the meiotic spindle. Based on the notion that the spindle undergoes depolymerization during exposure to low temperatures, thawed oocytes are cultured for a few hours, assuming that this is required to allow proper spindle reassembly. In many studies, the duration of this period of recovery is simply not reported, while in others it amounts to 3 h.19 Reports on the condition

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of the meiotic spindle after cryopreservation have long been controversial.38 Recently, confocal microscopy has confirmed that the overall structure of the meiotic spindle may be found unaltered after cryostorage. Stachecki et al,39 using a protocol involving choline as a substitute for sodium in association with a 0.2 mol/l concentration of sucrose, observed that frequency of oocytes with a normal barrel-shaped spindle and a regular chromosome alignment was not statistically different between fresh and frozen– thawed groups (76.7% vs 69.7% and 76.7% vs 71.2%, respectively). In a study involving a slow-cooling method, including 1.5 mol/l PrOH and different sucrose concentrations (either 0.1 or 0.3 mol/l) in the freezing solution, it was found that in a control unfrozen group, 73.1% of oocytes displayed normal bipolar spindles with equatorially aligned chromosomes. Spindle and chromatin organizations were significantly affected (50.8%) after cryopreservation involving lower sucrose concentration, whereas these parameters were unchanged (69.7%) using the 0.3 mol/l sucrose protocol.40 Little is known, though, of the actual kinetics of spindle depolymerization during freezing and thawing. In polarized light studies, it has been described that the spindle of human oocytes disappears either during the freezing–thawing process or the subsequent rehydration phase, reappearing within 3 h of culture at 37°C after completion of rehydration.41 However, this does not rule out that the spindle, assuming that it undergoes systematic depolymerization during freezing–thawing, can recover within a period shorter than 3 h. Suggestions in this direction derive from experiments conducted with mouse and human oocytes, which have shown that, following transient cooling at temperatures which causes microtubule depolymerization, significant, although not complete, spindle reorganization can occur after culture at 37°C for 1 h.42,43 Polarized light analysis also suggests that in human oocytes repolymerization can occur within 20 minutes after cooling at room temperature.44 This leaves open the question of the appropriate time of culture after thawing. In any case, the time of postthaw recovery should reflect also the necessity of inseminating at an appropriate time. This is required to avoid the oocyte undergoing aging in vitro, a phenomenon consisting of alterations in key oocyte factors (cell cycle kinases, intracellular calcium stores, cytoskeleton) that are still compatible with an apparently normal fertilization but that at the same time may have profound detrimental consequences on postimplantation development.45 The determination of an appropriate time of recovery should also take into account other structures and functional attributes of the oocyte (cortical actin, mechanism of calcium release, mitochondria) that may undergo transient disruption during freezing–thawing and be regained during a successive period of culture, but unfortunately nothing is known in this respect.

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Oocyte preparation for freezing At oocyte retrieval, the cumulus mass is formed by loosely connected somatic cells and a highly hydrated extracellular matrix. If left intact or only partially removed from around the oocyte, it cannot be ruled out a priori that its presence may influence the exchange of water and CPAs during oocyte dehydration and rehydration and thereby ultimately have an effect on the whole cryopreservation procedure. Gook et al.46 cryopreserved human oocytes after complete denudation or alternatively by leaving intact the entire cumulus mass found at retrieval. After freezing– thawing, using the Lassalle method,21 a higher survival rate was observed in the cumulus-free group (69% vs 48%). This has not been confirmed by Fabbri et al,14 who compared the survival rates of groups of oocytes frozen after complete denudation or mechanical removal of part of the cumulus mass. Irrespective of the concentration of sucrose contained in the freezing mixture (0.1, 0.2, or 0.3 mol/l), in all cases survival rates were comparable between the two categories of oocytes (39% vs 31%, 58% vs 60%, and 83% vs 81%, respectively), suggesting that the removal or maintenance of part of the cumulus mass is irrelevant to the oocyte’s ability to survive after thawing. More recently, in vitrification experiments involving 6.8 mol/l ethylene glycol as a penetrating CPA, Kuwayama et al47 observed that oocytes preserved with an intact cumulus mass gave rise to a higher survival rate (75.8% vs 30%). However, apart from the fact that controlled rate freezing and vitrification methods are not directly comparable, the very low numbers of oocytes in the cumulus-intact and cumulus-free groups (33 vs 10) attribute little significance to the test. Therefore, the evidence regarding a possible effect of the presence of the cumulus mass on postthaw viability remains controversial. Nonetheless, in the absence of a clear beneficial effect, the complete removal of cumulus and corona cells before freezing appears advisable. In fact, differences in the layers of cells surrounding the oocyte would introduce a source of variability that could affect the reproducibility of the method employed. In addition, the presence of cumulus cells makes very arduous the assessment of the oocyte meiotic status, leading to storage of material of varied nature.

Developmental performance of frozen–thawed oocytes Although not exhaustive, embryo morphology and pace of cell cleavage represent important clues for predicting the embryo developmental ability.48–51 Such embryonic parameters depend not only on the intrinsic gamete quality but also on extrinsic influences that may be inadvertedly inflicted by ex vivo manipulation. Embryo developmental ability may be

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affected by freezing conditions; also, the ability to resume the mitotic cycle has been recognized as a factor predicting the implantation potential of frozen– thawed embryos.52,53 Surprisingly, rather little information is available on the early development of embryos derived from cryopreserved oocytes. In general, studies on cryopreserved oocytes report the cleavage rate, intended as the proportion of fertilized oocytes which undergo at least one cell division by day 2. Comparisons between sibling fresh and frozen– thawed oocytes are rare. Embryos derived from oocytes frozen with the 0.3 mol/l sucrose protocol14 have been found to cleave with a significantly lower rate in comparison to sibling fresh controls (77.3% vs 91.1%).54 Such a relatively low cleavage rate in the frozen group is difficult to interpret. In fact, in various other studies conducted with the same freezing protocol, the proportion of cleaved embryos was 89– 91%,11–13,15 although fresh sibling control groups were not included. Recently, high cleavage (93.8%) has been reported also for oocytes stored with a protocol adopting differential sucrose concentrations at freezing (0.2 mol/l) and thawing (0.3 mol/l).24 Protocols based on sodium-depleted media have not been proven conclusively to support high cleavage rates. In one case, no information was given,28 while in another only 78% of fertilized oocytes cleaved,26 irrespective of high survival (90%) and fertilization (74%) rates. However, regardless of the absolute rate, it is important to note that the generic ability to undergo cell cleavage is not always fully informative of the developmental potential of cryopreserved human oocytes, as suggested by several experiences11–13 in which high rates of cleavage (90–93%) resulted in scarce implantation rates (5–6%). It is possible that, likewise the case of fresh embryos,51 the timing of the very first cleavage cycles, rather than the simple ability to undergo cell division after fertilization, may be revealing of the implantation potential of embryos developed from frozen–thawed oocytes. Oocytes frozen with the 0.3 mol/l sucrose protocol14 undergo early cleavage, which is a sign of developmental competence,55 with a very low rate in comparison to sibling fresh controls (7.1% vs 59.5%).56 The proportion of 4-cell embryos at 44–46 h postinsemination also appears insufficient (13.6%), although comparative data with fresh oocytes are not available. Such a delay in the initiation of the mitotic cycle may be indicative of freezing-induced damage, because oocytes stored with this method give rise to a poor implantation rate (5.2%).11 Conversely, a different protocol involving the use of 0.2 mol/l and 0.3 mol/l sucrose at freezing and thawing, respectively, has been found to produce a high frequency of 4-cell embryos at 44–46 h postinsemination (42.2%), as well as a higher implantation rate (13.5%).24 Concerning the morphological quality of embryos generated from frozen–thawed embryos, data are even scarcer. A high frequency (78–82%) of embryos with

good morphology (anucleated cytoplasmic fragments not exceeding 25% of the embryo mass) is associated with,24 but not necessarily sufficient11 for, the achievement of a high implantation rate. Data on embryo development beyond the day 3 stage are virtually lacking. The development of the blastocyst stage of embryos from frozen oocytes has been reported,57 but those results are of little current applicative value, having been produced with a traditional protocol whose use has been discontinued and with a culture system inadequate for blastocyst culture.

Clinical efficiency of oocyte cryopreservation As an IVF form of intervention, oocyte cryopreservation is influenced by a variety of well-known factors, such as type of infertility, female age, ovarian response to gonadotropins, ovarian stimulation, and laboratory strategies, which in the final analysis affect its efficiency. In addition, other more specific elements, pertaining especially to the methodology and procedure of cryopreservation, play a role in determining the perceived efficiency of oocyte cryostorage. Oocyte cryopreservation has been, and still is, seen with skepticism, as a result of the initial inability to recover the stored material with high rates of survival, as well as the persistence of preconceived opinions on the oocyte sensitivity to cryopreservation.58 This has restrained the efforts aimed at establishing oocyte cryostorage as a standard treatment option. Many studies are founded on very small number of patients15,33,59,60 and can merely provide a proof of principle rather than a realistic indication of efficiency. Clearly, although the impact of oocyte quality on oocyte storage has not been systematically investigated, it appears plausible that the use of oocytes of young donor patients and the transfer of a very high number of resulting embryos (in some cases more than five per recipient patient) may generate a high pregnancy rate.60 Generally, IVF treatment is offered in a rather different context, in which patients are old, oocyte quality may be affected by a number of factors, and the number of transferred embryos is appropriately much lower. Other more subtle aspects may influence the success rate of oocyte cryopreservation. The quality of embryos transferred, and as a consequence the rate of implantation, may change depending on whether the thawing strategy involves recovery of a relatively large number of oocytes and embryo selection before transfer61 or the insemination of a minimal number (three or less) of survived oocytes at each thawing and the transfer of all the resulting embryos, in compliance with existing laws.11,13,62 With reference to the use of a largely adopted protocol,14 the more numerically representative studies reported so far11–13,62 (Table 18.3) indicate that the rates of survival, fertilization, and cleavage are sufficiently high to ensure a suitable number of

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Table 18.3

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Recently reported rates of survival, fertilization, cleavage, and implantation following oocyte cryopreservation No. of thawed oocytes

Survival (%)

2PN (%)

Cleavage (%)

Implantation (%)

PrOH/0.3–0.3 mmol/l sucrose PrOH/Na-depleted PrOH/0.3–0.3 mmol/l sucrose

927 190 337

74.1 59.5 78.0

76.1 67.9 67.9

90.2 n.r. 77.3

5.2 15.9 5.6

PrOH/0.3–0.3 mmol/l sucrose PrOH/0.2–0.3 mmol/l sucrose PrOH/0.3–0.3 mmol/l sucrose

1087 325 396

69 75.1 71.2

67.5 77.3 80.4

89.1 93.0 91.0

5.7 16.7 5.7

Reference

Method

Borini et al, 200611 Boldt et al, 200628 Chamayou et al, 200654 Levi Setti et al, 200612 Bianchi et al, 200724 De Santis et al, 200713

ProH, propanediol; 2PN, two pronuclei; n.r., not recorded.

embryos available for transfer, even when at each thawing cycle the number of thawed oocytes is deliberately limited to 4–5. Under such conditions, up to 92% of thaws progress to the embryo transfer stage and in more than 80% of embryo transfers 2–3 embryos are available for replacement.11 Unfortunately, the same protocol which gives rise to these results produces apparently low rates of implantation (less than 6%). Proper control groups are often lacking, reducing the significance of these results, but at least in one case the implantation rate of embryos from cryopreserved oocytes (5.7%) has been found to be compromised in comparison to that of a control group (23.2%).12 It is unclear why oocyte storage may affect the implantation potential of the ensuing embryos. Nevertheless, it is interesting to note that, apart from possible detrimental effect on cell division (see above), the use of the protocol on which these studies are based is associated with a high incidence (68.9%) of frozen– thawed oocytes displaying cytoplasmic vacuoles,31 a phenomenon which may be considered a nonspecific response to cell injury63 and rarely observed in fresh material (6.2%). More recently developed protocols have suggested the possibility that oocyte cryopreservation may be compatible with higher implantation rates. In particular, a method based on sodium-depleted and HEPESbuffered freezing media appears to produce limited attrition at the thawing, fertilization, and cleavage stages and, at the same time, ensure an implantation rate of 15.9%28 (Table 18.3). The trial, though, apart from including only 23 patients, was carried out in the absence of a fresh control group. In another recent study24 in which differential sucrose concentrations were used in freezing (0.2 mol/l) and thawing (0.3 mol/l) solutions, it has been reported that in 62 patients younger than 39 years old, high frequencies of survival, fertilization, and 4-cell embryo on day 2 were accompanied by an implantation rate of 16.7% (Table 18.3). Notably, these results were obtained by thawing only a few oocytes and inseminating no more than 3 oocytes per thawing cycle, as prescribed by the Italian IVF law. Also, in a group of patients with comparable mean age and number of oocytes retrieved at pick-up (≥10), the

implantation rate of embryos developed from fresh oocytes was 17.3%. Therefore, it appears that under certain conditions embryos derived from fresh and frozen–thawed oocytes may implant with similar frequencies. This outcome obviously needs confirmation through larger studies, but it is not totally unexpected, considering that embryos which are found intact after freezing–thawing can implant with a frequency unaltered in comparison to fresh controls of equivalent developmental stage.64 Interestingly, when the efficiency of these recent studies24,28 is measured in terms of number of implantations per number of oocytes assigned to treatment (in these cases, per thawed oocytes), success rates are 5–6%. For comparison, embryo cryopreservation assures an implantation rate per oocyte of 4–5%,65 confirming the increasing competitiveness of oocyte freezing. Another possible key to the interpretation of the clinical outcome of oocyte cryopreservation consists of assessing its contribution to the cumulative pregnancy rate per cycle, when only a few oocytes are destined to the fresh treatment, while the remaining are frozen and used at later stages. This possibility is suggested from previous studies which illustrated the contribution of embryo cryopreservation to the clinical success rate,66 responding to the need to reduce the drawbacks of repeated ovarian stimulation treatments in terms of pharmacological and surgical risks, discomfort, psychological stress, cost, and time. Data relevant to cumulative pregnancy rates originating from cryopreserved oocytes are still very preliminary. Nevertheless, even the application of a freezing protocol that was found to be rather inefficient19 can raise the overall pregnancy rate from 30% (derived exclusively from fresh oocytes) to 47%.67 The importance of the contribution of oocyte freezing to the cumulative pregnancy rate has also been confirmed by data generated from the application of more recently developed protocols.68 This suggests that oocyte cryopreservation is destined to become a valid IVF strategy, considering the increased efficiency of recently developed protocols and its relative contribution to the overall clinical outcome in comparison to pronuclear stage69,70 and embryo cryopreservation.71

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Summary Initial failures to obtain high survival rates as well as competition from embryo cryopreservation, whose efficiency has long been recognized, have restrained the development of oocyte cryopreservation as a routine IVF procedure. Over the last several years, it has been shown that many aspects which account for the overall efficiency of oocyte cryopreservation can be improved. Diverse methods can assure survival rates comparable to those attained with frozen embryos. It appears that after cryostorage the rates of fertilization after ICSI and cleavage do not represent significant sources of attrition, in comparison to the performance of fresh controls. However, in general, detailed information on preimplantation development is lacking. Preliminary evidence suggests that certain oocyte cryopreservation protocols may affect the pace of the first two post-fertilization cell division cycles, an influence which is associated with low implantation ability. On the contrary, other freezing methods appear to have a smaller impact on pre- and postimplantation development, to an extent that oocyte cryopreservation is becoming increasingly used as an alternative to pronuclear stage and embryo storage. Adequately large, controlled, prospective studies are needed to assess objectively the overall efficiency of oocyte cryopreservation. The approach involving the concept of cumulative pregnancy rate may offer a significant contribution in pursuing this aim.

References 1. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eightcell embryo. Nature 1983; 305(5936): 707–9. 2. Edgar DH, Bourne H, Jericho H, McBain JC. The developmental potential of cryopreserved human embryos. Mol Cell Endocrinol 2000; 169(1–2): 69–72. 3. Coticchio G, Sereni E, Serrao L, et al. What criteria for the definition of oocyte quality? Ann N Y Acad Sci 2004; 1034: 132–44. 4. De Santis L, Cino I, Rabellotti E, et al. Polar body morphology and spindle imaging as predictors of oocyte quality. Reprod Biomed Online 2005; 11(1): 36–42. 5. Sereni E, Bonu MA, Borini A, et al. High survival rate after cryopreservation of human prophase I oocytes. Fertil Steril 2000; 74 (3S): 161. 6. Ruppert-Lingham CJ, Paynter SJ, Godfrey J, Fuller BJ, Shaw RW. Developmental potential of murine germinal vesicle stage cumulus–oocyte complexes following exposure to dimethylsulphoxide or cryopreservation: loss of membrane integrity of cumulus cells after thawing. Hum Reprod 2003; 18(2): 392–8. 7. Cekleniak NA, Combelles CM, Ganz DA, et al. A novel system for in vitro maturation of human oocytes. Fertil Steril 2001; 75(6): 1185–93. 8. Ford P, Merot J, Jawerbaum A, et al. Water permeability in rat oocytes at different maturity stages: aquaporin-9 expression. J Membr Biol 2000; 176(2): 151–8.

9. Karran G, Legge M. Non-enzymatic formation of formaldehyde in mouse oocyte freezing mixtures. Hum Reprod 1996; 11(12): 2681–6. 10. Carroll J, Wood MJ, Whittingham DG. Normal fertilization and development of frozen–thawed mouse oocytes: protective action of certain macromolecules. Biol Reprod 1993; 48(3): 606–12. 11. Borini A, Sciajno R, Bianchi V, et al. Clinical outcome of oocyte cryopreservation after slow cooling with a protocol utilizing a high sucrose concentration. Hum Reprod 2006; 21(2): 512–17. 12. Levi Setti PE, Albani E, et al. Cryopreservation of supernumerary oocytes in IVF/ICSI cycles. Hum Reprod 2006; 21(2): 370–5. 13. De Santis L, Cino I, Rabellotti E, et al. Oocyte cryopreservation: clinical outcome of slow-cooling protocols differing in sucrose concentration. Reprod Biomed Online 2007; 14(1): 57–63. 14. Fabbri R, Porcu E, Marsella T, et al. Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 2001; 16(3): 411–16. 15. Chen SU, Lien YR, Chen HF, et al. Observational clinical follow-up of oocyte cryopreservation using a slow-freezing method with 1,2-propanediol plus sucrose followed by ICSI. Hum Reprod 2005; 20(7): 1975–80. 16. Stachecki JJ, Cohen J, Willadsen S. Detrimental effects of sodium during mouse oocyte cryopreservation. Biol Reprod 1998; 59(2): 395–400. 17. Van Blerkom J, Davis PW. Cytogenetic, cellular, and developmental consequences of cryopreservation of immature and mature mouse and human oocytes. Microsc Res Tech 1994; 27(2): 165–93. 18. Tucker M, Wright G, Morton P, et al. Preliminary experience with human oocyte cryopreservation using 1,2-propanediol and sucrose. Hum Reprod 1996; 11(7): 1513–15. 19. Borini A, Bonu MA, Coticchio G, et al. Pregnancies and births after oocyte cryopreservation. Fertil Steril 2004; 82(3): 601–5. 20. Gook DA, Osborn SM, Bourne H, Johnston WI. Fertilization of human oocytes following cryopreservation; normal karyotypes and absence of stray chromosomes. Hum Reprod 1994; 9: 684–91. 21. Lassalle B, Testart J, Renard JP. Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil Steril 1985; 44(5): 645–51. 22. Paynter SJ, O’Neil L, Fuller BJ, Shaw RW. Membrane permeability of human oocytes in the presence of the cryoprotectant propane-1,2-diol. Fertil Steril 2001; 75(3): 532–8. 23. Paynter SJ, Borini A, Bianchi V, et al. Volume changes of mature human oocytes on exposure to cryoprotectant solutions used in slow cooling procedures. Hum Reprod 2005; 20(5): 1194–9. 24. Bianchi V, Coticchio G, Distratis V, et al. Differential sucrose concentration during dehydration (0.2 mol/l) and rehydration (0.3 mol/l) increases the implantation rate of frozen human oocytes. Reprod Biomed Online 2007; 14(1): 64–71. 25. Jericho H, Wilton L, Gook DA, Edgar DH. A modified cryopreservation method increases the survival of human biopsied cleavage stage embryos. Hum Reprod 2003; 18: 568–71.

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The human oocyte: controlled rate cooling 26. Stachecki JJ, Cohen J, Garrisi J, et al. Cryopreservation of unfertilized human oocytes. Reprod Biomed Online 2006; 13(2): 222–7. 27. Quintans CJ, Donaldson MJ, Bertolino MV, Pasqualini RS. Birth of two babies using oocytes that were cryopreserved in a choline-based freezing medium. Hum Reprod 2002; 17(12): 3149–52. 28. Boldt J, Tidswell N, Sayers A, Kilani R, Cline D. Human oocyte cryopreservation: 5-year experience with a sodium-depleted slow freezing method. Reprod Biomed Online 2006; 13(1): 96–100. 29. Carroll J, Depypere H, Matthews CD. Freeze–thawinduced changes of the zona pellucida explains decreased rates of fertilization in frozen–thawed mouse oocytes. J Reprod Fertil 1990; 90(2): 547–53. 30. Ghetler Y, Skutelsky E, Ben Nun I, et al. Human oocyte cryopreservation and the fate of cortical granules. Fertil Steril 2006; 86(1): 210–16. 31. Nottola SA, Macchiarelli G, Coticchio G, et al. Ultrastructure of human mature oocytes after slow cooling cryopreservation using different sucrose concentrations. Hum Reprod 2007; 22(4): 1123–33. 32. Porcu E, Fabbri R, Seracchioli R, et al. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997; 68(4): 724–6. 33. Li XH, Chen SU, Zhang X, et al. Cryopreserved oocytes of infertile couples undergoing assisted reproductive technology could be an important source of oocyte donation: a clinical report of successful pregnancies. Hum Reprod 2005; 20(12): 3390–4. 34. Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986; 1(8486): 884–6. 35. Al-Hasani S, Diedrich K, van der Ven H, Reinecke A, Hartje M, Krebs D. Cryopreservation of human oocytes. Hum Reprod 1987; 2(8): 695–700. 36. Stachecki JJ, Cohen J, Willadsen S. Detrimental effects of sodium during mouse oocyte cryopreservation. Biol Reprod 1998; 59(2): 395–400. 37. Cohen Y, Malcov M, Schwartz T, et al. Spindle imaging: a new marker for optimal timing of ICSI? Hum Reprod 2004; 19(3): 649–54. 38. Coticchio G, Bonu MA, Bianchi V, Flamigni C, Borini A. Criteria to assess human oocyte quality after cryopreservation. Reprod Biomed Online 2005; 11(4): 421–7. 39. Stachecki JJ, Munne S, Cohen J. Spindle organization after cryopreservation of mouse, human, and bovine oocytes. Reprod Biomed Online 2004; 8(6): 664–72. 40. Coticchio G, De Santis L, Rossi G, et al. Sucrose concentration influences the rate of human oocytes with normal spindle and chromosome configurations after slow-cooling cryopreservation. Hum Reprod 2006; 21(7): 1771–6. 41. Rienzi L, Martinez F, Ubaldi F, et al. Polscope analysis of meiotic spindle changes in living metaphase II human oocytes during the freezing and thawing procedures. Hum Reprod 2004; 19(3): 655–9. 42. Pickering SJ, Braude PR, Johnson MH, Cant A, Currie J. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990; 54(1): 102–8.

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43. Pickering SJ, Johnson MH. The influence of cooling on the organization of the meiotic spindle of the mouse oocyte. Hum Reprod 1987; 2(3): 207–16. 44. Wang WH, Meng L, Hackett RJ, Odenbourg R, Keefe DL. Limited recovery of meiotic spindles in living human oocytes after cooling–rewarming observed using polarized light microscopy. Hum Reprod 2001; 16(11): 2374–8. 45. Ducibella T. Biochemical and cellular insights into the temporal window of normal fertilization. Theriogenology 1998; 49(1): 53–65. 46. Gook DA, Osborn SM, Johnston WI. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum Reprod 1993; 8(7): 1101–9. 47. Kuwayama M, Vajta G, Kato O, Leibo SP. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005; 11(3): 300–8. 48. Giorgetti C, Terriou P, Auquier P, et al. Embryo score to predict implantation after in-vitro fertilization: based on 957 single embryo transfers. Hum Reprod 1995; 10(9): 2427–31. 49. Staessen C, Nagy ZP, Liu J, et al. One year’s experience with elective transfer of two good quality embryos in the human in-vitro fertilization and intracytoplasmic sperm injection programmes. Hum Reprod 1995; 10(12): 3305–12. 50. Van Royen E, Mangelschots K, De Neubourg D, et al. Characterization of a top quality embryo, a step towards single-embryo transfer. Hum Reprod 1999; 14(9): 2345–9. 51. Ziebe S, Petersen K, Lindenberg S, et al. Embryo morphology or cleavage stage: how to select the best embryos for transfer after in-vitro fertilization. Hum Reprod 1997; 12(7): 1545–9. 52. Edgar DH, Archer J, Bourne H. The application and impact of cryopreservation of early cleavage stage embryos in assisted reproduction. Hum Fertil (Camb) 2005; 8(4): 225–30. 53. Gabrielsen A, Fedder J, Agerholm I. Parameters predicting the implantation rate of thawed IVF/ICSI embryos: a retrospective study. Reprod Biomed Online 2006; 12(1): 70–6. 54. Chamayou S, Alecci C, Ragolia C, et al. Comparison of in-vitro outcomes from cryopreserved oocytes and sibling fresh oocytes. Reprod Biomed Online 2006; 12(6): 730–6. 55. Shoukir Y, Campana A, Farley T, Sakkas D. Early cleavage of in-vitro fertilized human embryos to the 2-cell stage: a novel indicator of embryo quality and viability. Hum Reprod 1997; 12(7): 1531–6. 56. Bianchi V, Coticchio G, Distratis V, Di Giusto N, Borini A. Early cleavage delay in cryopreserved human oocytes. 2005; 20(Suppl 1): i54. 57. Gook DA, Schiewe MC, Osborn SM, et al. Intracytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2propanediol. Hum Reprod 1995; 10(10): 2637–41. 58. Coticchio G, Bonu MA, Sciajno R, et al. Truths and myths of oocyte sensitivity to controlled rate freezing Reprod Biomed Online 2007; 15: 24–30. 59. Fosas N, Marina F, Torres PJ, et al. The births of five Spanish babies from cryopreserved donated oocytes. Hum Reprod 2003; 18(7): 1417–21.

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60. Barritt J, Luna M, Duke M, et al. Report of four donorrecipient oocyte cryopreservation cycles resulting in high pregnancy and implantation rates. Fertil Steril 2007; 87(1): 189 e13–17. 61. Porcu E, Fabbri R, Damiano G, et al. Clinical experience and applications of oocyte cryopreservation. Mol Cell Endocrinol 2000; 169(1–2): 33–7. 62. La Sala GB, Nicoli A, Villani MT, et al. Outcome of 518 salvage oocyte-cryopreservation cycles performed as a routine procedure in an in vitro fertilization program. Fertil Steril 2006; 86(5): 1423–7. 63. Ghadially FN. Ultrastructural Pathology of the Cell and Matrix, 2nd edn. London: Butterworths; 1982. 64. Edgar DH, Bourne H, Speirs AL, McBain JC. A quantitative analysis of the impact of cryopreservation on the implantation potential of human early cleavage stage embryos. Hum Reprod 2000; 15(1): 175–9. 65. Gook DA, Edgar DH. Cryopreservation of the human female gamete: current and future issues. Hum Reprod 1999; 14(12): 2938–40.

66. Jones HW Jr, Veeck LL, Muasher SJ, Gibbons WE. On reporting pregnancies by assisted reproductive technology. Fertil Steril 1993; 60(5): 759–61. 67. Borini A, Lagalla C, Bonu MA, et al. Cumulative pregnancy rates resulting from the use of fresh and frozen oocytes: 7 years’ experience. Reprod Biomed Online 2006; 12(4): 481–6. 68. Borini A, Bianchi V, Bonu MA, et al. Evidence-based clinical outcome of oocyte slow cooling. Reprod Biomed Online 2007; 15: 175–81. 69. FIVNAT-CH, Reproduktionsmedizin SGf, Reproduction SSdMdl. Annual report, 2002. 70. Damario MA, Hammitt DG, Session DR, Dumesic DA. Embryo cryopreservation at the pronuclear stage and efficient embryo use optimizes the chance for a liveborn infant from a single oocyte retrieval. Fertil Steril 2000; 73(4): 767–73. 71. Tiitinen A, Halttunen M, Harkki P, Vuoristo P, HydenGranskog C. Elective single embryo transfer: the value of cryopreservation. Hum Reprod 2001; 16(6):1140–4.

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19 The human oocyte: vitrification Masashige Kuwayama

Introduction The past 50 years have resulted in impressing breakthroughs in cryopreservation in reproductive biology. As typical in this discipline, techniques were initially established in experimental and domestic animals, and subsequently applied to humans. The first successes were achieved with spermatozoa,1 followed by cryopreservation of preimplantation embryos of different stage of development.2–4 Until recently, however, attempts to cryopreserve human oocytes (just like those of domestic animals) have mostly failed or remained far less efficient than required for practical application. Reasons to cryopreserve human oocytes are widely known and were summarized recently.5,6 Most frequent indications include diseases and treatments: i.e. preservation of reproductive competence of young cancer patients who need irradiation of the pelvic region or chemotherapy; or any surgical intervention before or during the reproductive age involving removal of ovaries. Ovary function-related problems include premature menopause, ovary hyperstimulation syndrome, or poor response to ovary stimulation. Legal, ethical, social, and practical problems may also require oocyte cryopreservation, including restrictions in embryo cryopreservation in several countries, wish to delay motherhood for various reasons, and lack of available semen after a successful oocyte retrieval. However, as discussed in detail recently,6 in broader terms oocyte cryopreservation is also needed to compensate For the marked age-related decline in fertility seen in women over 35 years. As in most mammalian species, women suffer more and sacrifice more for their offspring. On the other hand, their reproductive ability is seriously restricted in terms of quantity and duration. Males provide millions of sperm with one ejaculation, while only one or two oocytes are matured every 28 days. The reproductive age of men is almost unlimited, while that of women (without additional concerns) is restricted to a period of just 15–20 years. Assisted reproductive techniques did not help to eliminate this difference; in contrast, with the use of intracytoplasmic sperm injection (ICSI) and successful cryopreservation of sperm, the

gap has widened considerably. Apart from the practical goals, our moral duty is to help develop an efficient and safe oocyte cryopreservation method. Unfortunately, the task is rather demanding. Although the first pregnancy from a cryopreserved oocyte was achieved more than 20 years ago,7 the advancement was very slow until recently. Generally, inefficiency and lack of consistency have been the two main problems.8 Oocytes are special objects: their size, shape, lowest possible cell number, and general fragility explain most of the difficulties that occur during cryopreservation. Oocytes are usually referred to as being the largest cells in the mammalian body. Size seems to be an important factor in cryobiology. Viruses and bacteria may survive deep freezing without any special treatment (cryoprotectants or controlled rate cooling), causing a lot of potential problems also in embryology. Freezing of fibroblasts and epidermal cells is an easy and efficient routine task of tissue culture laboratories, and does not need any special instruments. Sperm cryopreservation can be efficiently performed with the use of a controlled-rate freezer. Early cleavage-stage embryos with cells with 10–50% of the original size of oocytes survive traditional slow-rate freezing and preserve their developmental competence. Preantral and primary follicles can also be frozen successfully, in contrast to the fully developed, MII phase, large oocytes. The almost completely spherical shape does not confer an advantage from the point of cryopreservation. During equilibration and dilution before and after cooling and warming, respectively, permeable cryoprotectants have to become distributed rapidly and equally in the ooplasm. From this point of view, a large spherical body is definitely a handicap compared with an elongated shape like a fibroblast or a neuron. The 1-cell stage also presents a handicapped situation, as there is no backup. Multicellular embryos may survive the death of more than 50% of their cells (this fact is clearly demonstrated by the success of embryo bisection in domestic animals), while the oocyte has to stake everything on one card. However, apart from the size, shape, and cell number, other factors may also play an important role. Germinal vesicle (GV)-stage oocytes and fertilized

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zygotes have almost exactly the same characteristics; however, zygotes are considerably more tolerant to cryoinjuries, whereas GV-stage oocytes are even more sensitive than matured ones. Known factors that further influence their sensitivity include chilling, serious deformation of shape during equilibration and dilution with cryoprotectants, and hardening of the zona pellucida. Chilling is probably the least understood type of injury at cryopreservation, involving damage to lipid droplets, lipid-rich membranes, and microtubules. The temperature zone where this injury occurs is rather high, between +15°C (in some biological objects +20°C) and −5°C.9 The damage to lipids is irreversible, and causes the death of the oocyte. Compared with other species, the lipid droplet content of human oocytes is relatively low. On the other hand, their sensitivity to chilling is still considerable, caused probably by the membrane damage and the depolymerization of microtubules, with all the subsequent consequences, including misalignment of chromosomes and aneuploidy,10–14 although the latter effect may be less detrimental than earlier supposed.14 Chilling damage to membranes in human matured oocytes seems to be much more serious than in the latter developmental stages (zygotes), a possible cause for the well-known stage-dependent sensitivity.15 Serious deformation of the shape may occur when oocytes are exposed to the cryoprotectant solutions as a result of the osmotic effect. However, in spite of the somewhat frightening morphological view, human oocytes seem to tolerate these deformations rather well. Careful addition of cryoprotectants may minimize the misshape and the possible damage. The other suggested strategy, addition of cytoskeleton relaxants,16 may not be required in the human. On the other hand, at dilution, the spherical shape allows only a minimal expansion, and therefore the accumulating water may disrupt the cell membrane.

Vitrification vs traditional freezing During the past two decades two major strategies for cryopreservation of oocytes and embryos in mammalian species have been developed (reviewed in detail in Vajta and Nagy17). Traditional slow-rate freezing establishes a delicate balance between various sources of injuries, while the principal goal of vitrification is to eliminate entirely ice crystal formation in the whole solution containing the embryos and oocytes. To achieve this ice-free glass-like solidification of solutions, which can also be defined as an extremely increased viscosity, high cryoprotectant concentration and/or very high cooling rates are required. To decrease the potential osmotic and toxic damage caused by the cryoprotectants, recent vitrification methods focus on increase of cooling and warming rates.18–21 Most of the successful vitrification methods are based on an extremely low amount of

Fig 19.1 The Cryotop vitrification device. The polypropylene strip (a) is attached to a rigid plastic handle (b). After vitrification, a plastic cover (c) is attached to protect the strip during storage in liquid nitrogen (d). (Reproduced from Kuwayama et al27 with permission)

solution containing the sample and a direct contact between this solution and the liquid nitrogen. One of these approaches, the minimum drop size (MSD) method was first applied by Arav,22 and further modified by Hamawaki et al.23 Based on these earlier achievements, a special method, the Cryotop vitrification technique, has been developed for cryopreservation of oocytes and embryos.24 The Cryotop has been applied successfully to a wide variety of mammalian species (reviewed in Kuwayama and Kato24), and has resulted in a considerable increase in the overall efficiency of cryopreservation of human oocytes and embryos.25–27

The use of Cryotop vitrification for cryopreservation of human MII phase oocytes Timing Oocytes should be vitrified between 2 and 6 hours (preferably 2 hours) after ovum pick up, and immediately after denudation (cumulus cell removal). ICSI can be performed in 2–4 hours (preferably 2 hours) after warming.

Device The Cryotop consists of a 0.4 mm wide, 20 mm long, 0.1 mm thick flexible filmstrip attached to a rigid plastic handle (Fig 19.1). To protect the filmstrip and the sample cryopreserved on it, a 30 mm long transparent plastic cap is also provided to cover this part during storage in liquid nitrogen. The device is sterilized, should be handled under aseptic conditions, and only for one cycle of vitrification.

Solutions Media for all phases of vitrification are listed in Table 19.1.

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Solutions used for Cryotop vitrification

Name

Basic medium

Permeable cryoprotectants

Nonpermeable cryoprotectants

Washing solution (WS)

M199 + 20% SSS

–

–

Equlibration solution (ES)

M199 + 20% SSS

7.5% EG, 7.5% DMSO

–

Vitrification solution (VS)

M199 + 20% SSS

15% EG, 15% DMSO

0.5 mol/l sucrose

Thawing solution (TS)

M199 + 20% SSS

–

1.0 mol/l sucrose

Dilution solution (DS)

M199 + 20% SSS

–

0.5 mol/l sucrose

M199, HEPES-buffered TCM 199 medium; SSS: synthetic serum substitute (Irvine Scientific, Santa Ana, CA, USA); EG, ethylene glycol; DMSO, dimethylsulfoxide. All chemicals, except indicated otherwise, are dervived from Sigma Chemical Co. (St. Louis, MO, USA).

Working environment and preparation steps The vitrification procedure has to be performed in a well-ventilated laboratory at 25–27°C room temperature. As all equilibration and dilution parameters were adjusted according to this temperature, it is very important to warm media to 25–27°C, as well, preferably by placing all the solutions and vials for more than 1 hour on a clean bench, preferably with a laminar hood, to reach this temperature. The only exception is the thawing solution (TS), which should be warmed to 37°C. Note that the basic solution apart from HEPES also contains bicarbonate buffer, and is adjusted to maintain the appropriate pH in air. Therefore, a carbon dioxide incubator is not required for warming of closed vials and empty dishes.

Additional tools Vitrification has to be performed in 35 mm diameter Falcon Petri dishes (Cat. No. 1008; Falcon, San Jose, CA, USA). For practical reasons a relatively small, thick-walled foam box (approximately 250 × 150 × 200 for length, width, and height) with minimum 3 cm thick walls and bottom is suggested, preferably with appropriate foam cover. The box should be placed on a stable surface with easy reach but minimal danger of accidental splitting or pouring off of the liquid nitrogen. All safety instructions related to work with liquid nitrogen should be strictly followed. Points for selection of optimal sources and possible pretreatment of liquid nitrogen will be discussed later. The foam box should also contain plastic racks for temporary storage of the device. Cryotop vitrification requires delicate handling of oocytes and embryos. For vitrification and warming, a relatively simple stereomicroscope equipped with zoom and capable of providing sharp, contrasted views is appropriate. Except for special purposes, there is no need for conventional or inverted light microscopes or fluorescent equipment. There is no need to restrict illumination if sources are filtered for UV light. Use microscope lights only when required.

Fig 19.2 The arrangement of drops in the lid of a Petri dish, and the procedure of equilibration. See text for details.

Equilibration and cooling Mix gently prewarmed washing solution (WS), TS, and vitrification solution (VS) vials (one vial from each) with up and down movement three times. Label three Petri dishes with WS, equilibration solution (ES), and VS, respectively. Pour the entire content of the vials into the proper Petri dish. For equilibration, place one drop of WS and 3 drops of ES, 20 µl each, in the lid of one Petri dish. The arrangement of drops is shown in Fig 19.2. The distance between the WS, ES1, and ES2 drops should be approximately 1 mm. The ES3 drop should be made further from the other drops. Before starting the vitrification procedure, check oocyte quality, and perivitelline space (compare with the thickness of the zona pellucida) and record any characteristic that might affect oocyte survival. The equilibration and vitrification procedure consists of the following steps (see also Fig 19.2): 1. Place the oocytes in the center of the WS drop. 2. With the pipette, merge the WS and ES1 drops. Oocytes are displaced by the ES1 drop toward the left side. Shrinking of oocytes should occur at latest 90 seconds after merging. Wait for 3 minutes. Place oocytes in the middle of WS + ES1, then merge ES2 with WS + ES1. Oocytes are displaced again by the ES2 drop. Wait for another 3 minutes. Full re-expansion of oocytes should occur; during this period the perivitelline space should be the same as before equilibration. If the recovery of oocytes is incomplete, continue equilibration in WS + ES1 + ES2 for another minute. 3. Pick up the oocytes with the pipette in a minimum amount of solution, and place them on the top of

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the ES3 drop. Just do nothing. Equilibrate the oocytes for an additional 9 minutes) and check for oocyte re-expansion afterwards. Turn the oocytes with the pipette to attain a 3D vision. If the recovery is complete, you may proceed to the next step. If not, wait until 15 minutes. Normal oocytes should completely regain their original appearance after equilibration. The recovery period may be used to prepare the liquid nitrogen container and to label the Cryotops. Pick up the oocytes with the pipette in a minimum amount of solution and place them on the top of the 4.5 ml VS solution in the Petri dish. Set the timer for 60 seconds. Discard immediately the ES3 solution that has remained in the pipette and flush the pipette with VS solution. Immediately discard the excess of ES3 solution. Wash the oocytes repeatedly by removing the surrounding ES3 medium (visible under the stereomicroscope due to the different osmolarity). Submerge the oocytes several times at different areas of the dish. Proper washing is a critical step of the procedure. Extreme shrinkage of oocytes should be evaluated as a positive sign. Although 60 seconds is the recommended period for the equilibration in VS, a slight increase (up to 90 seconds) or decrease (minimum 40 seconds) may not be detrimental. Pick up the oocytes again with the pipette in a minimum amount of solution and place them on the strip of the Cryotop near the black mark (Fig 19.3). Excess of media must be completely avoided or removed with the pipette after expelling oocytes. Remove any excess by using the pipette; media will enter by capillary. Submerge the Cryotop in liquid nitrogen vertically, followed by rapid horizontal movements to obtain the maximum cooling rate (>23 000°C/min). Repeat steps 5, 6, and 7 if there are more oocytes in the ES3 drop. While keeping it submerged in liquid nitrogen, cover the strip of the Cryotop with the plastic cap using tweezers.

Warming and dilution The unopened TS vial and one Petri dish marked with TS should be prewarmed to 37°C in an incubator for at least 1 hour. All other solutions should be kept at room temperature, i.e. 25–27°C. Mix gently prewarmed dilution solution (DS) and WS vials (one and two vials, respectively), with up and down movement three times. Label three Petri dishes with DS, WS1, and WS2, respectively. Pour the entire content of the vials into the proper Petri dish. Use the same procedure for the TS; however, prepare the dish just before the thawing to maintain its temperature to obtain the required warming rate (42 000°C/min). The warming and dilution procedure consists of the following steps (dilution is also shown in Fig 19.4):

Fig 19.3 Cryotop.

Arrangement of the oocyte on the filmstrip of the

1. By using tweezers, remove the plastic cap of the Cryotop while still submerged in liquid nitrogen. This manipulation can be performed easily if the foam box is filled almost entirely with liquid nitrogen. The container should be positioned close to the microscope to avoid delay when transferring the Cryotop. The microscope has to be focused to the center of the TS dish with low magnification. 2. Hold the Cryotop and look for the black mark maintaining the tip submerged. Remove the Cryotop with a rapid and straight movement from the liquid nitrogen and place the tip immediately in the middle of the S plate. 3. Localize the oocytes and start counting for 10 seconds (handling of a timer is not practical for such a short period when parallel tasks have to be performed, either). Subsequently, remove oocytes from the surface of the strip with the pipette, applying a gentle aspiration, and place them into the TS dish for 60 seconds. Follow all movements of oocytes continuously, as they become transparent in this phase of the work and it is easy to lose them. Later, they will regain their normal appearance. 4. After 60 seconds, pick up oocytes into the capillary and also aspirate an additional 2 mm long TS column to the end of the capillary. Transfer the capillary into the DS dish and expel contents gently to the bottom: first, the TS media, allowing a small mound to form, then the oocytes on the top of this mound. Just do nothing. Wait for 3 minutes. The dilution period can also be extended to 5 minutes without significant harm to the oocytes. 5. Subsequently, the same method of transfer should be applied, but with different solutions: oocytes are placed on the top of the mound formed from DS medium in the WS1 dish for 5 minutes, without any stirring or mixing of the media. 6. After 5 minutes, place the oocytes onto the surface of the WS2 plate and wait for an additional 5 minutes. 7. Finally, oocytes are transferred to the culture dish and their morphology is controlled under the stereomicroscope. ICSI can be performed after a recovery period of 2 hours. A practical suggestion: as the time spent in washing dishes is not very critical if there are several groups of oocytes, wait until the last group has completed

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Fig 19.4 The dilution procedure. After warming in a Petri dish filled with thawing solution (TS), oocytes are transferred into dilution solution (DS) for 3 minutes, then into washing solution (WS) 1 and 2, for 5 and 5 minutes, respectively. During transfer to DS and WS1, a 2 mm column of the previous media is aspirated into the capillary, and expelled first into the new dish. This creates a small mound of the previous medium on the bottom of the new dish: oocytes are placed on the top of this mound and sink slowly inside. This simple manipulation decreases osmotic shock during the dilution procedure.

5 minutes, then move all the oocytes to WS2, wait for 1 minute, and then transfer all the oocytes to a culture dish.

The danger of liquid nitrogenmediated disease transmission Safety issues regarding open methods of vitrification have been discussed recently in detail.5,17,28,29 Liquid nitrogen may become infected by pathogenic agents and can transmit these agents to other samples stored in the same batch. Under experimental conditions, transmission has also been demonstrated between embryological samples.30,31 Although during the past 30 years no disease transmission related to liquid nitrogen-mediated infection and embryo transfer has been reported in human or animal fields, a theoretical danger exists and should be minimized with rational measures. According to most observations, hermetic isolation of the sample from the liquid nitrogen or medium during cooling and thawing, respectively, decreases cooling and warming rates considerably and, as a consequence, survival of oocytes. The reasonable solution for the problem is to separate cooling and thawing from storage. Cooling can be performed in liquid nitrogen that is directly provided from the factory, has not been in contact with any other sample, and is filtered before use.32,33 For storage, samples may be sealed into a precooled, hermetically isolated container: e.g. 1 ml diameter CBS straw (IMV, L’Aigle, France). An analog of the system was successfully

applied for open pulled straw (OPS) vitrification32 and the required instrument is commercially available (VitSet, Minitüb, Landshut, Germany29). At warming, the end of the 1 ml straw may be cut with sterile scissors while the rest of the straw is still submerged into liquid nitrogen, and the Cryotop can be quickly removed with a narrow forceps for immersion into the proper medium. Recently, the safety of this system (applied for OPS vitrification) has been proved by independent investigation.34

Results achieved with Cryotop vitrification of human oocytes Although the first baby born after oocyte vitrification was achieved with the OPS technique,35 success with the Cryotop vitrification soon followed.25,26 In Japan, only 20 babies were born with this method, as a result of legal restrictions for oocyte donation. However, impressive results have been achieved in Colombia, Mexico, and very recently in Spain. Results achieved in Colombia have been published recently,36 but further successes have also been obtained, including a 83.5% post-thaw survival rate, 82.8% fertilization, and 94.7% cleavage rate after ICSI. The blastocyst rate is 49%, and transfer of blastocysts results in 42.3% pregnancy and 48% implantation, eventually leading to a birth rate of 62.8% of those implanted. At the time of submission of this manuscript, 107 live births and 43 ongoing pregnancies have been achieved (Professor Elkin Lucena, pers commun).

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In Mexico, out of 849 thawed oocytes, 87% survived the procedure. The fertilization and cleavage rates were 83 and 82%, respectively. One hundred and seventy-two transfers (3 embryos transferred into one patient) resulted in 47 pregnancies (27.3%), comprising 37 babies and 10 ongoing pregnancies (Dr Ruvalcaba, pers comm). A recent paper34 has described in detail achievements in Spain: the survival of the 231 thawed oocytes was 96.9%, and the fertilization and cleavage rates were 76.3 and 94.2%, respectively. Transfer of 49 embryos into 23 patients resulted in a 38.2% implantation rate, and included 5 twin pregnancies and 3 miscarriages; the ongoing pregnancy rate is 11/23 (47.8%) (Dr Cobo, pers commun).

Conclusion Cryopreservation of oocytes is regarded as one of the most demanding tasks of human-assisted reproduction. With proper application of the latest vitrification techniques, the efficiency has increased remarkably. The Cryotop vitrification method has resulted in high survival, fertilization, pregnancy, and birth rates, comparable to those achieved with nonvitrified control oocytes. The technique may be useful in diverse situations where oocyte storage is required or considered.

Acknowledgments The author is very grateful to Professor Gábor Vajta for the critical reading of the manuscript and to Dr Noriko Kagawa for her helpful advice.

References 1. Polge C, Smith AY, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature (London) 1949; 164: 666. 2. Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to −196°C and −269°C. Science 1972; 178: 411–14. 3. Wilmut I. The effect of cooling rate, warming rate, cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing. Life Sci 1972; 11: 1071–9. 4. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing, and transfer of an eightcell embryo. Nature 1983; 305: 707–9. 5. Kuwayama M. Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method. Theriogenology 2007; 67: 73–80. 6. Kuwayama M, Cobo A, Vajta G. Vitrification of oocytes: general considerations and the use of the Cryotop method. In: Tucker MJ, Liebermann J, eds. Vitrification in Assisted Reproduction. London: Informa Healthcare, 2007. 7. Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986; 1(8486): 884–6.

8. Liebermann J, Tucker MJ. Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertil Steril 2006; 86: 20–6. 9. Leibo SP, Martino A, Kobayashi S, Pollard JW. Stagedependent sensitivity of oocytes and embryos to low temperatures. Anim Reprod Sci 1996; 42: 45– 53. 10. Magistrini M, Szollosi D. Effects of cold and of isopropyl N-phenylcarbamate on the second meiotic spindle of mouse oocytes. Eur J Cell Biol 1980; 22: 699–707. 11. Sathananthan AH, Ng SC, Trounson AO, et al. The effects of ultrarapid freezing on meiotic and mitotic spindles of oocytes and embryos. Gam Res 1998; 21: 385–401. 12. Pickering SJ, Braude PR, Johnson MH, Cant A, Currie J. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte. Fertil Steril 1990; 54: 102–8. 13. Fabbri R, Porcu E, Marsella T, et al. Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 2001; 16: 411–16. 14. Stachecki JJ, Munné S, Cohen J. Spindle organization after cryopreservation of mouse, human, and bovine oocytes. Reprod Biomed Online 2004; 8: 664–72. 15. Ghetler Y, Yavin S, Shalgi R, Arav A. The effect of chilling on membrane lipid phase transition in human oocytes and zygotes. Hum Reprod 2005; 20: 3385–9. 16. Dobrinsky JR, Pursel VG, Long CR, Johnson LA. Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification. Biol Reprod 2000; 62: 564–70. 17. Vajta G, Nagy PZ. Are programmable freezers still needed in the embryo laboratory? Review on vitrification. Reprod Biomed Online 2006; 12: 779–96. 18. Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 1996; 54: 1059–69. 19. Vajta G, Holm P, Kuwayama M, et al. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998; 51: 53–8. 20. Lane M, Schoolcraft WB, Gardner DK. Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil Steril 1999; 72: 1073–8. 21. Lane M, Bavister BD, Lyons EA, Forest KT. Containerless vitrification of mammalian oocytes and embryos. Nat Biotechnol 2001; 17: 1234–6. 22. Arav A. Vitrification of oocytes and embryos. In: Lauria A, Gandolfi F, eds. New Trends in Embryo Transfer. Cambridge, UK: Portland Press, 1992: 255–64. 23. Hamawaki A, Kuwayama M, Hamano S. Minimum volume cooling method for bovine blastocyst vitrification. Theriogenology 1999; 51: 165. 24. Kuwayama M, Kato O. All-round vitrification method for human oocytes and embryos. J Assist Reprod Genet 2000; 17: 477. 25. Katayama P, Stehlik J, Kuwayama M, Kato O, Stehlik E. High survival rate of vitrified human oocytes results in clinical pregnancy. Fertil Steril 2003; 80: 223–4.

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The human oocyte: vitrification 26. Kuwayama M, Vajta G, Kato O, Leibo S. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005; 11: 300–8. 27. Kuwayama M, Vajta G, Ieda S, Kato O. Vitrification of human embryos using the CryoTip™ method. Reprod Biomed Online 2005; 11: 608–14. 28. Vajta G, Kuwayama M. Improving cryopreservation systems. Theriogenology 2006; 65: 236–44. 29. Vajta G, Kuwayama M, Vanderzwalmen P. Disadvantages and benefits of vitrification. In: Tucker MJ, Liebermann J, eds. Vitrification in Assisted Reproduction. London: Informa Healthcare, 2007: 33–45. 30. Bielanski A, Nadin-Davis S, Sapp T, Lutze-Wallace C. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 2000; 40: 110–16. 31. Bielanski A, Bergeron H, Lau PC, Devenish J. Microbial contamination of embryos and semen

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during long-term banking in liquid nitrogen. Cryobiology 2003; 46: 146–52. Vajta G, Lewis IM, Kuwayama M, Greve T, Callesen H. Sterile application of the Open Pulled Straw (OPS) vitrification method. Cryo-Letters 1998; 19: 389–92. Cobo A, Kuwayama M, Pérez S, et al. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method. Fertil Steril 2007; Sept 21 [Epub ahead of print]. Bielanski A. Non-cross-contamination of bovine embryos with microbes using the OPS vitrification system. Reprod Fertil Dev 2007; 19: 232. Kuleshova L, Gianaroli L, Magli C, Trounson A. Birth following vitrification of a small number of human oocytes. Hum Reprod 1999; 14: 3077–9. Lucena E, Bernal DP, Lucena C, et al. Successful ongoing pregnancies after vitrification of oocytes. Fertil Steril 2006; 85: 108–11.

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20 The human embryo: slow freezing Lucinda L Veeck Gosden, Rosemary Berrios, Richard Bodine, Robert N Clarke, Nikica Zaninovic

Overview Since the birth of the first in vitro fertilization (IVF) baby in 1978, there have been numerous advances in the field of assisted reproductive technologies (ART), including improvements in hormonal stimulation regimens, the formulation of optimal embryo culture media, and refinements in embryo replacement techniques. As a result, it has become a common occurrence in many ART programs to have patients with large numbers of good-quality embryos or blastocysts available for transfer. In addition, there are times when patients with certain medical conditions may need to freeze all conceptuses in lieu of transfer. Thus, the need for an adequate embryo cryopreservation program in most centers has evolved from one of luxury to one of necessity. Until the mid-1990s, the number of ART centers performing cryopreservation and the pregnancy results following the transfer of cryopreserved– thawed conceptuses were relatively low. By way of comparison, in 1989, Fugger1 reported results from 25 IVF member institutions of the Society of Assisted Reproductive Technology (SART) with cryopreservation experience. In these centers, the total number of cleaved embryos and blastocysts frozen was 4460 and 341, respectively. The average clinical pregnancy rate per transfer was 13.4%. In contrast, 2001 results reported by the SART registry from 368 ART programs in the United States performing cryopreservation indicated that over 14 000 cryo–thaw cycles were initiated, resulting in an overall clinical pregnancy rate per transfer of 29.3% and a delivery rate per transfer of 23.5%.2 While these percentages are far lower than those reported for fresh IVF cycles (38.0 and 31.6%, respectively), many centers have more recently experienced higher cryopreservation– thaw success rates, almost equivalent to their fresh counterparts. Indeed, advances in these techniques and procedures have made cryopreservation an important adjunct treatment for many ART patients. This is reflected in a 1999 report from Hoffman et al,3 who estimated that nearly 400 000 embryos were frozen and stored in ART centers

in the United States. This number today is most likely much higher, perhaps even doubled. Before cryopreservation methods were common in human ART practice, a patient with multiple oocytes harvested had few options for optimizing her IVF attempt, and most of these options resulted in either clinical or ethical dilemmas. Patients would sometimes choose to inseminate only the number of oocytes equal to the number of embryos they were willing to transfer. Since there was no guarantee of 100% fertilization and some embryos would surely be of poor quality, suboptimal transfers were commonplace in these situations. Alternatively, patients choosing to inseminate all available oocytes were often faced with the possibility of having to discard or donate potentially viable conceptuses after transfer. Since the birth of the first children following the transfer of a cryopreserved human embryo in 19834 and human blastocyst in 1985,5 much work has been performed by embryologists, clinicians, and animal researchers to optimize methods for freezing and thawing human conceptuses. These studies have focused primarily on method development and/or identification of clinical or laboratory variables which affect potential clinical outcome following cryopreservation. Method development studies have focused on the formulation of cryopreservation media (including type and concentration of cryoprotectant and the addition of dehydrating agents), cryopreservation equipment, and post-thaw culture conditions. Clinical cryopreservation reports have attempted to identify factors that affect cryopreservation–thawing success and outcome, including patient age, stimulation regimens, embryo quality considerations, timing of thawed conceptus transfer, and the methods for endometrial preparation for transfer. It is safe to say that, in general, patient variables that affect clinical outcome of fresh cycles will similarly alter the success of cryopreserved cycles. Similar to fresh cycles, advanced patient age has a negative effect on pregnancy rate following the transfer of thawed conceptuses.6 In addition, Toner et al7 found basal follicle-stimulating hormone (FSH) levels and age to affect embryo cryopreservation outcome. It is well accepted that the transfer of good-quality fresh

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embryos results in higher pregnancy rates than the transfer of morphologically poor or developmentally delayed ones. Similarly, the transfer of good-quality frozen and thawed conceptuses results in more optimal clinical outcomes.6,8–10 There appears to be no effect of patient stimulation regimen on subsequent clinical outcome following cryopreservation.11 There are conflicting reports on the effect of other clinical factors on cryopreservation success, including the role of intracytoplasmic sperm injection (ICSI), the method of endometrial preparation, and the timing of transfer. While Oehninger et al11 reported no difference between ICSI or inseminated frozen–thawed embryos in terms of survival or subsequent clinical pregnancy rates, another report12 demonstrated higher post-thaw mortality and pregnancy losses in ICSI frozen–thawed embryos. Similarly, Oehninger et al11 reported no difference between natural or programmed replacement thaw cycles, while Loh and Leong13 found endometrial preparation to be the most important factor in determining post-thaw clinical success. In the Cornell ART program, we have found neither method of fertilization nor method of endometrial preparation to be critical factors in subsequent post-thaw clinical success. Much of the improved success in clinical outcome of thawed human cycles can be traced to overall improvements in embryo culture conditions, which have resulted in better-quality fresh embryos and blastocysts available for cryopreservation. Formulation of improved culture media and more optimal culture conditions over recent years have contributed to higher clinical success rates for fresh and frozen conceptuses. Many of these improvements have stemmed from earlier animal experiments, including the use of ultramicrofluorometry in determining very subtle changes in mouse embryo metabolism during embryo culture.14 More recently, Lane et al15 showed that the addition of ascorbate to mouse embryo culture media resulted in lower hydrogen peroxide accumulation and increased development of the inner cell mass of resulting blastocysts. Whether or not these types of medium additives prove beneficial to the culture of human fresh and frozen conceptuses remains unclear. The type of cryoprotectant and the use of dehydrating agents such as sucrose are important considerations in a cryopreservation program. Most ART centers are now using either 1,2-propanediol (PROH) or dimethylsulfoxide (DMSO) as the cryoprotectant of choice for embryos, while glycerol is routinely used when freezing human blastocysts. Sucrose has been added in many cases to both systems to aid in cellular dehydration and reduce osmotic shock. Method of cooling (slow freezing versus rapid freezing) is also an important consideration. Many ART laboratories are still using slow-cooling methods for freezing human conceptuses, based on the original work of Testart et al,16,17 while most of the human blastocyst freezing protocols have evolved from the earlier work of Ménézo et al18,19 Many laboratories have modified

these original protocols over the years, and some have found that very subtle changes in freezing procedures can improve post-thaw results. Gardner et al20 showed that changing the starting temperature and cooling rate in a slow-freezing protocol significantly increased human blastocyst post-thaw viability. Rapid-freezing techniques (vitrification) have gained popularity recently, and a growing number of births have been reported to date.21–24

Methods The primary goal in establishing an appropriate freezing protocol is to do as little damage as possible while exposing specimens to nonphysiologic ultralow temperatures. Popular protocols essentially freeze-dry or dehydrate blastocysts to prevent intracellular ice from forming. The formation of intracellular ice crystals can mechanically damage specimens by disrupting and displacing organelles, or slicing through membranes. This is why freezing techniques use cryoprotective agents and control ice formation at critical temperatures. It has been shown that when human cells are placed into a medium that contains an intracellular cryoprotective agent, intracellular water readily exits the cell as a result of the higher extracellular concentration of cryoprotectant. This causes some cell shrinkage until osmotic equilibrium is reached by the slower diffusion of the cryoprotectant into the cell. Once equilibrium is reached, the cell resumes a normal appearance. The rate of permeation of cryoprotectant and water is dependent on temperature; equilibrium is achieved faster at higher temperatures. However, some cryoprotectants such as DMSO are toxic at elevated concentrations, and must be used at lower temperatures to reduce adverse effects. Cryoprotectants are also beneficial in their ability to lower the freezing point of a solution. Solutions may remain unfrozen at −5 to −15°C because of supercooling (cooling to well below the freezing point without extracellular ice formation). When solutions supercool, cells do not dehydrate appropriately since there is no increase in osmotic pressure from the formation of extracellular ice crystals. To prevent supercooling, an ice crystal is introduced in a controlled fashion in a process called seeding. This contributes to intracellular dehydration, as water leaves the cell to achieve equilibrium with the extracellular environment. If the rate of cooling is too rapid, water cannot pass quickly enough from the cell, and as the temperature continues to drop, it reaches a point when the intracellular solute concentration is not high enough to prevent the formation of ice crystals. Membrane permeability by cryoprotectants varies between developmental stages. While DMSO and PROH are frequently used for freezing early-cleavagestage embryos, propylene glycol (glycerol) is commonly used for blastocysts. All three intracellular

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agents have fairly small molecules that permeate cell membranes easily. In addition to these agents there are several extracellular substances that help dehydrate and protect cells. The most frequently used is sucrose, which possesses large, nonpermeating molecules and exerts an osmotic effect to aid in accelerated cell dehydration. Sucrose cannot be used alone but is often used in conjunction with standard permeating, intracellular cryoprotectants. On the other hand, most blastocyst freezing protocols have evolved from the published work of Yves Ménézo and co-workers.18,19 Cornell methods utilize PROH for embryo stages, and glycerol for blastocysts; sucrose aids cell dehydration. All specimens have been frozen in sterile cryovials within a cryoprotectant medium volume of 0.3 ml. A Planer series III biologic freezer (Kryo10-1.7; TS Scientific, Perkasie, PA) is utilized. The Cornell protocols have been amended from the early-published work in several ways to fit our current needs. Modifications include: 1. The base medium is a phase I sequential formulation, modified by 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES) buffers. 2. Extra macromolecules (protein) are added in the form of 0.5 g/l human serum albumin (5% HSA solution) and ~20% Plasmanate. 3. For blastocysts, the freezing cryoprotectant concentration is elevated to 10% and additional dilutions are included for the thawing process.

Embryo freezing Embryos are exposed to increasing concentrations of cryoprotective medium at room temperature: 0.5 mol/l PROH for 5 min, 1.0 mol/l PROH for 5 min, 1.5 mol/l PROH for 10 min, 1.5 mol/l PROH/0.2 mol/l sucrose for 10 s. They are then loaded into cryovials containing 1.5 mol/l PROH/0.2 mol/l sucrose. Cryovials are equilibrated for 15 min at room temperature before being cooled at a rate of −2.0°C/min until −7.0°C. They are held for 5 min, manual seeding is performed, and they are held for an additional 5 min. Cooling is continued at a rate of −0.3°C/min until −30°C. Cryovials are then plunged into liquid nitrogen.

Embryo thawing Cryovials are warmed in a 30°C water bath for 30–90 s and then held for 5 min at room temperature before embryos are removed. Embryos are taken through decreasing concentrations of cryoprotective medium: 1.0 mol/l PROH + 0.2 mol/l sucrose for 3 min, 0.75 mol/l PROH + 0.2 mol/l sucrose for 3 min, 0.5 mol/l PROH + 0.2 mol/l sucrose for 3 min, 0.25 mol/l PROH + 0.2 mol/l sucrose for 3 min (no PROH) + 0.2 mol/l sucrose for 3 min. Specimens are then washed thoroughly and incubated until intrauterine transfer.

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Blastocyst freezing Blastocysts are exposed to two concentrations of cryoprotective medium at room temperature: 5% glycerol solution for 10 min and 10% glycerol/0.2 mol/l sucrose solution for 10 min. They are then loaded into cryovials and cooled at a rate of −2.0°C/min until −7.0°C. Cryovials are held for 5 min, manual seeding is performed, and they are held for an additional 10 min. Cooling is continued at −0.3°C/min until −38°C. Cryovials are then plunged into liquid nitrogen.

Blastocyst thawing Cryovials are thawed at room temperature for 60 s before being warmed in a 30°C waterbath for 30–90 s (until all ice is removed). Blastocysts are removed from the cryovials and taken through decreasing concentrations of cryoprotective medium: 10% glycerol + 0.4 mol/l sucrose for 30 s, 5% glycerol + 0.4 mol/l sucrose solution for 3 min, 0.4 mol/l sucrose solution (no glycerol) for 3 min, 0.2 mol/l sucrose solution (no glycerol) for 2 min, and 0.1 mol/l sucrose solution (no glycerol) for 1 min. Specimens are then washed thoroughly and incubated until transfer. Blastocysts frozen on day 5 are incubated overnight; blastocysts frozen on day 6 are transferred the same day as thawing.

Replacement strategies in Cornell At Cornell, frozen–thawed conceptuses are replaced in either natural or programmed cycles. Natural cycles are not supplemented with progesterone unless there is an overwhelming reason to do so, and all women are treated in a prophylactic manner for 4 days with antibiotics and corticosteroids.

Natural cycle replacement (used in ovulatory cycles with normal concentrations of luteal phase progesterone) Supplemental progesterone is not administered unless medically indicated or unless the patient experienced a previous pregnancy failure using a nonsupplemented protocol. If administered, 200 mg of micronized progesterone is given vaginally twice or three times a day and continued until a negative pregnancy test 12–14 days after replacement or through week 7 if pregnant and confirmed by ultrasound investigation. Medrol (methylprednisolone; 16 mg/ day) and tetracycline (250 mg, four times a day) are administered for 4 days, beginning on the day of the luteinizing hormone (LH) surge for preembryos or beginning 2 days before transfer for blastocysts. Embryos are thawed 1 day after ovulation (2 days after LH peak and/or day after estradiol dip) and transferred on the day of thaw. Blastocysts are thawed 4 days after the LH peak and transferred on the following day (day-5 blastocysts) or

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thawed 5 days after LH peak and transferred on the same day (day-6 blastocysts).

Programmed cycle replacement (adequate suppression confirmed on day 2 of cycle) Luteal suppression is accomplished using 0.2 mg gonadotropin-releasing hormone agonist (GnRHa). This dosage is reduced to 0.1 mg starting on the predetermined day 1 of the cycle and maintained until day 15. Transdermal estrogen patches (Climara, 0.1 mg patch) are administered as follows: days 1–4, 0.1 mg every other day; days 5–8, 0.2 mg every other day; days 9–10, 0.3 mg every other day (depending on estradiol concentrations); days 11–14, 0.4 mg every other day; days 15+, 0.2 mg (two patches every other day, 7 weeks). Progesterone (50 mg intramuscularly) is administered beginning on day 15 after evaluating estrogen, progesterone, and endometrial parameters and judging them to be adequate. Progesterone is continued through 12 weeks’ gestation (weaned down starting week 9–11, depending on serum concentrations). Medrol (16 mg/day) and tetracycline (250 mg, four times a day) are administered beginning on day 17 for 4 days. Embryos are thawed on day 17 and transferred the same day; blastocysts frozen on day 5 are thawed on day 19 and transferred the following day; blastocysts frozen on day 6 are thawed on day 20 and transferred the same day.

Fig 20.1 Three embryos that were frozen longer than 5 years. Upon thawing, two of the three were completely intact (lower and right). The third displayed three surviving blastomeres of four total. The degenerative blastomere was removed through micromanipulative procedures. Following the transfer of these three conceptuses to a 42-year-old woman who had conceived in her fresh cycle, implantation failed to occur.

Results Embryos Embryos freeze well and implant at acceptable rates after thaw and transfer. Almost any cleavage-stage specimen can be frozen successfully, from 2-cell to blastocyst. Freezing the embryo is fairly convenient because there are no urgent timing considerations. In addition, information is known about both morphology and growth rate, allowing the selection of potentially viable conceptuses for either fresh transfer or storage. It has become extremely common in the past decade to choose embryos with the best morphology for fresh transfer, and to freeze others with acceptable morphology only after fresh selection has been made. Sometimes survival after thaw is difficult to evaluate because not all blastomeres endure the rigors of freezing and thawing. Dying blastomeres may be present amongst living ones, but these can be removed easily by aspirating them out through an artificial hole in the zona pellucida. Generally, an embryo possessing >50% viable blastomeres upon thaw is considered a survivor (Figs 20.1–20.6). There is no convincing evidence to suggest that the loss of one or two blastomeres is detrimental to 8-celled human or mouse embryos.25–27 Nonetheless, it has been reported that fully intact embryos demonstrate higher implantation rates than do partially intact ones.28

Fig 20.2 In this example, two of four embryos, stored for 861 days, survived thawing with all blastomeres intact. The remaining two conceptuses lost a single blastomere during the process. Degenerative cells were removed before intrauterine transfer to a 35-year-old woman. A singleton pregnancy was established and a healthy male child delivered.

Blastocysts Blastocysts have the advantage of possessing many cells. The loss of a few during freezing and thawing will not compromise the integrity of the entire specimen. This may be one reason why blastocysts have been frozen and thawed so successfully over the years

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Fig 20.5 Seven healthy appearing conceptuses photographed before freezing on day 3 after harvest. Fig 20.3 Four embryos photographed on day 3 after harvest, just before freezing was carried out.

Fig 20.6 Upon thawing four of the conceptuses shown in Fig 20.5 after 122 days, all survived. Fig 20.4 The same four embryos as in Fig 20.3 photographed a few hours after thawing, 68 days later. Two of these had degenerative blastomeres and fragments removed before the photograph was taken. The 37-year-old patient became pregnant after the transfer of these conceptuses, with two sacs and one fetal heart by ultrasound. A healthy female child was delivered.

in domestic animals for both research and commercial purposes. Blastocyst cryopreservation in the human was first reported by Cohen et al,5 using glycerol in a series of 10 increasing concentrations. Following that initial report, blastocyst freezing was only occasionally incorporated into clinical protocols because of the difficulties involved with maintaining high rates of blastocyst development in vitro. Through the 1990s, reports of clinical pregnancy after blastocyst thaw fell in the range of 10–30% per transfer,29 percentages not significantly better than results with earlier stages. Although several groups

reported freezing blastocysts quite successfully, early attempts often relied on coculture systems to support embryo growth.18,30,31 Today, the availability of sequential media has led to a dramatic increase in the practice of blastocyst freezing, and pregnancy rates well over 50% have been reported following the replacement of thawed blastocysts. Few reports have been published detailing the efficiency of blastocyst freezing after culture in sequential media. Langley et al describe a comparison of thawed day 3 embryos versus blastocysts during a 30month period.32 In this study, the survival rate was higher for blastocysts and the implantation rate was doubled (21.9 versus 10.1%, 72 blastocyst cycles). In 2002, Behr et al reported a 36% clinical pregnancy rate and 16% implantation rate for thawed blastocysts from 64 cycles.33 Given these few peer-reviewed reports generated after extended culture in sequential media, there may not be adequate evidence to support

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Table 20.1 Pregnancy and implantation by stage of development*

Stage transferred

Clinical pregnancies/ transfer (%)

Implanted/ no transfer (%)

Embryos only Blastocysts only

369/886 (41.7)a 457/868 (52.7)b

590/3493 (16.9)c 623/1792 (34.8)d

p <0.0001 in favor of blastocysts for comparisons a versus b, c versus d. *Embryos and blastocysts frozen January 1995 to December 2006 (chi square).

the concept that blastocysts are now optimal for human freezing trials. Nonetheless, the Cornell program has benefited greatly from the adoption of blastocyst freezing protocols (Table 20.1). While acceptable clinical pregnancy rates of >40% have been realized after freezing and thawing cleavage-stage embryos, much higher rates have been established using blastocysts (>50%) without any concomitant drop in the number or proportion of patients having conceptuses frozen. Nearly one in four women under age 40 have had blastocysts frozen after undergoing day-3 transfers, and 60% of women undergoing day-5 transfers have had at least one blastocyst cryopreserved on day 5 or day 6. Well over 7000 blastocysts have been frozen in the last 6 years, though only one-quarter of these have been thawed, since so many of the patients involved have not returned for a second child after becoming pregnant from their fresh cycles. Most of the blastocysts frozen in the Cornell program are generated following the fresh transfer of day-3 conceptuses. After intrauterine transfer, the remaining viable embryos are examined each day for 2 or 3 additional days to evaluate their suitability for freezing. This has been termed the post-transfer observation period. Blastocysts forming on either day 5 or day 6 are cryopreserved for future use. Only rarely and under special circumstances have day-7 conceptuses been frozen. The survival rate for thawed blastocysts is very stable at 77%. Clinical pregnancy per cycle with only blastocysts thawed and replaced is 53%; the ongoing or delivered rate is 45% and the implantation rate is 35%. Pregnancy rates are not different whether blastocysts are replaced in either natural or programmed cycles. Furthermore, pregnancy rates with blastocysts are stable across all maternal ages; 41/90 women (46%) over the age of 41 have established clinical pregnancies, although their miscarriage rate is more than double that observed for younger women. It is generally assumed that blastocysts that develop in a timely manner in vitro are of better quality than those that develop more slowly. However, this study and an earlier retrospective analysis of blastocyst thaw outcomes from our program demonstrate otherwise. In 154 consecutive patients returning for

thawed blastocysts, 60 patients received a transfer of day-5 frozen–thawed blastocysts and 94 patients underwent transfer with day-6 blastocysts. No significant differences were observed between groups for patient age, blastocyst survival rates, average number of blastocysts replaced, morphology of thawed blastocysts, clinical pregnancy rates, ongoing pregnancy rates, or implantation rates. These findings are identical to those presented in an earlier study from this center34 and others.10,33 While it is intuitive to assume that embryos reaching the blastocyst stage faster (day 5) might be ‘healthier’ than their day-6 counterparts, these data suggest that the rate of development may not be crucial to subsequent post-thaw success. Surprisingly, this is in direct conflict with reports of fresh transfer using day-5 and day-6 blastocysts, where pregnancy has been observed to be significantly lower with slower-growing day-6 conceptuses.35 Also, in contrast to our work, Marek et al carried out a study comparing outcomes from 127 thawed blastocyst cycles where blastocysts were frozen on day 5 or day 6.36 Survival rates postthaw were good for both groups, but the clinical pregnancy rate per thaw (50% vs 29%, respectively), ongoing pregnancy rate per thaw (43% vs 23%), and implantation rate (34% vs 15%) were all significantly higher for day-5 blastocysts. Why these results are so different from our own is not clear. We, like others, observed that blastocysts with a high probability of survival after thaw acted as perfect osmometers, shrinking, re-expanding, and swelling in accordance with their osmotic environment.37 One uneasy task immediately after thawing was to determine that a blastocyst had indeed survived, since it often presented a contracted state for up to several hours after reincubation in culture medium. It has been our experience that blastocysts that shrink appropriately in response to cryoprotective agents and exhibit contracted, healthy-appearing cells after thaw do quite well in their ability to survive the rigors of freezing and thawing (Figs 20.7–20.13). All pregnancies were established with blastocysts that had at least begun to re-expand. Two cycles involving the freezing of noncavitated morulae failed to generate pregnancies.

Calculating pregnancy potential from embryo and blastocyst stages Of the many tribulations associated with running a cryopreservation program, one of the most frustrating is that embryologists cannot reap the fruits of their labor (pregnancy after thawing) until months or years have passed. It is common for patients to wait for some time before returning for a thaw attempt after a negative fresh cycle, or to delay 2 or more years after the birth of a child. This situation gives rise to special problems in tracking results during a given freezing period, and makes it difficult to identify the efficiency of a new protocol.

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Fig 20.7 Nonviable inner cell mass despite blastocoel reexpansion after thaw.

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Fig 20.9 Same blastocyst as in Fig 20.8 after thawing 56 days later; most cells appear viable. This blastocyst was transferred and implanted, and a healthy female child was delivered. Note that the blastocyst was thawed almost 1 full day before replacement, and that the trophectoderm appears quite different after prolonged culture, being made up of many more cells.

Fig 20.8 Expanded blastocyst immediately before freezing; inner cell mass is large and trophectoderm is sparse.

There are three common ways to analyze freezing–thawing results:

Fig 20.10 Blastocyst known to have implanted after freeze, thaw, and transfer. After cryostorage for 57 days, this blastocyst was thawed and led to the birth of a healthy female child.

1. By calculating pregnancy rate per thaw attempt. 2. By calculating pregnancy rate per cycle with transfer of thawed conceptuses. 3. By calculating an augmented pregnancy rate per cycle with freezing based on fresh pregnancy plus thawed pregnancy.

In the last analysis, augmented pregnancy rate refers to the actual cumulative pregnancy rate achieved by patients upon combining pregnancies established from both fresh and thawed transfers:

This last method has been discussed in detail in numerous publications.38–42

1. The base fresh pregnancy rate is defined as the number of clinical pregnancies established after

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Fig 20.11 Blastocyst known to have implanted after freeze, thaw, and transfer. After cryostorage for 558 days, an ongoing pregnancy was established with one fetal heart.

the transfer of noncryopreserved embryos over the number of noncryopreserved (fresh) transfer cycles; i.e. (250/500) × 100 = 50%. 2. The augmented pregnancy rate is defined as the actual number of clinical pregnancies generated by the transfer of noncryopreserved embryos plus the actual number of clinical pregnancies generated by the transfer of thawed embryos in cycles failing to become pregnant with fresh transfer, over the number of transfer cycles; i.e. [(250 + 125)/500] × 100 = 75%. 3. A projected augmented pregnancy rate can be defined as the actual number of clinical pregnancies generated by the transfer of noncryopreserved embryos plus the actual number of clinical pregnancies generated by the transfer of thawed embryos in cycles failing to become pregnant with fresh transfer, plus the number of clinical pregnancies expected from the potential transfer of conceptuses still in cryostorage for patients not yet pregnant from fresh or thawed attempts (this last calculation uses the thawed pregnancy rate established to date) over the total number of cycles with a transfer; i.e. [(250 + 12 + 525)/500] × 100 = 80%. The validity of reporting this last projected cumulative pregnancy rate is open to criticism because of its reliance on past performance and assumptions that future results will be similar. Using the augmented pregnancy model described here, Cornell results are shown in Table 20.2 where blastocysts appear to be an optimal stage for freezing.

Children born following cryopreservation and thawing Fig 20.12 Two blastocysts known to have implanted after freeze, thaw, and transfer. After cryostorage for 84 days, two healthy male children were subsequently delivered.

Cryopreservation has no apparent negative impact on perinatal outcome and does not appear to affect adversely the growth or health of children during infancy or early childhood.43 Furthermore, the available data do not indicate an elevation in congenital malformations for children born after freeze–thaw procedures.44–46 While it remains unclear if freezing

Table 20.2 Cumulative pregnancies, January 1995 to December 2006

Fig 20.13 Two blastocysts known to have implanted after freeze, thaw, and transfer. After cryostorage for 68 days, two fetal hearts were documented for this pregnancy that was ongoing at the time of writing.

Fresh base clinical pregnancies/transfer True augmented cumulative Projected total cumulative *p <0.05 (chi square).

Embryos only

Blastocysts only

frozen (%)

frozen (%)

68.5

66.9

73.3

76.4

74.5*

81.3*

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poses long-term risks to children so conceived, there is no direct evidence thus far to raise concern.

General considerations Before beginning a cryopreservation program in an ART setting, a few general considerations should be taken into account. First and foremost is the adequate training of personnel. This training should be administered by an experienced embryologist who is skilled and fully versed in cryopreservation techniques. The trainee should already have a basic background in clinical embryology and should be comfortable with handling human oocytes and embryos under the stereoscope. Whenever possible it is also a good idea to have a back-up biologic freezer. This will prevent loss of specimens in the unfortunate event that a machine malfunctions. Furthermore, in today’s busy laboratory, it is not uncommon to undertake day-1, day-3, and day-5 freezes all in the same day. With additional biologic freezers, time spent in the laboratory becomes more efficient as well as reducing the wear and tear on any individual machine. It is important to have at least one back-up source for liquid nitrogen delivery. If for some reason a primary source fails (faulty valve or level indicator), or one experiences delivery problems from the vendor, it is a good idea to have an alternative. In case of a fire or other type of unforeseen disaster, an emergency or hazard plan should be outlined and put into place. While it may be impossible to save conceptuses that are in the process of being cryopreserved or thawed, it is important to preserve the larger number in storage. Such a plan should detail the location of storage tanks, exactly what they look like, what they contain, their count, and how they should be evacuated safely from a dangerous location. Copies of the plan should be given to the program administrator, building maintenance personnel, and any security staff involved with building evacuation. Planning in advance will make it easier for rescue workers to find and identify the storage tanks in the event of true hazard. An emergency power supply system is also a serious consideration. The need for an expensive back-up power supply may depend on the facility (some buildings have back-up power sources built-in) and the reliability of the local power company. For most laboratories it is wise to invest in an uninterrupted power supply (UPS) system that sits next to the biologic freezer(s). This system consists of a rechargeable battery that is connected to the freezer and will automatically power on if there is an electrical outage. One of the more mundane but essential tasks in running a cryopreservation program is the scheduling of daily inspection and documentation of liquid nitrogen levels in the storage tanks. These inspections should be carried out two to three times a week at regular intervals. Performing the task on the same days each week enables one to detect easily any leaks that

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tanks develop over time. It is also important, and in most IVF programs mandatory, to install alarm systems to monitor the levels of liquid nitrogen in storage tanks. If liquid nitrogen levels drop dangerously during off hours, the system should be able to alert a predefined list of people by phone or beeper. It is also a good idea to have at least one empty, fully charged tank at the ready in case of such an emergency.

Quality control issues A good-quality control program is essential to every aspect of assisted reproductive technologies, and cryopreservation is no exception.

Preventive maintenance A good preventive maintenance schedule is essential. We recommend having a specialist examine each biologic freezer and perform recalibration procedures twice a year. These duties are usually part of a service contract provided by the dealer.

Testing of freezing and thawing solutions Each new batch of culture medium, whether prepared in-house or purchased from outside, must pass rigid quality control testing. This is achieved by means of endotoxin testing, a human sperm survival bioassay, and a mouse embryo bioassay. Any problematic batch of medium, medium component, or lot of plasticware is immediately discarded and replaced.

Endotoxin testing and interpretation Bacterial endotoxins are common contaminants of materials and solutions used in culture. A sensitive assay to quantitate endotoxin contamination levels is necessary. Evidence suggests that endotoxins may be responsible for much of the variability in cell culture that is often associated with changes in batch or formulation of media. The ubiquitous Gram-negative bacteria that produce these endotoxins can and do contaminate a variety of materials used to cultivate cells in vitro. A sample of each culture medium is sent to an independent testing center each week. For our purposes, only values <0.03 EU are acceptable when using the limulus amebocyte lysate (LAL) assay.

Human sperm survival assay Each batch of culture medium is tested using human sperm (the same donor is used for each assay). The sperm sample is split into as many fractions as the number of assays to be performed, and prepared using a culture medium lot currently in use (control) and the new lot to be tested (test). A 24-hour and a 48-hour motility and progression assessment are performed on a sample prepared by standard swim-up procedures.

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A motility of >70% and a progression of >40% type ‘a’ motility are considered acceptable. A comparison should be made between the control sample and the test sample; any significant difference between the two should be interpreted as problematic.

Mouse embryo bioassay The in vitro development of mouse 1-cell or 2-cell embryos to the hatched blastocyst stage has traditionally been used as a quality control system in IVF laboratories. Based on a large body of evidence, we believe this test to be less reliable than the human sperm survival assay, and the results unrelated to the pattern of development of human embryos in vitro. Mouse embryos easily reach the blastocyst stage in the presence or absence of exogenous protein, and the great majority of these blastocysts hatch when provided with protein. Slight variations in culture media composition or minute amounts of endotoxin do not appear to affect the rate of blastocyst formation or hatching. As a result, mouse embryo quality control is used in our laboratories only as a broad toxicity testing method.

Logging seeding temperatures It is important to keep a log of seeding temperatures for each individual freezing run. Tracking these values helps to alert personnel if the machine’s seeding temperature begins to drift out of an acceptable range.

Complications Trouble-shooting procedures; i.e. ‘What if…’ What if the source liquid nitrogen tank empties or malfunctions during a freezing run? If the liquid nitrogen source tank runs dry or does not possess adequate pressure to supply the freezing unit, most biologic freezers will sound an alarm. On our Planer unit (K10–1.7; TS Scientific, Perkasie, PA), an audible alarm is triggered and the display panel flashes ‘control deviation.’ The first thing one should do is examine the display to determine if the chamber temperature is beginning to rise, and check the plotted temperature graph. If the unit is not receiving liquid nitrogen, the bursts of sound normally heard as liquid nitrogen rushes into the chamber will be quite different. If the temperature is rising or the unit sounds much different, first inspect the feed tank. If it is empty, change it immediately. The Planer will automatically adjust and bring the chamber to its proper temperature once the flow of liquid nitrogen is restored. If the tank is full, check the pressure gauge. It is possible that the safety valve that allows excess pressure to escape will become frozen in the open position. Once the pressure drops too low, the tank will not be able to feed the unit. In this case, again, change the tank immediately. One may be able to

thaw the stuck valve by applying hot water, but all malfunctions should be documented and reported.

What if an embryo cannot be located after thawing a cryovial or straw? Thoroughly re-examine the contents of the freezing vessel. Pay special attention to any small bubbles floating on the surface as embryos may become attached. It is also helpful to adjust the contrast on the stereomicroscope and gently tap the sides of the vessel and receiving dish while looking. If the specimen still cannot be visualized, flush the straw or fill the empty cryovial with medium kept at room temperature. It is preferable to flush with the medium which is used for the first thawing dilution. Fill the cryovial using a pipette and wash the contents up and down, being careful not to create bubbles. Withdraw the contents using the pipette, transfer contents to a fresh Petri dish, and check for the missing specimen. If this fails, one can try half-filling the cryovial, capping it, and gently agitating. More aggressive agitation follows if the embryo is still not located. Doing so may release an embryo stuck to its inner walls.

What should one do if after loading an embryo into a cryovial it cannot then be visualized under the stereomicroscope? First, try flicking the cryovial gently with a finger to set its contents in motion. This should enable easier visualization of the embryo. If this is unsuccessful, examine the inner walls of the cryovial for small medium droplets: occasionally, if the embryo is too close to the opening of the pipette and the inner wall is touched with the tip, the embryo will be dislodged along with a droplet of the medium. If the embryo is located on the inner wall, it may be washed off or picked up with the pipette, or the cryovial can be filled and emptied. Should none of these methods be successful for locating the embryo, be sure to check the walls of the loading pipette visually.

What if one loses track of an embryo as it is moved through freezing or thawing dilutions? Tapping the side of the vessel and adjusting the contrast on the stereomicroscope may assist visualization. Examine any bubbles that might be in the vessel and rinse and check the pipette. It is also helpful to ask another experienced embryologist to examine the materials. If all else fails, draw the entire contents of the dish/well into a fresh pipette and transfer to a large Petri dish for examination.

What if a cryovial drops into the freezing unit during seeding or during a freezing or thawing run? When putting cryovials into the freezing unit, make certain that they fit tightly onto their freezing canes.

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With extended use, the canes do not grasp the vials as firmly as before, and there is a danger of vials dropping into the chamber during a run. To avoid this happening, simply pinch (remold) the clasping portion of the cane so that it holds the vial more tightly. Also, when placing canes into the chamber and when removing them, a good rule to follow is always to slowly slide the back of the cane along the edge of the opening. This ensures that a vial will not catch on the chamber edge and possibly dislodge it. However, if a cryovial does become dislodged from its cane, there are steps to take to resolve the problem. If the cryovial has already been seeded and it is resting on the bottom without interfering with the mechanics of the machine, it may be prudent to wait until the end of the run to retrieve it. This is especially true if other samples are being frozen or if another patient’s samples are being cryopreserved simultaneously. However, if the cryovial has not yet been seeded, it is mandatory to retrieve it, as failure to introduce the seed will destroy the specimen. This is a very tricky procedure and should only be attempted by an experienced embryologist. With the freezing unit continuing to run, unlock the cover and remove the lid. It may be easier if one person lifts the lid while another person attempts to retrieve the lost cryovial. The best tool to use is a long thin grabber which can be purchased at most hardware stores. Needless to say, this should be purchased in advance of setting up the cryopreservation program. The cryovial should be retrieved as quickly as possible since the remaining specimens will warm rapidly. The difficulty in retrieval lies in the fact that at the bottom center of the chamber (Planer unit) there is a spinning fan blade. Touching the blade with any instrument may damage the blade and compromise the remainder of the run. Reaching in with a hand is not advised as the blade can seriously injure fingers. Once the cryovial is retrieved, it must be placed securely back onto the freezing cane and the temperature of the chamber should be checked and documented. It may take a little extra time for the chamber to cool to the seeding temperature. When it does, manually seed the vial and any others still not properly seeded.

Exploding cryovials During the freezing process the O-ring that sits between the cryovial and the cap shrinks, becomes rigid, and may cause the seal to loosen. Under these circumstances, a slight vacuum is formed within the cryovial that allows air or liquid nitrogen vapor to enter. As the cryovial is warmed during a subsequent thaw, the O-ring expands and becomes more malleable, creating a tighter seal. The cold air/vapor within the vial begins to expand with the rising temperature. If the pressure inside the vial becomes too great for the O-ring to contain, the vial may expel a burst of air. If this happens while the vial is sitting in

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a rack waiting to be examined, it may ‘jump’ out of the rack, allowing the medium inside the vial to splash around and making the embryo much more difficult to locate. One way to avoid this situation is to loosen the cap of the vial immediately after it has been thawed. This should release any built-up pressure inside the vial without disrupting its contents.

Tips for success Preparing media: appropriate delivery of cryoprotective solutions It is important to ensure that the appropriate volume of cryoprotectant is delivered to freezing and thawing media made on-site. Failing to prepare solutions properly can result in reduced survival and pregnancy rates after thawing. Cryoprotectants, especially glycerol, are very viscous and tend to adhere to the inside and outside walls of the delivery pipette. To ensure that all the cryoprotectant solution is delivered to the medium one must rinse the pipette repeatedly. Even a relatively small reduction in the amount of cryoprotectant can influence the success of a frozen–thawed cycle.

Moving through dilutions When moving from one dilution to another, aspirate an appropriate volume of medium into the pipette before picking up the conceptus in an effort to avoid creating bubbles, but not so much as to interfere with the dilution concentrations. Make sure to wash the conceptus several times in each new dilution. This also applies when moving thawed embryos into fresh culture media. Washing them in a few different droplets before placing them in a clean, fresh droplet will help to ensure that most of the cryoprotectant has been removed.

Safety: cryopreserving multiple patients simultaneously When cryopreserving multiple patients it is best to prepare a separate rack for each. Each rack holds one patient’s dilutions, culture dishes, and cryovials. This helps to ensure that samples stay separated and will make multiple freezes more manageable. Never place culture dishes or dilutions from different patients on the microscope stage simultaneously. During each step in the process one should verify a patient’s name and repeat it aloud.

Summary Most highly successful ART centers in the United States and around the world offer as part of their treatment services a well-organized and successful cryopreservation program. Organization and success are

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most often reflected in a high overall cumulative pregnancy rate, which takes into account both fresh and frozen–thawed pregnancies per cycle.47 While the benefits of such a program to the ART patient are obvious, there are other smaller groups of patients who also benefit greatly from cryopreservation. Patients at risk for ovarian hyperstimulation can have all of their conceptuses frozen, which greatly reduces their risk of severe clinical symptoms should they become pregnant.48,49 In addition, patients with breast or other cancers can opt for IVF followed by cryopreservation to preserve fertility.50,51 Final consideration must be given to the reduction of costs associated with frozen embryo transfer compared with fresh cycles.52 As medical costs escalate and costs associated with newborn care increase, particularly in cases of multiple births, cryopreservation offers a valuable and less expensive alternative for ART patients. Using embryo and blastocyst freezing, patients can choose to reduce the number of embryos transferred during a fresh cycle, thus reducing their chances and risks of multiple pregnancy. Adding to this benefit, the overall costs of a thaw cycle are considerably lower than those of a fresh cycle in that cryo–thaw patients do not usually incur extensive hospital, clinical, and laboratory fees. For these reasons, the addition of cryopreservation as an alternative for ART patients in today’s world has become of primary importance. The value of cryopreserving embryos and blastocysts for future thaw and transfer is an important consideration of every IVF program. The convergence of two factors, a higher pregnancy rate and a lower multiple gestation rate, can be managed effectively through the establishment of a successful cryopreservation program.

References 1. Fugger EF. Clinical status of human embryo cryopreservation in the United States of America. Fertil Steril 1989; 52: 986–90. 2. Society for Assisted Reproductive Technology; American Society for Reproductive Medicine. Assisted reproductive technology in the United States: 2001 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology registry. Fertil Steril 2007; 87: 1253–66. 3. Hoffman DI, Zellman GL, Fair CC, et al. Cryopreserved embryos in the United States and their availability for research. Fertil Steril 2003; 79: 1063–9. 4. Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eightcell embryo. Nature (London) 1983; 305: 707–9. 5. Cohen J, Simons RF, Edwards RG, et al. Pregnancies following the frozen storage of expanding human blastocysts. J In Vitro Fert Embryo Transf 1985; 2: 59–64. 6. Karlstrom PO, Bergh T, Forsberg AS, et al. Prognostic factors for the success rate of embryo freezing. Hum Reprod 1997; 12: 1263–6.

7. Toner JP, Veeck LL, Muasher SJ. Basal follicle-stimulating hormone level and age affect the chance for and outcome of pre-embryo cryopreservation. Fertil Steril 1993; 59: 664–7. 8. Mandelbaum J, Junca AM, Plachot M, et al. Human embryo cryopreservation, extrinsic and intrinsic parameters of success. Hum Reprod 1987; 2: 709–15. 9. Schalkoff ME, Oskowitz SP, Powers RD. A multifactorial analysis of the pregnancy outcome in a successful embryo cryopreservation program. Fertil Steril 1993; 59: 1070–4. 10. Shoukir Y, Chardonnens D, Campana A, et al. The rate of development and time of transfer play different roles in influencing the viability of human blastocysts. Hum Reprod 1998; 13: 676–81. 11. Oehninger S, Mayer J, Muasher S. Impact of different clinical variables on pregnancy outcome following embryo cryopreservation. Mol Cell Endocrinol 2000; 169: 73–7. 12. Van den Abbeel E, Camus M, Joris H, Van Steirteghem A. Embryo freezing after intracytoplasmic sperm injection. Mol Cell Endocrinol 2000; 169: 49–54. 13. Loh SK, Leong NK. Factors affecting success in an embryo cryopreservation programme. Ann Acad Med Singapore 1999; 28: 260–5. 14. Gardner DK, Leese HJ. Assessment of embryo viability prior to transfer by the noninvasive measurement of glucose uptake. J Exp Zool 1987; 242: 103–5. 15. Lane M, Maybach JM, Gardner DK. Addition of ascorbate during cryopreservation stimulates subsequent embryo development. Hum Reprod 2002; 17: 2686–93. 16. Testart J, Lassalle B, Belaisch-Allart J, et al. High pregnancy rate after early human embryo freezing. Fertil Steril 1986; 46: 268–72. 17. Testart J, Lassalle B, Belaisch-Allart J, et al. Cryopreservation does not affect future of human fertilised eggs. Lancet 1986; 2: 569. 18. Ménézo Y, Nicollet B, Herbaut N, Andre D. Freezing cocultured human blastocysts. Fertil Steril 1992; 58: 977–80. 19. Ménézo YJ, Nicollet B, Dumont M, et al. Factors affecting human blastocyst formation in vitro and freezing at the blastocyst stage. Acta Eur Fertil 1993; 24: 207–13. 20. Gardner DK, Lane M, Stevens J, Schoolcraft WB. Changing the start temperature and cooling rate in a slow-freezing protocol increases human blastocyst viability. Fertil Steril 2003; 79: 407–10. 21. Choi DH, Chung HM, Lim JM, et al. Pregnancy and delivery of healthy infants developed from vitrified blastocysts in an IVF–ET program. Fertil Steril 2000; 74: 838–9. 22. Yokota Y, Sato S, Yokota M, et al. Successful pregnancy following blastocyst vitrification: case report. Hum Reprod 2000; 15: 1802–3. 23. Yokota Y, Sato S, Yokota M, et al. Birth of a healthy baby following vitrification of human blastocysts. Fertil Steril 2001; 75: 1027–9. 24. Mukaida T, Nakamura S, Tomiyama T, et al. Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertil Steril 2001; 76: 618–20. 25. Veiga A, Calderon G, Barri PN, Coroleu B. Pregnancy after the replacement of a frozen–thawed embryo

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26.

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with less than 50% intact blastomeres. Hum Reprod 1987; 2: 321–3. Hartshorne GM, Elder K, Crow J, et al. The influence of in vitro development upon post-thaw survival and implantation of cryopreserved human blastocysts. Hum Reprod 1991; 6: 136–41. Rulicke T, Autenried P. Potential of two-cell mouse embryos to develop to term despite partial damage after cryopreservation. Lab Anim 1995; 29: 320–6. Van den Abbeel E, Camus M, Van Waesberghe L, et al. Viability of partially damaged human embryos after cryopreservation. Hum Reprod 1997; 12: 2006–10. Kaufman RA, Ménézo Y, Hazout A, et al. Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles. Fertil Steril 1995; 64: 1125–9. Freitas S, Le Gal F, Dzik A, et al. Value of cryopreservation of human embryos during the blastocyst stage. Contracept Fertil Sex 1994; 22: 396–401. Ménézo YJ, Ben Khalifa M. Cytogenetic and cryobiology of human cocultured embryos: a 3-year experience. J Assist Reprod Genet 1995; 12: 35–40. Langley MT, Marek DM, Gardner DK, et al. Extended embryo culture in human assisted reproduction treatments. Hum Reprod 2001; 16: 902–8. Behr B, Gebhardt J, Lyon J, Milki AA. Factors relating to a successful cryopreserved blastocyst transfer program. Fertil Steril 2002; 77: 697–9. Clarke RN, Bodine R, Zaninovic N. A comparison of post-thaw survival and pregnancy rates in day 5 and 6 frozen–thawed human blastocysts. Presented at the 58th Annual Meeting of the American Society for Reproductive Medicine, Seattle, WA, October 2002, 78(Suppl), 1001, S12. Shapiro BS, Richter KS, Harris DC, Daneshmand ST. A comparison of day 5 and day 6 blastocyst transfers. Fertil Steril 2001; 75: 1126–30. Marek DM, Langley MT, McKean C, et al. Frozen embryo transfer FET of day 5 blastocyst embryos compared to transfer of day 6 blastocyst embryos. Fertil Steril 2000; 74: S52–3. Kaidi S, Donnay I, Lambert P, et al. Osmotic behavior of in vitro produced bovine blastocysts in cryoprotectant solutions as a potential predictive test of survival. Cryobiology 2000; 41: 106–15. Veeck LL, Amundson CH, Brothman LJ, et al. Significantly enhanced pregnancy rates per cycle through cryopreservation and thaw of pronuclear stage oocytes. Fertil Steril 1993; 59: 1202–7.

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39. Jones HW Jr, Veeck LL, Muasher SJ. Cryopreservation: the problem of evaluation. Hum Reprod 1995; 10: 2136–8. 40. Jones HW Jr, Jones D, Kolm P. Cryopreservation: a simplified method of evaluation. Hum Reprod 1997; 12: 548–53. 41. Jones HW Jr, Out HJ, Hoomans EH, et al. Cryopreservation: the practicalities of evaluation. Hum Reprod 1997; 12: 1522–4. 42. Schnorr JA, Muasher SJ, Jones HW Jr. Evaluation of the clinical efficacy of embryo cryopreservation. Mol Cell Endocrinol 2000; 169: 85–9. 43. Wennerholm UB, Albertsson-Wikland K, Bergh C, et al. Postnatal growth and health in children born after cryopreservation as embryos. Lancet 1998; 351: 1085–90. 44. Wada I, Macnamee MC, Wick K, et al. Birth characteristics and perinatal outcome of babies conceived from cryopreserved embryos. Hum Reprod 1994; 9: 543–6. 45. Tarlatzis BC, Grimbizis G. Pregnancy and child outcome after assisted reproduction techniques. Hum Reprod 1999; 14(Suppl 1): 231–42. 46. Wennerholm WB. Cryopreservation of embryos and oocytes: obstetric outcome and health in children. Hum Reprod 2000; 15(Suppl 5): 18–25. 47. Veeck LL. Does the developmental stage at freeze impact on clinical results post-thaw? Reprod Biomed Online 2003; 6: 367–74. 48. Pattinson HA, Hignett M, Dunphy BC, Fleetham JA. Outcome of thaw embryo transfer after cryopreservation of all embryos in patients at risk of ovarian hyperstimulation syndrome. Fertil Steril 1994; 62: 1192–6. 49. Queenan JT Jr. Embryo freezing to prevent ovarian hyperstimulation syndrome. Mol Cell Endocrinol 2000; 169: 79–83. 50. Brown JR, Modell E, Obasaju M, King YK. Natural cycle in vitro fertilization with embryo cryopreservation prior to chemotherapy for carcinoma of the breast. Hum Reprod 1996; 11: 197–9. 51. Oktay K, Buyuk E, Davis O, et al. Fertility preservation in breast cancer patients: IVF and embryo cryopreservation after ovarian stimulation with tamoxifen. Hum Reprod 2003; 18: 90–5. 52. Van Voorhis BJ, Syrop CH, Allen BD, et al. The efficacy and cost effectiveness of embryo cryopreservation compared with other assisted reproductive techniques. Fertil Steril 1995; 64: 647–50.

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21 The human embryo: vitrification Zsolt Peter Nagy, Gábor Vajta, Ching-Chien Chang, Hilton Kort

Overview Embryo cryopreservation is an essential tool in assisted reproduction. The option to freeze gametes and embryos provides unique possibilities for patients with various indications. After a review on some of the technical details of embryo freezing the idea of vitrification compared to slow freezing is discussed. It is recognized that the slow freezing technique is still overwhelmingly applied in most human in vitro fertilization (IVF) centers, but there is mounting evidence demonstrating the benefit of vitrification. Theoretical and practical examples are now sufficient to convince the embryologist that the time has arrived to switch to the vitrification technique.

Introduction The cryopreservation technology has existed for a long time and has been applied successfully in the context of reproductive cell and embryo preservation. There is a well-demonstrated difference among different species; many innovative vitrification techniques have been introduced and predominantly used in some animals, while in humans the more ‘traditional’ approaches are usually applied. Additionally, there are significant differences related to the gender of origin of the reproductive tissue: male gametes are readily freezable and provide excellent outcomes, whereas female gametes perform much poorer during cryopreservation. Fundamental morphological and physiological differences explain the wide gap in the outcome; however, in humans, it is mainly women who would truly need and benefit from an efficient gamete cryopreservation technology. Embryo cryopreservation, on the other hand, is regarded as relatively efficient, although it still requires further improvement. To resolve technical problems, considerable efforts have been expended in oocyte and embryo cryopreservation, both in the animal and human fields. Initial diverse approaches and inconsistent results have eventually converged and led to an alternative and

very promising strategy. Primarily, slow freezing, in spite of its well-known limitations, has become highly standardized with a considerable industrial and commercial background. Recently, however, the alternative technique for cryopreservation, i.e. vitrification, is gaining well-deserved attention. This chapter attempts to summarize the main features of this progress and to outline the future perspectives. According to the authors’ own experiences and the evidence provided by the scientific literature, it appears that slow freezing has little if any future in the field of embryology. Terms and definitions used in cryobiology are reviewed excellently by Shaw and Jones.1 The principles of cryobiology in reproductive medicine have been reviewed by Fuller and Paynter2 and others.3–5

Approaches for embryo cryopreservation The first successes of mammalian embryo cryopreservation occurred in the 1970s6–10 and the first human pregnancies were achieved relatively soon after.11,12 During the past decades, two major groups of methods can be separated: slow freezing and vitrification. Storage, warming, and rehydration, i.e. removal of cryoprotectants, differ only slightly between the two procedures (with some exceptions), the main difference being in the addition of cryoprotectants and cooling rates. To demonstrate clearer the principles of vitrification, slow freezing is first reviewed briefly. Slow freezing creates a delicate balance between various damaging factors, including ice crystal formation, fracture, and toxic and osmotic damage. Embryos and oocytes are typically equilibrated in 1–2 mol/l solutions of permeable and nonpermeable cryoprotectants, loaded into 0.25 ml straws, sealed, and cooled relatively rapidly to −6°C by placing the straws into the controlled-rate freezer. At −6°C, seeding, i.e. ice crystal formation, is induced in the solution, preferably far from the embryo. The subsequent steps are entirely or almost entirely performed by machine. There are slight variations in the subsequent cooling rates, but values are between 0.3 and 1°C/min. The controlled rate cooling then continues to around

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−30°C. At these temperatures, straws are immersed in liquid nitrogen for final cooling and storage. In slow freezing, the toxic and osmotic damage caused by the relatively low concentration of cryoprotectant solutions may not be too serious. However, this concentration is insufficient to avoid ice crystal formation; therefore, an additional manipulation is required to minimize the damage. It is the slow cooling and seeding that result in controlled growth of ice in the extracellular solution; consequently, a considerable increase of the concentration of ions, macromolecules, and other components, including cryoprotectants, occurs in the remaining fluid. The slow rate of the procedure allows solution exchange between the extracellular and intracellular fluids without serious osmotic effects and deformation of the cells (this fact is reflected in the other name of the procedure – equilibrium freezing13). Vitrification is the alternative approach to slow freezing. It may be regarded as a radical approach, as one of the main sources of injuries, ice crystal formation, is entirely eliminated. However, a negative consequence of this strategy is the increased probability of other forms of injury except for those caused by ice crystal formation. To achieve vitrification of solutions, a radical increase of either the cooling rates or the concentration of cryoprotectants (or both) is required. The higher the cooling rate, the lower the required cryoprotectant concentration is, and vice versa. The balance required in vitrification is between (1) establishment of a safe system for maximal and reliable cooling (and warming) rates while avoiding consequent damage, including fracture of the zona pellucida or the cells, and (2) elimination or minimization of the toxic and osmotic effects of high cryoprotectant concentrations needed to obtain and maintain the glass-like solidification. Cell shrinkage caused by nonpermeable cryoprotectants and the incomplete penetration of permeable components may cause a relative increase of intracellular concentration of macromolecules that is enough to hamper intracellular ice formation. Accordingly, vitrification belongs to the group of nonequilibrium cryopreservation methods. It should also be mentioned that there is a small, rather controversial group of cryopreservation techniques called ultrarapid freezing where ultrarapid cooling is applied with cryoprotectant concentrations insufficient to establish vitrification.7,14–16 This approach has been established entirely empirically and does not meet the supposed requirements of cryopreservation in embryology. In spite of the definite signs of ice formation in the solution, under certain circumstances embryos and oocytes may survive and develop.17,18 The general explanation for this phenomenon is that extracellular ice formation does not necessarily result in intracellular freezing, and that the former is less harmful for the cells; even a moderate ice formation inside the embryos or oocytes may be tolerated and may allow normal development following regeneration. Probably as the consequence of the uncontrollable processes, the results of ultrarapid freezing techniques are generally

less predictable than those of slow freezing or vitrification, and due to this inconsistency, application is rather restricted. On the other hand, according to Kasai and Mukaida,3 some of the early experiments described as ultrarapid freezing were in fact vitrifications, including those published by Barg et al19 and Feichtinger et al20 for human embryos.

Cryopreservation-related injury and options for prevention Injuries may occur at all phases of the cryopreservation procedure. Understanding the causes and mechanisms of damage may help the development of cryopreservation methods to avoid lethal or irreversible injuries. During cooling, three types of damage may be distinguished, according to the different temperature ranges the cells pass through: 1. At relatively high temperatures between +15 and −5°C, the chilling injury is the major factor, damaging predominantly the cytoplasmic lipid droplets and microtubules, including the meiotic spindle.21–24 While the latter damage may be reversible, the former is always irreversible and contributes to much of the death of cryopreserved lipid-rich oocytes and embryos of some species. 2. Between −5 and −80°C, extracellular or, predominantly, intracellular ice crystal formation is the main source of injury. 3. Temperatures between −50 and −150°C can cause fracture damage to the zona pellucida or the cytoplasm25 and are postulated to occur, although the mechanism and the actual temperature of occurrence is not entirely defined. However, it is unlikely that zona fracture could occur as a simple consequence of osmotic stress, as suggested by Smith and Silva.4 Storage below −150°C (typically in liquid nitrogen, at −196°C) is probably the least dangerous phase of the cryopreservation procedure. Importantly, accidental warming is probably the most frequent form of injury. The effect of background irradiation seems to be less harmful than supposed, and is not a significant source for DNA injury in a realistic time interval, i.e. years, decades, or even centuries.26 However, there is increasing concern regarding possible disease transmission between the stored samples (mediated by the liquid nitrogen), even though there are no reported cases in the literature involving embryos. At warming, the same types of injuries may occur as at cooling, obviously in reverse order. Apart from these processes, there are some partially understood injuries such as damage to intracellular organelles, the cytoskeleton, and cell-to-cell contacts.27–29

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Typically, embryos suffer considerable damage during cooling and warming. Fortunately, they also have a remarkable, sometimes surprising, ability to repair fully or partially this damage, and (in the best case) to continue normal development. Almost all cryopreservation strategies are based on two factors: cryoprotectants and cooling−warming rates. The common feature of cryoprotectants is their ability to decrease cryoinjuries. A wide range of materials may be used, including low-molecular-weight solvents such as ethanol, or complex, partially undefined biological compounds such as sera or egg yolk. They may either enter the cell (permeable cryoprotectants) or remain outside (nonpermeable cryoprotectants). The main supposed effect of permeable cryoprotectants is to minimize ice formation. A similar mechanism, osmotic dehydration, may play a considerable role in the protective effect of nonpermeable cryoprotectants, although there are very few examples in which nonpermeable cryoprotectants actually protect cells against freezing damage. However, both permeable and nonpermeable cryoprotectants may also have additional protective mechanisms, e.g. to stabilize intracellular structures and the cell membrane. Unfortunately, most cryoprotectants have some negative effects such as toxicity and osmotic injuries. Toxicity is usually proportional to the concentration of the substance and to the time of exposure (at physiological temperatures). Cooling rates during vitrification may be extremely high, e.g. 20 000 to 100 000°C/min, in sharp contrast to the ‘slow cooling’ method where it can take 3–4 min to decrease temperature by 1°C (in some specific stages).3,26 Warming may be also performed stepwise – with highly controlled or just slightly delayed increase of the temperature – or (more typically) rapidly, including the commonly achievable highest rates of temperature change described above. Cryoprotectant composition, addition, concentration, and removal, as well as warming rates, are more or less determined by the selected cooling rates. The mechanism and reasons for damage during cryopreservation as well as the precise protective mechanisms of cryoprotectants are poorly understood at present. Morphological observations of the intracellular structures during the actual phase of cooling (especially at subzero temperatures) are difficult; functional analysis of specific processes at a given moment is almost impossible. The most frequently applied approaches are to investigate the effect of cryoprotectants without cooling and warming, or make retrospective conclusions based on the damage that can be observed after warming. However, the effects of a given cryoprotectant may substantially differ at physiological and at low temperatures; thus, the retrospective analysis of damage may result in faulty conclusions. Considering these uncertainties, it is not surprising that the vast majority of existing cryopreservation techniques were established empirically, based on rough morphological changes observed

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under a stereomicroscope, and have been justified by the outcome, i.e. in vitro and in vivo survival. This is valid for the development and perfection of the vitrification technique, but also stands for the slow freezing technology. A very recent development has been the use of highly sophisticated diagnostics such as protein expression to detect gene expression, to assess freezing conditions.30

Vitrification Cryoprotectants Because of the need for high concentrations of cryoprotectants in vitrification, research was focused on decreasing the toxic and osmotic damage. Consequently, ethylene glycol has become an almost standard part of all present vitrification protocols. Another typical approach was to use a combination of two, or frequently three cryoprotectants, to decrease the individual specific toxicity. At least one of these cryoprotectants had to be permeable (for that, ethylene glycol was the obvious choice), and one or two impermeable. Other permeable components, including propylene glycol, acetamide, glycerol, raffinose, and dimethylsulfoxide (DMSO), were tested in various combinations;3,31 eventually, the mixture of ethylene glycol and DMSO came to be used frequently.32–34 According to some studies, the permeability of the mixture is higher than that of the individual components.35 For the nonpermeable cryoprotectants, mono- and disaccharides such as sucrose, trehalose, glucose, and galactose were the primary candidates.36–38 Recently, sucrose has become almost a standard component of vitrification mixtures, although nearly all comparative investigations have proved the superiority of trehalose. Sucrose, along with other sugars, may not have any toxic effects at low temperatures, but may compromise embryo survival when applied extensively to counterbalance embryo swelling after warming,39–41 although this effect has not always been demonstrated.42 Although different polymers were also suggested for the purpose, including polyvinyl pyrrolidine, polyethylene glycol, Ficoll, dextran, and polyvinyl alcohol,43–48 so far the only widely used compound is Ficoll, predominantly in combination with ethylene glycol and sucrose.49 Various forms of protein supplementation have also been used, including egg yolk, but its optically dense appearance makes the microscopic manipulation rather difficult. High concentrations of sera of different origin as well as serum albumin preparations50 are common additives. In the bovine model, recombinant albumin and hyaluronan were also effective.51 During the past 15 years, many reports were published on the positive effects of antifreeze proteins isolated from arctic animals or analog synthetic compounds having a specific potential to decrease ice crystal formation;52–54 however, these components have not entered into routine use.

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Table 21.1

Various vitrification techniques in embryology

System

Reference

Direct dropping into liquid nitrogen Electron microscopic grids Open-pulled straw (OPS) Glass micropipettes (GMP) Super-finely pulled OPS (SOPS) Gel-loading tips Sterile stripper tip Flexipet denuding pipette (FDP) Fine diameter plastic micropipette 100 ml pipetting tip Closed-pulled straw (CPS) Sealed open-pulled straws Cryotip Cryoloop Nylon mesh Minimum drop size (MDS) Minimum volume cooling (MVC) Hemi-straw system (HSS) Cryotop Vitmaster Solid surface vitrification (SSV)

Landa and Tepla63 Martino et al23 Vajta et al72 Kong et al147 Isachenko et al151 Tominaga and Hamada152 Kuleshova and Lopata100 Liebermann et al98 Cremades et al150 Hredzak et al148 Chen et al146 Lopez-Bejar and Lopez-Gatius149 Kuwayama et al61 Lane et al80 Matsumoto et al86 Arav87 Hamawaki et al88 Vanderzwalmen et al89 Kuwayama et al60 Arav et al74 Dinnyes et al91

Reprinted from Vajta and Nagy168 with permission from Reproductive Healthcare Ltd.

Other strategies to minimize toxic effects are the stepwise addition of cryoprotectants, and the use of lower temperatures (about 4°C) at high levels of cryoprotectant.50,55,56,57 Typically, a two-step strategy has been used, where the first solution contained 20–50% of the final cryoprotectant concentration. Regarding incubation time, recently, a longer (5−15 min) preincubation with a considerably diluted first cryoprotectant solution, followed with an approximately 1 min incubation in the final solution, has been used.58–61 This approach may increase slightly the toxic effect, but provides a much better protection for the whole cell, and may be especially beneficial in the case of large objects with a low surface/volume ratio, including oocytes or early-stage embryos.

Traditional tools Plastic insemination straws or cryovials were used initially for vitrification experiments. These tools were not designed for the special purpose of vitrification, had a thick wall, and required a relatively large amount of solution for safe loading. Accordingly, the cooling and warming rates were quite limited (approximately 2500°C/min for straws,62 and even less for cryovials). This relatively low rate was still hazardous to perform, as direct immersion into liquid nitrogen at cooling, and transfer to a water bath at warming, induced extreme pressure changes in the closed system and frequently led to the collapse or explosion of the straws and loss of the sample. One of the other consequences of these manipulations were

the decreased and inconsistent rates: the temperature of the vapor of liquid nitrogen is variable, depending on many factors, and the definition of ‘room temperature’ laboratory air may mean 5–7°C differences, even at the same place on the same day. Consequently, a minimum 5−7 mol/l cryoprotectant concentration was required, and chilling injury could not be lowered to the level occurring at slow freezing.

Minimal volume and direct contact The logical way to increase the cooling rate is to use the smallest possible volume of cryoprotectant medium surrounding the embryo and to establish a direct contact (without any thermoinsulating layer) between the solution and the liquid nitrogen. The small volume may also offer a special advantage: it prevents heterogeneous ice formation.50 There is a truly wide variety of choices for tools that can be used to place the embryo for vitrification (summarized in Table 21.1; examples in Fig 21.1). One of the earliest documented attempts used the simplest way, and dropped the sample without any container directly into the liquid nitrogen.58,59,63–65 Additionally, this vapor coat did not allow the sample to sink, decreasing the cooling rate even further. Copper electron microscopic grids as a carrier for the sample was the first method to fully utilize the enormous potential of the small sample–direct contact approach.66–69 The size of the drop surrounding the sample was extremely small, as, after loading, most of it was removed by placing the grid on a filter membrane.

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(a) (a)

(b) (b)

(c) (c)

(d)

(d)

Fig 21.1 Examples of commercially available tools used as carriers for high-speed vitrification. (a) Open-pulled straw, OPS (Minitüb, Landshut, Germany); (b) McGill Cryoleaf (MediCult, Jyllinge, Denmark); (c) Cryotop (Kitazato, Tokyo, Japan); (d) Cryoloop (Hampton Research, Aliso Viejo, California, USA). Bars represent 2 mm.

The thermoconductive metal grid also increased the cooling and warming rates. Surprisingly, the solidified cryoprotectant solution fixed the sample safely to the grid during cooling and storage, and released it easily after warming.70 However, it is the straw that is most widely used in this field. That is probably why the OPS (open-pulled straw) technology has achieved widespread attention and (together with its analogues) is probably the most widely used approach for ultrarapid vitrification.71–79 The idea of the OPS is great and the technique is easy and simple. Because the diameter and the wall thickness of the straw decreased to approximately half of the original while the required amount of solution to form a safe column decreased from 5 µl to less than 1 µl, this led eventually to a 10-fold increase in the achievable cooling rate, thus allowing a 30% decrease in cryoprotectant concentration required for safe vitrification. An additional benefit was related to the open system: no explosion of the straws occurred and the fracture damage (with some precautions) could be entirely eliminated. The Cryoloop is another approach using the small volume–direct contact principle: a small nylon loop is attached to a holder and equipped with a container.

It has been used for cryopreservation in crystallography and is now used widely for oocyte and embryo cryopreservation.80–83 The solution film bridging the hole of the loop is strong enough to hold the oocyte or the embryo, and with this minimal solution volume, the achievable cooling rate may be extremely high, up to an estimated 700 000°C/min.84 Using this tool, safe cryopreservation can be achieved even in the vapor of liquid nitrogen.85,86 The minimum drop size (MDS) method of Arav87 consists of placing a small droplet of vitrification solution containing the oocyte or embryo on a solid surface that is immersed in liquid nitrogen. The approach was used later with some modifications and called the minimum volume cooling (MVC)88 or in the hemi-straw system (HSS),89 where the carrier tool was a cut-open straw. The flow chart of a typical high-speed vitrification procedure is summarized in Fig 21.2.

Avoiding vapor The vapor coat that arises around the sample in the liquid nitrogen is a concern that can prevent a truly high rate of cooling; therefore, eliminating vapor may be important. One possibility is to use liquid nitrogen

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Equilibration in concentrated solution of permeable and nonpermeable CPs (0.5–2 min)

Loading to a carrier tool

Cooling by submerging into LNM2

available form has been produced (CMV; Cryologic, Australia). The few comparative data do not provide entirely convincing evidence regarding the superiority of these vapor-minimizing or vapor-free approaches compared with the other vitrification procedures.

Transmission of infectious agents One of the concerns regarding the use of vitrification in human embryology is the potential risk of liquid nitrogen-mediated disease transmission. To understand better the issues, we need to consider the following: 1.

2.

Storage

3.

Warming in concentrated solution of an impermeable CP

Dilution of cryoprotectants in decreasing concentration of an impermeable CP (3x5 min)

Direct transfer to recipients

4.

5. 6. Transfer to recipients

In vitro culture (2–24 h)

Semen and embryo collection, processing, and cryopreservation protocols are not sterile procedures;92 consequently, the contents of virtually all stored straws and cryovials may be a source of infection. In human embryology, liquid nitrogen may also be contaminated by the surface of straws, cryovials, racks, and other tools that are usually not handled fully aseptically. Accordingly, the presence of infective agents is not strictly related to leaky or open containers. Seemingly sterile containers may not be as safe as supposed. Infection may occur in common straws in slow freezing (through the holes of incomplete sealing, or pores of the plastic walls), and most cryovials do not have secure caps. A possible source of infection may also be the inappropriate decontamination of the outer walls of straws before loading and expelling. Liquid nitrogen in storage tanks likely contains a number of commensal and potentially pathogenic environmental microorganisms.92 There are documented cases of liquid nitrogenmediated disease transmission.93–95 According to the experiments of Bielanski et al,96 cross-contamination may also occur during storage between OPS straws if one of them is artificially infected. This is, without doubt, applicable to any open systems.

Transfer to recipients

Fig 21.2 Flow chart of a typical high-speed vitrification procedure. CP, cryoprotectant; LN2, liquid nitrogen.

slush instead of liquid nitrogen for cooling. At 10°C below its boiling point, liquid nitrogen behaves differently: the nitrogen escapes from the fragile boiling temperature zone, and the sample immersed into the semi-solid slush creates just a minimal evaporation; consequently, the cooling rate gets considerably higher.74,75,90 The other possibility of eliminating the vapor is the use of pre-cooled metal surfaces instead of liquid nitrogen for cooling. Originally, a metal block immersed in liquid nitrogen was used,91 but eventually a commercially

In conclusion, the danger of liquid nitrogen-mediated disease transfer exists and traditional tools and methods of cryopreservation are vulnerable, as well. On the other hand, no documented case of liquid nitrogen-mediated disease transmission related to embryo transfer activities has been reported. The few published disease transmissions occurred between blood specimens. Some of the recent techniques do not even need the direct contact with the liquid nitrogen. The Cryoloop method can be successfully performed by cooling in the vapor of liquid nitrogen,85 the ‘sealed open-pulled straw,’ the closed-pulled straw (CPS), and the Cryotip techniques are basically closed systems, and the methods using metal surfaces for cooling such as CMV, do not expose samples to liquid nitrogen. Tools and containers are now commercially available for

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easy application. Subsequently, similar approaches were also applied for the super-finely pulled OPS (SOPS), HSS, and flexipet denuding pipette (FDP) methods, as well.97–99 Consequently two major achievements of the recent vitrification methods, the elimination of chilling injury and the low cryoprotectant concentration (i.e. less toxic and osmotic injury), are sacrificed for biosafety. It should be mentioned that there is a considerable difference between the observations of the two groups: while Isachenko still emphasizes the importance of rapid warming, Kuleshova and Lopata100 state that straws can be kept ‘inside a second protective container throughout vitrification, storage and warming’; consequently, authors do not regard it as important to maintain the high rate of temperature changes at warming, either. One may doubt if this method is really the optimal compromise between the various requirements in vitrification. Concerns may also be raised regarding the applicability in cases of chilling sensitive objects such as cattle oocytes and early-stage embryos, porcine blastocysts, or human oocytes,101 regarding not just in vitro survival rates but also in vitro development, pregnancies, and birth of healthy offspring.

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Factors influencing outcome Species and genotype There are well-demonstrated but poorly-understood differences in sensitivity to cryoinjuries between different species in mammals. It appears that transparent oocytes and embryos are usually more resistant; dense dark ones are more fragile, due to the increased lipid content. Accordingly, cryopreservation of light mouse embryos is a relatively easy task, darker bovine embryo is a more difficult task, and the cryopreservation of dense pig embryos is truly a challenge in cryobiology. In parallel with the lighter appearance of the cytoplasm, considerably increased survival rates were detected after both slow freezing108 and vitrification.109–113 This approach also improves in vitro survival of vitrified porcine blastocysts produced by somatic cell nuclear transfer.113,114 It should be also noted that apart from the differences between species, in mouse, differences between genotypes in the ability to develop after vitrification were also observed.115,116

Developmental stage: size matters Warming Warming after vitrification is performed almost the same way as after most slow freezing procedures, i.e. by direct immersion into a solution at the core temperature of the given species (in humans, at 37°C), although this may not be fundamentally necessary, since Rall50 demonstrated that, depending on the cryoprotectant, high survival of embryos can be achieved even with rather slow warming. Generally, it seems to be advisable to keep the samples for 1−3 s in air to avoid fracture damage caused by gas bubbles occurring in the toorapidly immersed samples. Closed systems are usually immersed into water baths, while open systems can be directly submerged into the medium; in this way, the warming and the first dilution is performed in a single step. Although a slight devitrification (occurrence of ice crystals) may occur, especially when the cryoprotectant level is kept at the minimum level, this transitional change is usually restricted to a part of the embryo-containing medium and most probably does not involve intracellular crystal formation and, consequently, does not cause significant harm to the embryos or oocytes.102 In routine warming protocols of vitrified embryos, the dilution is a multistep procedure, with decreasing concentration of osmotic buffers (usually sucrose) to counterbalance the swelling caused by the permeable cryoprotectant that leaves the cells relatively slowly. Additional to the multistep warming, the one-step dilution without significant decrease of in vitro survival was reported in some animal species, including cattle41,56,103,104 and pigs.105 Direct transfer or analogue methods after ultrarapid vitrification of embryos resulted in offspring after transfer in cattle106 and sheep.107

The change in the size and shape of the cells is unprecedented in the first 5–6 days of mammalian development. A relatively simple spherical shape protected by an acellular outer layer develops to a complicated multicellular structure without external protection. Predictably, the extreme differences in morphology also result in considerable differences in sensitivity to cryoinjuries. Generally, the earlier the development stage (starting from the germinal vesicle stage), the more sensitive oocytes and embryos are. However, although there is only a minimal difference between the size and shape, the immature oocytes are usually more sensitive to cryopreservation than mature (metaphase II [MII]) oocytes.22,101,117 Additionally, a very remarkable difference exists between the chilling sensitivity of unfertilized and fertilized human oocytes. A possible explanation for this phenomenon is the increased chilling sensitivity of membranes: the lipid phase transition at room temperature storage in human germinal vesicle and MII stage oocytes is 10 times higher than that of human pronuclear embryos.101 In the human, the survival rates after slow freezing are not significantly different between zygotes, cleavage-stage embryos, and blastocysts (between 75 and 80% for each).118,119 The complex structure of blastocysts may give rise to additional problems. An artificial elimination of the blastocoel in mouse with high atmospheric pressure improves survival.120 In humans, mechanical reduction of the blastocoel by puncturing or repeated pipetting improved survival and pregnancy rates.121–123 The usual explanation is that the large blastocoel may not be protected appropriately from ice crystal formation.121 However, other feasible mechanisms

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Table 21.2

Examples in mammalian embryology where first success in cryopreservation was achieved by vitrification

Species, stage, system

Reference

Bovine immature oocytes for IVF Bovine in vitro matured oocytes for IVF Bovine in vitro matured oocytes for somatic cell nuclear transfer Bovine cytoplasts for embryonic cell nuclear transfer Bovine early-stage IVF embryos Bovine zona-included blastocysts generated by somatic cell nuclear transfer Bovine zona-free blastocysts generated by somatic cell nuclear transfer Bovine transgenic blastocysts generated by somatic cell nuclear transfer Ovine zona-included embryos generated by nuclear transfer Porcine immature oocytes for ICSI Porcine in vitro matured oocytes for ICSI Porcine in vivo derived blastocysts Porcine in vivo derived morulae Porcine in vitro produced blastocysts Equine in vivo matured oocytes European polecat in vivo derived morulae and blastocysts Siberian tiger in vivo derived embryos Minke whale immature oocytes for maturation

Vieria et al153 Martino et al72; Vajta et al67 Hou et al154 Booth et al155 Vajta et al73; in vitro study French et al156 Tecirlioglu et al106 French et al157 Peura et al158 Fujihira et al159; in vitro study Fujihira et al160; in vitro study Kobayashi et al161 Berthelot et al162 Men et al163; in vitro study Maclellan et al164 Piltty et al165 Crichton et al166; in vitro study Iwayama et al167; in vitro study

Embryos and oocytes were not treated mechanically or chemically to prepare them for the vitrification. Full-term developments were reported except where otherwise indicated. IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection. Reprinted from Vajta and Nagy168 with permission from Reproductive Healthcare Ltd.

may include the inappropriate dilution of the accumulated cryoprotectants after warming (Vajta, unpublished work) or increased protection against cryoinjuries due to stress-induced biochemical changes.120

In vitro and in vivo produced The origin of the embryos may also be an important factor. In domestic animals, in vivo produced embryos are generally more resistant to injuries, including cryoinjuries, than in vitro fertilized or somatic cell cloned ones.28 There might be some correlation between the increased lipid content of embryos produced in some in vitro systems. In general, the less morphological difference from the in vivo counterpart is detectable in the in vivo produced embryos, the smaller the expected difference in survival after cryopreservation. Although total elimination of these differences is still impossible, according to the joint conclusion of many publications, vitrification seems to be especially appropriate to counterbalance this handicap.

Evidences in favor of vitrification Domestic, experimental, and wild animals There is an extensive literature of comparative experiments between slow freezing and vitrification.80,81,115,116,124–127 The overwhelming majority of these papers prove the superiority of vitrification for the given purpose. Probably less than 10% of the studies

did not find significant differences, and, according to our knowledge, no publication stated that results achieved by vitrification were significantly worse than those obtained by slow freezing. Moreover, there are situations where vitrification is uniquely or predominantly suitable to achieve the goal: most of these areas are summarized in Table 21.2. A special application where vitrification was part of a complex procedure to produce somatic cell cloned pigs from cryopreserved blastocysts is shown on Fig 21.3.

Human embryos In humans, the clinical pregnancy rate from embryo transfer after slow freezing is approximately twothirds that from the fresh transfer of embryos,128 although new techniques have recently been introduced to restore (cleavage-stage) embryo viability.129,130 The theoretical possibility for improvement is supported by the results obtained in cattle, where the difference is no more than 10–15%. Until recently, the published sporadic results based on relatively low numbers proved only the feasibility and potential competitiveness but not the superiority of vitrification in this field.3,19,20,68,69,77,78,82,83,89,97,99,121,122,126,131–141 In 2005, however, three comparative investigations were published, and all three concluded that vitrification was a more efficient way for cryopreservation of human embryos than slow-rate freezing. Zheng et al142 performed an in vitro experiment with biopsied nontransferable human embryos. Three versions of slow

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(a)

(b)

(c)

(d)

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Fig 21.3 Potential application of vitrification in a complex procedure including delipation of porcine oocytes and somatic cell nuclear transfer. (a) Delipation of in vitro matured and enucleated porcine oocytes (cytoplasts) by partial digestion of the zona pellucida and high-speed centrifugation. L, lipid droplets; DC, delipated cytoplast. (b) Cryotop vitrified and warmed day-5 porcine blastocyst produced by somatic cell cloning with fusion of a delipated zona-free cytoplast and a fetal fibroblast. (c) The same blastocyst after fixation, Hoechst nuclear staining, and UV illumination. (d) Three piglets (in front) born after transfer of vitrified–warmed somatic cell cloned zona-free blastocysts after delipation of cytoplasts. (a–c), inverted microscopic pictures; (c), in UV light; bars represent 50 µm. Courtesy of Dr Yutao Du.

freezing were compared with vitrification, and survival rates were the highest after vitrification. Stehlik et al143 used slow freezing vs Cryotop vitrification for cryopreservation of supernumerary human blastocysts, and observed a significant difference in pregnancy rates after 44 transfers (16.7 vs 50%, respectively). The largest comparative investigation between the effect of slow freezing vs vitrification was published by Kuwayama et al61 based on cryopreservation of more than 16 000 human embryos at different stages. Cryotop vitrification was found superior for pronuclear embryo cryopreservation in regard to survival, cleavage, and developmental rates. Survival rates of 4-cell stage and blastocyst-stage human embryos were also significantly higher than those after slow freezing. Pregnancy and birth rates after cryopreservation with the two methods were not significantly different. Accordingly, this representative comparison has proved that vitrification is at least as

efficient as slow-rate freezing for cryopreservation of human embryos in all developmental stages. Summarizing the application fields of different vitrification methods, two technologies have obtained the greatest attention: the OPS, predominantly in animals; and the Cryotop, for humans. It should be emphasized, however, that the differences do not necessarily mean an unbreakable frontier. The OPS is more robust and is easier to perform even under compromised conditions, while the delicate Cryotop method may be the choice where extremely high cooling rates are the primary objectives. However, if properly applied, both OPS and Cryotop methods seem to be suitable for the given purposes. A good example to support this statement is that very healthy piglets were born after OPS vitrification and transfer of the extremely sensitive somatic cell cloned blastocyst derived from delipated oocytes114 (Rongfen Li, University of Missouri-Columbia, Missouri, Columbia, USA: pers comm).

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Table 21.3 Outcomes of freezing/thawing comparing vitrification with slow freezing at different stages of human embryonic development (cryopreservation embryo transfer cycles were performed during 2006 and 2007 both for slow freezing and vitrification) Stage

Slow freezing

Vitrification

p

2PN embryos (day 1 freezing) Thaw cycles Embryos thawed Survived Day 2 Cell No. Clinical pregnancy rate

134 667 568 (85%) 3.0 41%

28 178 172 (97%) 3.1 60%

<0.01 NS NS

Cleaved embryos (day 3 freezing) Thaw cycles Embryos thawed Survived (>50% intact) % of intact cells/total cells Clinical pregnancy rate

136 682 512 (75%) 67.3% 42%

18 69 62(90%) 93.2% 56%

<0.01 <0.01 NS

Blastocyst (day 5/6 freezing) Thaw cycles Embryos thawed Survived Expanded in 1 h after thaw Clinical pregnancy rate

45 145 110 (76%) 55 (38%) 53%

59 171 159 (93%) 128 (75%) 61%

<0.01 <0.01 NS

Most current results using the Cryotop vitrification approach on sibling embryos have demonstrated equal or better outcomes than slow freezing (Table 21.3).

Conclusions In the hundreds of publications, strong concern against the use of vitrification was found in only one manuscript, and even this paper referred only to theoretical dangers that were not supported by experimental or clinical results. Yves Ménézo144 stated that the impact of vitrification, especially when ethylene glycol is used, has to be carefully evaluated before its use on a large scale. Ethylene glycol and its metabolites can be toxic at a very low concentration.145 Ménézo also states that ‘the balance between permeable and non-permeable cryoprotectants has to be re-evaluated probably in favour of non-permeable ones.’ However, survival, development, pregnancy, and healthy offspring rates have demonstrated the limits of the nonpermeable vs permeable cryoprotectant approach. Regarding toxicity as a concern, the result of combined application of different approaches to minimize the toxicity is that the concentration of ethylene glycol can be as low as 15% (2.2 mol/l), and it is applied for a very short period before and after deep cooling. This concentration is similar to that used at the industrial level for traditional slow freezing of cattle embryos, where no increase of developmental abnormalities or other toxic effects have been reported so far. Similarly, no reports of abnormal pregnancy or children born with malformations have been published following the vitrification protocol in humans.

The overwhelming majority of the studies/publications support the application of vitrification by emphasizing its advantages: this simple, inexpensive, and rapid procedure leads to higher survival and developmental rates than those achievable with alternative methods. Concerns regarding disease transmission are partially justified, but safer methods are now available to lessen this danger. Convincing results, like the emerging breakthrough in human oocyte vitrification and the excellent (and improving) results on embryo cryopreservation, may helping to eliminate these obstacles and the remaining concerns. Recently achieved results using vitrification seem to convince more and more professionals about the advantage of the technique, reflected by the increasing number of publications and also by the number of introduced (or soon to be introduced) commercial kits for vitrification. In the future, standardization should be attempted after comparing different protocols of vitrification, which also should be adjusted to the different stages of embryo development.

APPENDIX Embryo/blastocyst vitrification protocol Vitrification Materials •

Equilibration solution (ES) is a HEPES-buffered medium, 7.5% (v/v) of DMSO and ethylene glycol and 20% (v/v) serum protein substitute.

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•

Vitrification solution (VS) is a HEPES-buffered medium, 15% (v/v) of DMSO and ethylene glycol and 20% (v/v) serum protein substitute and 0.5 mol/l sucrose.

Cryolock, Biodesign, Columbia

Procedures 1. Bring one vial of both ES and VS to room temperature (20–27°C) for at least 30 min prior to freezing embryos. 2. Fill the liquid nitrogen reservoir with liquid nitrogen. 3. Determine the number of embryos to be vitrified. 4. Label each Cryolock with necessary information. 5. Prepare a 4-well dish with 1.0 ml ES and 1.0 ml VS in each well. 6. Transfer the embryos to ES for 15 min. 7. Transfer the embryos to VS for 1 min. 8. Load the embryos onto the Cryolock with a minimal volume. 9. Plunge the Cryolock into liquid nitrogen (cooling at a rate of −12 000°C/min). 10. Move the plunged Cryolock to the liquid nitrogen freezer for long-term storage.

Warming Materials • • •

Thawing solution (TS) is a HEPES-buffered medium, 1.0 mol/l sucrose and 20% (v/v) serum protein substitute. Dilution solution (DS) is a HEPES-buffered medium, 0.5 mol/l sucrose and 20% (v/v) serum protein substitute. Washing solution (WS) is a HEPES-buffered medium and 20% (v/v) serum protein substitute.

Procedures 1. Bring one vial of each TS, DS, and WS to room temperature (20–27°C) for at least 30 min prior to thawing embryos. 2. Fill the liquid nitrogen reservoir with liquid nitrogen. 3. Determine the number of embryos to be thawed. 4. Take the Cryolock out of the liquid nitrogen and quickly transfer embryos into TS (3 ml at 37°C), where embryos should stay for 1 min. 5. Transfer the embryos into 1.0 ml DS for 3 min at room temperature. 6. Transfer the embryos into 1.0 ml WS for 10 min at room temperature. 7. Transfer the embryos into pre-equilibrated culture medium.

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21. Aman RR, Parks JE. Effects of cooling and rewarming of the meiotic spindle and chromosomes of in vitro matured bovine oocytes. Bio Reprod 1994; 50: 103–10. 22. Leibo SP, Martino A, Kobayashi S, Pollard JW. Stagedependent sensitivity of oocytes and embryos to low temperatures. Anim Reprod Sci 1996; 42: 45–53. 23. Martino A. Pollard JA, Leibo SP. Effect of chilling bovine oocytes on their developmental competence. Mol Reprod Devt 1996a; 45: 503–12. 24. Zenzes MT, Bielecki R, Casper RF, et al. Effects of chilling to 0°C on the morphology of meiotic spindles in human metaphase II oocytes. Fertil Steril 2001; 75: 769–77. 25. Rall WF, Meyer TK. Zona fracture damage and its avoidance during the cryopreservation of mammalian embryos. Theriogenology 1989; 31: 683–92. 26. Rall WF. Cryopreservation of mammalian embryos, gametes and ovarian tissues. Current issues and progress. In: Wolf DP, Zelinski-Wooten M, eds. Assisted Fertilization and Nuclear Transfer in Mammals. Totowa, NJ: Humana Press, 2001: 173–87. 27. Vincent C, Johnson, JH. Cooling cryoprotectants and the cytoskeleton of the mammalian oocyte. Oxf Rev Reprod Biol 1992; 14: 73–100. 28. Massip A, Mermillod P, Dinnyés A. Morphology and biochemistry of in-vitro produced bovine embryos: implications for their cryopreservation. Hum Reprod 1995; 10: 3004–11. 29. Dobrinsky JR. Cellular approach to cryopreservation of embryos. Theriogenology 1996; 45: 17–26. 30. Gardner DK, Sheehan CB, Rienzi L, et al. Analysis of oocyte physiology to improve cryopreservation procedures. Theriogenology 2007; 67: 64–72. 31. dela Peña EC, Takahashi Y, Atabay EC, et al. Vitrification of mouse oocytes in ethylene glycol–raffinose solution: effects of preexposure to ethylene glycol or raffinose on oocyte viability. Cryobiology 2001; 42: 103–11. 32. Ishimori H, Takahashi Y, Kanagawa H. Viability of vitrified mouse embryos using various cryoprotectant mixtures. Theriogenology 1992a; 37: 481–7. 33. Ishimori H, Takahashi Y, Kanagawa H. Factors affecting survival of mouse blastocysts vitrified by a mixture of ethylene glycol and dimethylsulfoxide. Theriogenology 1992b; 38: 1175–85. 34. Ishimori H, Saeki K, Inai M, et al. Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide. Theriogenology 1993; 40: 427–33. 35. Vicente JS, Garcia-Ximenez F. Osmotic and cryoprotective effects of a mixture of DMSO and ethylene glycol on rabbit morulae. Theriogenology 1994; 42: 1205–15. 36. Ali J, Shelton JN. Design of vitrification solutions for the cryopreservation of embryos. J Reprod Fertil 1993; 99: 471–7. 37. Kasai M. Vitrification: refined strategy for the cryopreservation of mammalian embryos. J Mammal Ova Res 1997; 14: 17–28. 38. Wright DL, Eroglu A, Toner M, et al. Use of sugars in cryopreserving human oocytes. Reprod Biomed Online 2004; 9: 179–86. 39. Kasai M. Nonfreezing technique for short-term storage of mouse embryos. J In Vitro Fert Embryo Transf 1986; 3: 10–14.

40. Kasai M, Nishimori M, Zhu SE, et al. Survival of mouse morulae vitrified in an ethylene glycolbased solution after exposure to the solution at various temperatures. Bio Reprod 1992; 47: 1134–9. 41. Vajta G, Holm P, Greve T, et al. Survival and development of in vitro produced bovine blastocysts following assisted hatching, vitrification and in-straw direct rehydration. J Reprod Fertil 1997; 111: 65–70. 42. Kuleshova LL, MacFarlane DR, Trounson AO, et al. Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have a low toxicity to embryos and oocytes. Cryobiology 1999; 38: 119–30. 43. Leibo SP, Oda K. High survival of mouse zygotes and embryos cooled rapidly or slowly in ethylene glycol plus polyvinylpyrrolidone. Cryo Letters 1992; 14: 133–44. 44. Ohboshi S, Fujihara N, Yoshida T, et al. Usefulness of polyethylene glycol for cryopreservation by vitrification of in vitro-derived bovine blastocysts. Anim Reprod Sci 1997; 48: 27–36. 45. Shaw JM, Kuleshova LL, MacFarlane DR, Trounson AO. Vitrification properties of solutions of ethylene glycol in saline containing PVP, Ficoll, or dextran. Cryobiology 1997; 35: 219–29. 46. Naitana S, Ledda S, Loi P, et al. Polyvinyl alcohol as a defined substitute for serum in vitrification and warming solutions to cryopreserve ovine embryos at different stages of development. Anim Reprod Sci 1997; 48: 247–56. 47. Kuleshova LL, Shaw JM, Trounson AO. Studies on replacing most of the penetrating cryoprotectant by polymers for embryo cryopreservation. Cryobiology 2001; 43: 21–31. 48. Asada M, Ishibashi S, Ikumi S, et al. Effect of polyvinyl alcohol (PVA) concentration during vitrification of in vitro matured bovine oocytes. Theriogenology 2002; 58: 1199–208. 49. Kasai M, Komi JH, Takakamo A. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J Reprod Fertil 1990; 89: 91–7. 50. Rall WF. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 1987; 24: 387–402. 51. Lane M, Maybach JM, Hooper K, et al. Cryo-survival and development of bovine blastocysts are enhanced by culture with recombinant albumin and hyaluronan. Mol Reprod Dev 2003; 64: 70–8. 52. Rubinsky B, Arav A, Devries AL. The cryoprotective effect of antifreeze glycopeptides from Antarctic fishes. Cryobiology 1992; 29: 69–79. 53. Eto TK, Rubinsky B. Antifreeze glycoproteins increase solution viscosity. Biochem Biophys Res Commun 1993; 197: 927–31. 54. Wowk B, Leitl E, Rasch CM, et al. Vitrification enhancement by synthetic ice blocking agents. Cryobiology 2000; 40: 228–36. 55. Vanderzwalmen P, Touati K, Ectors FJ, et al. Vitrification of bovine blastocysts. Theriogenology 1989; 31: 270. 56. Saha S, Takagi M, Boediono A, et al. Direct rehydration of in vitro fertilised bovine embryos after vitrification. Vet Rec 1994; 134: 276–7.

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The human embryo: vitrification 57. Széll A, Shelton NJ. Survival of vitrified sheep embryos in vitro and in vivo. Theriogenology 1994; 42: 881–9. 58. Papis K, Shimizu M, Izaike Y. The effect of gentle pre-equilibration on survival and development rates of bovine in vitro matured oocytes vitrified in droplets. Theriogenology 1999; 51: 173. 59. Papis K, Shimizu M, Izaike Y. Factors affecting the survivability of bovine oocytes vitrified in droplets. Theriogenology 2000; 54: 651–8. 60. Kuwayama M, Vajta G, Kato O, et al. Highly efficient vitrification method for cryopreservation of human oocytes. Reprod Biomed Online 2005; 11: 300–8. 61. Kuwayama M, Vajta G, Ieda S, Kato O. Vitrification of human embryos using the CryoTip™ method. Reprod Biomed Online 2005; 11: 608–14. 62. Palasz AT, Mapletoft RJ. Cryopreservation of mammalian embryos and oocytes: recent advances. Biotechnol Adv 1996; 14: 127–49. 63. Landa V, Tepla O. Cryopreservation of mouse 8-cell embryos in microdrops. Folia Biol (Praha) 1990; 36: 153–8. 64. Riha J, Landa V, Kneissl J, et al. Vitrification of cattle embryos by direct dropping into liquid nitrogen and embryo survival after nonsurgical transfer. Zivoc Viroba 1994; 36: 113–20. 65. Yang BS, Leibo SP. Viability of in vitro-derived bovine zygotes cryopreserved in microdrops. Theriogenology 1999; 51: 178. 66. Steponkus PL, Myers SP, Lynch DV, et al. Cryopreservation of Drosophila melanogaster embryos. Nature 1990; 345: 170–2. 67. Martino A, Songsasen N, Leibo SP. Development into blastocysts of bovine oocytes cryopreserved by ultrarapid cooling. Bio Reprod 1996; 54: 1059–69. 68. Choi DH, Chung HM, Lim JM, et al. Pregnancy and delivery of healthy infants developed from vitrified blastocysts in an IVF-ET program. Fertil Steril 2000; 74: 838–9. 69. Cho HJ, Son WY, Yoon SH, et al. An improved protocol for dilution of cryoprotectants from vitrified human blastocysts. Hum Reprod 2002; 17: 2419–22. 70. Son WY, Lee SY, Chang MJ, et al. Pregnancy resulting from transfer of repeat vitrified blastocysts produced by in vitro matured oocytes in patient with polycystic ovary syndrome. Reprod Biomed Online 2005; 10: 398–401. 71. Vajta G, Holm P, Greve T, et al. Vitrification of porcine embryos using the open pulled straw (OPS) method. Acta Vet Scand 1997b; 38: 349–52. 72. Vajta G, Kuwayama M, Holm P, et al. Open pulled straw vitrification: a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 1998; 51: 53–8. 73. Vajta G, Lewis IM, Kuwayama M, Greve T, Callesen H. Sterile application of the open pulled straw (OPS) vitrification method. Cryo Letters 1998; 19: 389–92. 74. Arav A, Zeron Y, Ocheretny A. A new device and method for vitrification increases the cooling rate and allows successful cryopreservation of bovine oocytes. Theriogenology 2000; 53: 248. 75. Arav A, Yavin S, Zeron Y, et al. New trends in gamete’s cryopreservation. Mol Cell Endocrinol 2002; 187: 77–81.

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76. Chen SU, Lien YR, Chen HF, et al. Open pulled straws for vitrification of mature mouse oocytes preserve patterns of meiotic spindles and chromosomes better than conventional straws. Hum Reprod 2000; 15: 2598–603. 77. El-Danasouri I, Selman H. Successful pregnancies and deliveries after a simple vitrification protocol for day 3 human embryos. Fertil Steril 2001; 76: 400–2. 78. Selman H, El-Danasouri I. Pregnancies derived from vitrified human zygotes. Fertil Steril 2002; 77: 422–3. 79. Isachenko V, Selman H, Isachenko E, et al. Modified vitrification of human pronuclear oocytes: efficacy and effect on ultrastructure. Reprod Biomed Online 2003; 7: 211–16. 80. Lane M, Forest KT, Lyons EA, et al. Live births following vitrification of hamster embryos using a novel containerless technique. Theriogenology 1999; 51: 167. 81. Lane M, Schoolcraft WB, Gardner DK. Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil Steril 1999; 72: 1073–8. 82. Mukaida T, Nakamura S, Tomiyama T, et al. Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertil Steril 2001; 76: 618–20. 83. Mukaida T, Takahashi K, Kasai M. Blastocyst cryopreservation: ultrarapid vitrification using cryoloop technique. Reprod Biomed Online 2003; 6: 221–5. 84. Isachenko E, Isachenko V, Katkov II, et al. Vitrification of mammalian spermatozoa in the absence of cryoprotectants: from past partial difficulties to present success. Reprod Biomed Online 2003; 6: 191–200. 85. Larman MG, Sheenan CB, Gardner DK. Vitrification of mouse pronuclear oocytes with no direct liquid nitrogen contact. Reprod Biomed Online 2006; 12: 66–9. 86. Matsumoto H, Jiang JY, Tanaka T, et al. Vitrification of large quantities of immature bovine oocytes using nylon mesh. Cryobiology 2001; 42: 139–44. 87. Arav A. Vitrification of oocytes and embryos. In: Lauria A, Gandolfi F, eds. New Trends in Embryo Transfer. Cambridge: Portland Press, 1992: 255–64. 88. Hamawaki A, Kuwayama M, Hamano S. Minimum volume cooling method for bovine blastocyst vitrification. Theriogenology 1999; 51: 165. 89. Vanderzwalmen P, Bertin G, Debauche V, et al. In vitro survival of metaphase II oocytes (MII) and blastocysts after vitrification in an hemi-straw (HS) system. Fertil Steril 2000; 7: 4, S215–16. 90. Huang CC, Lee TH, Chen SU, et al. Successful pregnancy following blastocyst cryopreservation using super-cooling ultra-rapid vitrification. Hum Reprod 2005; 20: 122–8. 91. Dinnyes A, Dai Y, Jiang S, et al. High developmental rates of vitrified bovine oocytes following parthenogenetic activation, in vitro fertilization, and somatic cell nuclear transfer. Bio Reprod 2000; 63: 513–18. 92. Bielanski A, Bergeron H, Lau PCK, et al. Microbial contamination of embryos and semen during longterm banking in liquid nitrogen. Cryobiology 2003; 46: 146–52. 93. Tedder RS, Zuckerman MA, Goldstone AH, et al. Hepatitis B transmission from contaminated cryopreservation tank. Lancet 1995; 346: 137–40.

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94. Fountain DM, Ralston M, Higgins N, et al. Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components. Transfusion 1997; 37: 585–91. 95. Berry ED, Dorsa WJ, Siragusa GR, et al. Bacterial cross-contamination of meat during liquid nitrogen immersion freezing. J Food Prot 1998; 61: 1103–8. 96. Bielanski A, Nadin-Davis S, Sapp T, et al. Viral contamination of embryos cryopreserved in liquid nitrogen. Cryobiology 2000; 40: 110–16. 97. Jelinkova L, Selman HA, Arav A, et al. Twin pregnancy after vitrification of 2-pronuclei human embryos. Fertil Steril 2002; 77: 412–14. 98. Liebermann J, Tucker MJ, Graham JR, et al. Blastocyst development after vitrification of multipronuclear zygotes using the Flexipet denuding pipette. Reprod Biomed Online 2002; 4: 146–50. 99. Vanderzwalmen P, Bertin G, Debauche Ch, et al. Vitrification of human blastocysts with the hemistraw carrier: application of assisted hatching after thawing. Hum Reprod 2003; 18: 1501–11. 100. Kuleshova LL, Lopata A. Vitrification can be more favorable than slow cooling. Fertil Steril 2002; 78: 449–54. 101. Ghetler Y, Yavin S, Shalgi R, et al. The effect of chilling on membrane lipid phase transition in human oocytes and zygotes. Hum Reprod 2005; 20: 3385–9. 102. Shaw JM, Kola I, MacFarlane DR, et al. An association between chromosomal abnormalities in rapidly frozen 2-cell mouse embryos and the ice-forming properties of the cryoprotective solution. J Reprod Fertil 1991; 91: 9–18. 103. Kuwayama M. In straw dilution of bovine IVF−blastocysts cryopreserved by vitrification. Theriogenology 1994; 41: 231. 104. Vajta G, Murphy C, Macháty Z, et al. In straw dilution of in vitro produced bovine blastocysts after vitrification with the open pulled straw (OPS) method. Vet Rec 1999; 144: 180–1. 105. Cuello C, Gil MA, Parrila I, et al. In vitro development following one-step dilution of OPS vitrified porcine blastocysts. Theriogenology 2004; 62: 1144–52. 106. Tecirlioglu RT, French AJ, Lewis IM, et al. Birth of a cloned calf derived from a vitrified cloned embryo. Reprod Fertil Dev 2004; 15: 361–6. 107. Isachenko V, Alabart JL, Dattena M, et al. New technology for vitrification and field (microscope free) warming and transfer of small ruminant embryos. Theriogenology 2003; 59: 1209–18. 108. Nagashima H, Kashiwazaki N, Ashman RJ, et al. Removal of cytoplasmic lipid enhances the tolerance of porcine embryos to chilling. Biol Reprod 1994; 51: 618–22. 109. Dobrinsky JR, Nagashima H, Pursel VG, et al. Cryopreservation of swine embryos with reduced lipid content. Theriogenology 1999; 51: 164. 110. Nagashima H, Cameron R, Kuwayama M. Survival of porcine delipated oocytes and embryos after cryopreservation by freezing or vitrification. J Reprod Dev 1999; 45: 167–76. 111. Beebe LFS, Cameron RDA, Blackshaw AW, et al. Piglets born from centrifuged and vitrified early and peri-hatching blastocysts. Theriogenology 2002; 57: 2155–65.

112. Esaki R, Ueda H, Kurome M, et al. Cryopreservation of porcine embryos derived from in vitro-matured oocytes. Bio Reprod 2004; 71: 432–7. 113. Du Y, Kragh PM, Zhang X, et al. Successful vitrification of parthenogenetic porcine blastocysts produced from delipated in vitro matured oocytes. Reprod Fertil Dev 2006; 18: 153. 114. Li R, Lai L, Wax D, et al. Cryopreservation of porcine embryos derived from somatic cell nuclear transfer. Reprod Fertil Dev 2006; 18: 159. 115. Dinnyes A, Carolan C, Lonergan P, et al. In vitro survival of IVF bovine embryos frozen or vitrified by techniques suitable for direct transfer. Theriogenology 1995; 43: 197. 116. Dinnyes A, Wallace GA, Rall WF. Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing methods. Mol Reprod Dev 1995; 40: 429–35. 117. Men H, Monson RL, Rutledge JJ. Effect of meiotic stage and maturation protocols on bovine oocyte’s resistance to cryopreservation. Theriogenology 2002; 57: 1095–103. 118. Veeck LL. Does the developmental stage at freeze impact on clinical results post-thaw? Reprod Biomed Online 2003; 6: 367–74. 119. Pool TB, Leibo SP. Cryopreservation and assisted human conception. Introduction. Reprod Biomed Online 2004; 9: 132–3. 120. Pribenszky C, Molnár M, Cseh S, et al. Improving post-thaw survival of cryopreserved mouse blastocysts by hydrostatic pressure challenge. Anim Reprod Sci 2005; 87: 143–50. 121. Vanderzwalmen P, Bertin G, Debauche Ch, et al. Births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod 2002; 17: 744–51. 122. Son WY, Yoon SH, Yoon HJ. Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Hum Reprod 2003; 18: 137–9. 123. Hiraoka K, Hiraoka K, Kinutani M, et al. Blastocoele collapse by micropipetting prior to vitrification gives excellent survival and pregnancy outcomes for human day 5 and 6 expanded blastocysts. Hum Reprod 2004; 19: 2884–8. 124. Mahmoudzadeh AR, van Soom A, Ysebaert MT, et al. Comparison of two-step vitrification versus controlled freezing on survival of in vitro produced cattle embryos. Theriogenology 1994; 42: 1387–97. 125. Wurth IA, Reinders JMC, Rall WF, et al. Developmental potential of in vitro produced bovine embryos following cryopreservation and singleembryo transfer. Theriogenology 1994; 42: 1275–84. 126. Reinders JMC, Wurth YA, Kruip TAM. From embryo to a calf after embryo transfer, a comparison of in vivo and in vitro produced embryos. Theriogenology 1995; 43: 306. 127. Agca Y, Monson RL, Northey DL, et al. Transfer of fresh and cryopreserved IVP bovine embryos: normal calving, birth weight and gestation lengths. Theriogenology 1998; 50: 147–62. 128. Check JH, Choe JK, Nazari A, et al. Fresh embryo transfer is more effective than frozen for donor oocyte recipients but not for donors. Hum Reprod 2001; 16: 1403–8.

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The human embryo: vitrification 129. Nagy ZP, Taylor T, Elliott T, et al. Removal of lysed blastomeres from frozen–thawed embryos improves implantation and pregnancy rates in frozen embryo transfer cycles. Fertil Steril 2005; 84: 1606–12. 130. Elliott TA, Colturato LF, Taylor TH, et al. Lysed cell removal promotes frozen–thawed embryo development. Fertil Steril 2007; 87: 1444–9. 131. Vanderzwalmen P, Delval A, Chatrziparasidou A, et al. Pregnancy after vitrification of human day 5 embryos. Hum Reprod 1997; 12(Suppl): 98. 132. Mukaida T, Wada M, Takahashi K, et al. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Hum Reprod 1998; 13: 2874–9. 133. Park SP, Kim EY, Oh JH, et al. Ultra-rapid freezing of human multipronuclear zygotes using electron microscope grids. Reproduction 2000; 15: 1787–90. 134. Saito H, Ishida GM, Kaneko T, et al. Application of vitrification to human embryo freezing. Gynecol Obstet Invest 2000; 49: 145–9. 135. Yokota Y, Sato S, Yokota M, et al. Successful pregnancy following blastocysts vitrification. Hum Reprod 2000; 15: 1802–3. 136. Yokota Y, Sato S, Yokota M, et al. Birth of a healthy baby following vitrification of human blastocysts. Fertil Steril 2001; 75: 1027–9. 137. Liebermann J, Tucker MJ. Effect of carrier system on the yield of human oocytes and embryos as assessed by survival and developmental potential after vitrification. Reproduction 2002; 124: 483–9. 138. Reed ML, Lane M, Gardner DK, Jensen NL, Thompson J. Vitrification of human blastocysts using the cryoloop method: successful clinical application and birth of offspring. J Assist Reprod Genet 2002; 19: 304–6. 139. Son WY, Yoon SH, Park SJ, et al. Ongoing twin pregnancy after vitrification of blastocysts produced by in-vitro matured oocytes retrieved from a woman with polycystic ovary syndrome: case report. Hum Reprod 2002; 17: 2963–6. 140. Isachenko V, Montag M, Isachenko E, et al. Aseptic technology of vitrification of human pronuclear oocytes using open-pulled straws. Hum Reprod 2005; 20: 492–6. 141. Liebermann J, Tucker MJ. Vitrifying and warming of human oocytes, embryos, and blastocysts: vitrification procedures as an alternative of conventional cryopreservation. Methods Mol Biol 2004; 254: 345– 64. 142. Zheng WT, Zhuang GL, Zhou CQ, et al. Comparison of the survival of human biopsied embryos after cryopreservation with four different methods using non-transferable embryos. Hum Reprod 2005; 20: 1615–18. 143. Stehlik E, Stehlik J, Katayama KP, et al. Vitrification demonstrates significant improvement versus slow freezing of human blastocysts. Reprod Biomed Online 2005; 11: 53–7. 144. Ménézo YJR. Blastocyst freezing. Eur J Obstet Gynaecol Reprod Biol 2004; 1158: S12–15. 145. Klug S, Merker HJ, Jackh R. Effects of ethylene glycol and metabolites on in vitro development of rat embryos during organogenesis. Toxicol In Vitro 2001; 15: 635–42.

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146. Chen SU, Lien YR, Cheng YY, et al. Vitrification of mouse oocytes using closed pulled straws (CPS) achieves a high survival and preserves good patterns of meiotic spindles, compared with conventional straws, open pulled straws (OPS) and grids. Hum Reprod 2001; 11: 2350–6. 147. Kong IK, Lee SI, Cho SG, et al. Comparison of open pulled straw (OPS) vs glass micropipette (GMP) vitrification in mouse blastocysts. Theriogenology 2000; 53: 1817–26. 148. Hredzak R, Ostro A, Zdilova V. Clinical experience with a modified method of human embryo vitrification. Ceska Gynekol 2005; 70: 99–103 [in Slovakian]. 149. Lopez-Bejar M, Lopez-Gatius F. Nonequilibrium cryopreservation of rabbit embryos using a modified (sealed) open pulled straw procedure. Theriogenology 2002; 58: 1541–52. 150. Cremades N, Sousa M, Silva J. Experimental vitrification of human compacted morulae and early blastocysts using fine diameter plastic micropipettes. Hum Reprod 2004; 19: 300–5. 151. Isachenko V, Alabart JL, Vajta G, et al. Double cryopreservation of rat embryos at different developmental stages with identical vitrification protocol: the not properly understood phenomenon. In: Abstracts of the Winter Meeting of Society for the Study of Fertility, Utrecht, Holland. J Reprod Fertil 2000; 26 (Abstract Series): 10. 152. Tominaga K, Hamada Y. Gel-loading tips as container for vitrification of in vitro-produced bovine embryos. J Reprod Dev 2001; 47: 259–65. 153. Vieria AD, Mezzalira A, Barieri DP, et al. Calves born after open pulled straw vitrification of immature bovine oocytes. Cryobiology 2002; 45: 91–4. 154. Hou YP, Dai YP, Zhu SE, et al. Bovine oocytes vitrified by the open pulled straw method and used for somatic cell cloning supported development to term. Theriogenology 2005; 64: 1381–91. 155. Booth PJ, Vajta G, Høj A, et al. Full-term development of nuclear transfer calves produced from open-pulled straw (OPS) vitrified cytoplasts. Theriogenology 1999; 51: 99–1006. 156. French AJ, Hall VJ, Korfiatis NT, et al. Viability of cloned bovine embryos following OPS vitrification. Theriogenology 2002; 57: 413. 157. French AJ, Lewis IM, Ruddock NT, et al. Generation of aS1 casein gene transgenic calves by nuclear transfer. Biol Reprod 2003; 68: 240. 158. Peura TT, Hartwich KM, Hamilton HM, et al. No differences in sheep somatic cell nuclear transfer outcomes using serum starved or actively growing donor granulosa cells. Reprod Fertil Dev 2003; 15: 157–65. 159. Fujihira T, Kishida R, Fukui Y. Developmental capacity of vitrified immature porcine oocytes following ICSI: effects of cytochalasin B and cryoprotectants. Cryobiology 2004; 49: 286–90. 160. Fujihira T, Nagai H, Fukui Y. Relationship between equilibration times and the presence of cumulus cells, and effect of taxol treatment for vitrification of in vitro matured porcine oocytes. Cryobiology 2005; 51: 339–43. 161. Kobayashi S, Takei M, Kano M, et al. Piglets produced by transfer of vitrified porcine embryos after stepwise dilution of cryoprotectants. Cryobiology 1998; 36: 20–31.

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162. Berthelot F, Martinat-Botté F, Perreau, et al. Birth of piglets after OPS vitrification and transfer of compacted morula stage embryos with intact zona pellucida. Reprod Nutr Dev 2004; 1: 267–72. 163. Men H, Agca Y, Critser E, et al. Beneficial effects of serum supplementation during in vitro production of porcine embryos on their ability to survive cryopreservation by the open pulled straw vitrification. Theriogenology 2005; 64: 1340–9. 164. Maclellan LJ, Carnevale EM, Coutinho da Silva MA, et al. Pregnancies from vitrified equine oocytes collected from super-stimulated and non-stimulated mares. Theriogenology 2002; 58: 911–19. 165. Piltty K, Lindeberg H, Aalto J, et al. Live cubs born after transfer of OPS vitrified–warmed embryos in

the farmed European polecat (Mustela putorius). Theriogenology 2004; 61: 811–20. 166. Crichton EG, Bedows E, Miller-Lindholm AK, et al. Efficacy of porcine gonadotropins for repeated stimulation of ovarian activity for oocyte retrieval and in vitro embryo production and cryopreservation in Siberian tigers (Panthera tigris altaica). Biol Reprod 2003; 68: 105–13. 167. Iwayama H, Hochi S, Kato M, et al. Effects of cryodevice type and donors’ sexual maturity on vitrification of minke whale (Balaenoptera bonaerensis) oocytes at germinal vesicle stage. Zygote 2004; 12(4): 333–8. 168. Vajta G, Nagy ZP. Are programmable freezers still needed in the embryo laboratory? Reprod Biomed Online 2006; 12: 779–96.

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22 Managing the cryopreserved embryo bank Phillip Matson

Introduction The cryopreservation of embryos certainly increases the chance of pregnancy from the initial oocyte collection, and several recipes for the freezing process have been described to optimize embryo survival. However, the correct identification of frozen embryos and their effective management once in storage is just as important in assuring that difficulties do not arise because embryos are lost, stored illegally, or used inappropriately. Accurate records and documentation are essential in this time of increased accountability to professional peers, legislators, patients, and society as a whole.

Legislation There are many countries now with legislation in place, and some aspects of the legislation can be helpful and others quite frustrating. However, the fact remains that any legislation must be obeyed or severe penalties may be incurred such as fines, revocation of licenses, or even imprisonment. Examples of pieces of legislation relevant to the cryopreservation of embryos include prohibition of the freezing 1 of cleaved embryos in Germany and the limit of storage of embryos in the first instance of 3 years in Western Australia.2 One should also be mindful of the existence of local legislation, as exists in several states in Australia, rather than having a single federal law, and the extent to which one law may affect activity elsewhere (e.g. local legislation prohibits the creation of embryos in Western Australia if those embryos are to be moved somewhere else to perform a procedure forbidden under the West Australian Act). The introduction of new legislation is always going to cause some problems and upset the status quo. However, the largest difficulty encountered thus far seems to be when legislation is introduced governing in vitro fertilization (IVF) and related techniques, and this new law is then applied to embryos in storage that were frozen under a different set of rules before the act came into being. The most glaring example was that of the Human Fertilisation and Embryology

Act 1990 in the UK. The new act came into force on August 1, 1991, and one of the effects of the act was to introduce a maximum storage period of 5 years to prevent embryos from being held in storage after contact was lost from the genetic parents. All patients having embryos frozen after this time were told of the 5-year limit. However, embryos frozen before August 1, 1991 had the 5-yearcountdown begin on that day, whether patients knew about the introduction of the act or not. By the time the 5 years had passed for embryos frozen before the act, there were still some remaining in storage, and without the formal consent of the genetic parents for continued storage. According to the act, it was then unlawful for clinics to store the embryos after August 1, 1996, and some 3000–4000 embryos were destroyed.3 There was much heated debate over this,4,5 although some workers felt that more effort could have been made earlier to avert the disaster6 and that effective consents regarding the possible fate of the stored embryos completed at the time of treatment would have also been useful.7 The lawful operation of a cryopreserved embryo bank is paramount. Measures must therefore be in place to check that the law is being adhered to. This will help to protect both the operators of the embryo bank and the patients whose embryos are in storage from either honest mistakes or fraudulent activity.8

Laboratory accreditation In line with legislation as mentioned above, several countries have Codes of Practice or similar regulations that require a laboratory to maintain minimal standards. Failure to comply with these minimal standards will prevent the laboratory from being authorized to undertake IVF and associated techniques including embryo storage. Apart from matters directly affecting the stored embryos, this regulation usually extends to issues such as the qualifications of staff, occupational health and safety, suitability of equipment to perform the nominated tasks, and appropriate laboratory manuals and documentation. All laboratories wishing to store embryos must ensure that they comply with local regulations. Similarly, any accredited laboratory

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the donor could only withdraw consent prior to the primary use of the donated semen, i.e. before the fertilization of oocytes, but not thereafter.

sending embryos to another laboratory must ensure that the receiving laboratory is also accredited and complying with their local regulations.

Consents

Timing of thawing

The volume and complexity of information sheets and consent forms for an IVF procedure these days can be very daunting. However, one should see the signing of consent forms as a way of documenting the wishes of patients and securing some protection from future disaster, even though their worth in a court of law may be limited. Points that require consideration include the need for:

A decision should be made upon the strategy used for the timing of the replacement relative to either a luteinizing hormone (LH) surge, the triggering of ovulation with human chorionic gonadotropin (hCG), or within an artificial cycle stimulated with exogenous steroids. This decision should then apply to all cases. Once the day of embryo transfer has been decided upon, a decision then needs to be made about the timing of the thaw. Options then depend upon:

1. 2.

3.

4.

Both partners of a couple to sign the consent forms prior to treatment. A new consent form to be signed at the beginning of each treatment cycle, including cycles for the replacement of frozen embryos. This is illustrated by a local case in which a woman became pregnant from the transfer of thawed embryos and then separated from her husband. He then claimed that he did not know that she had had the embryos transferred, would not have agreed, and therefore should be exempt from the financial maintenance of the child. It was confirmed in court that he had signed a consent form and therefore had to fulfill his parental obligations, but it might have been different if the form had not been completed. Equally, it would have been unfair if the man had not wished the embryos to be used when he had some genetic investment in them. Several clinics continue to use the original IVF consent form to cover the transfer of frozen–thawed embryos generated in that cycle, even though the transfer of frozen embryos can take place several months or even years later, and the limitation of that approach should not be underestimated. Such forms to be witnessed. Many patients do not like to ask family members, neighbors, or colleagues to sign such forms because of the private nature of the treatment. Legal opinion should therefore be sought regarding the validity of the couple witnessing each other’s signature, probably in the absence of a clinic staff member. The consents of donors to be clarified within the local legal framework. Fuscaldo9 describes the example of how in Victoria, Australia, the consent of a sperm donor was given for the use of the donated sperm but was later withdrawn. This seemed reasonable, regarding the use of semen for insemination, but the problem arose because of frozen embryos that had been created with the donated sperm and were still in storage. The donor then requested that the embryos not be used, and this was in conflict with the wishes of the couple in whose name the embryos were stored. The latest interpretation of Victorian law would suggest that

1.

2.

3.

4.

The stage of the embryos in storage. Embryos can be frozen at a number of developmental stages, including the pronucleate, 4-cell (i.e. day 2 after oocyte collection), 8-cell (i.e. day 3), or blastocyst stage. Hence, pronucleate oocytes would need to be thawed 24 hours earlier than say a group of 4-cell embryos, and so on. Accurate records of the stage of freezing are therefore required, particularly if the embryos are being imported from another clinic. The philosophical decision of whether the embryos should be in phase or not with the endometrium. Some laboratories thaw out the embryos so that they are ahead of the endometrium, presumably in the belief that the embryo can take a few hours to resume its function, whereas others find synchronous transfers to give better pregnancy rates.10 The requirement to see division of the thawed embryos as part of the survival criteria. Most laboratories would thaw out the pronucleate oocytes and culture at least overnight, as with the original IVF cycle. However, some laboratories culture day-2 embryos before transfer,11,12 whereas others simply thaw out on the day of transfer. There appears to be no hard evidence as to which strategy is best, and the individual needs of the clinic may become the overriding factors. For example, if clinicians routinely perform embryo transfers very early in the morning because of other work commitments, then the laboratory may favor thawing the embryos out the day before. The survival of thawed embryos. Consideration should be given to the definition of survival, and the possible need to thaw out additional embryos if one or more has not survived. Damage to earlycleavage embryos does seem to reduce the potential for implantation.13 However, if it is only on the next day following culture that the damage is discovered (as can be the case with either pronucleate or early-cleavage embryos), the thawing of more embryos may not be a simple option if the ones remaining in storage are then out of phase. This problem has led some laboratories to try to freeze a

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mixture of pronucleate oocytes and early-cleavage embryos in an IVF cycle, so that the pronucleates can be thawed first and then the early-cleavage embryos can be thawed the next day if one of the pronucleate oocytes has not divided.14

Management of embryos in storage Embryos stored in liquid nitrogen seem to be fairly stable providing that the storage conditions are well kept, suggesting that the survival and functional capacity of embryos are not compromised by prolonged storage.15 The main concerns regarding embryo storage would therefore seem to relate to matters of housekeeping and legal constraints.

Record keeping and labeling Before setting up a storage bank, whether it be embryos or gametes, it is important to ensure that there is an efficient system in place for labeling the storage item, whether it be a straw or ampule. The labeling must clearly and accurately indicate the name and unique identifying code for the owner of the stored material, and this labeling must be stable and not erode with time. The location within the storage Dewar must also be recorded so that the items can be found easily and with the minimum disruption to other stored material.

Stocktaking The number of embryos placed in storage within any IVF laboratory will inevitably increase with time. Conversely, there will be removal of a number of embryos for use, transport to another location, donation, or disposal. Regular stocktaking is therefore an important part of the management of a cryobank to reconcile what is actually in storage with the records. During such stocktaking, straws or ampules that have become dislodged from their storage location can be identified and returned safely to their correct position.

Duration of storage Many places have local legislation that puts a limit on the time of storage of embryos before an extension is required from the local regulatory body, such that the storage of embryos by the clinic beyond the allowed date is an offence. Examples of that include the Human Fertilisation and Embryology Act in the UK (5 years’ storage in the first instance), and the Human Reproductive Technology Act in Western Australia (3 years’ storage initially). In such cases, it is important to obtain a directive from the patients that can confirm that an extension is required, and so clinics in these countries will usually contact patients before the expiry of the storage period, e.g. 6 months prior to the expiry date.16 Postal communication with patients

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appears the easiest method, but there will be a group of patients who do not respond17 and will require other means of contact.

Unwanted embryos Upon the completion of a family or the decision not to have any further treatment, patients may well have embryos remaining in storage that they do not wish to use. The fate of such stored embryos has been the subject of many reports, usually with a view to the possibility of embryos that are no longer wanted being a source of donated embryos. Recipients of donated eggs seem more likely to donate their embryos.18 However, more IVF patients who do not wish to retain the embryos would choose to discard them rather than donate them to another couple16,19,20 or research. A method of disposal of embryos should therefore be documented and approved by the local regulatory group, even if it is the institutional ethics committee that oversees the running of the clinic.

Liquid nitrogen levels The maintenance of a sufficiently low temperature in the storage Dewar relies upon a minimum level of liquid nitrogen being maintained. Regular topping up of the Dewars with liquid nitrogen is therefore imperative. Alarms to detect low levels of nitrogen are also strongly recommended, or even mandatory in some countries. Such alarms use a probe which warms in the absence of liquid nitrogen and sounds an alarm (Fig 22.1). These alarms can be connected on-line to page a staff member for immediate attention. The maintenance of a low temperature must also occur when embryos are moved from one Dewar to another, and so it is prudent to keep the straws or vials holding the embryos under nitrogen during their handling. This can be achieved by having a small bath or similar containing liquid nitrogen, which is used as an interim holding vessel while the embryos are removed from or placed in the main storage vessel. Warming of embryos during handling can result in reduced survival, and this may well be even more important with embryos that have been vitrified in the absence of ice crystal formation.

Transport of embryos Many people now migrate to new cities or countries because of changes in their career or lifestyle, and so transportation of their frozen gametes and embryos is becoming more common. Thus, IVF clinics worldwide encounter the trials and tribulations of transporting frozen embryos. A ‘dry shipper’ is used for such transportations (Fig 22.2). This is a transportation vessel with a vacuumsealed inner core that absorbs liquid nitrogen. The shipper is ‘charged’ by pouring liquid nitrogen into it over consecutive days until the level of liquid inside

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Fig 22.1

Alarm to detect low level of liquid nitrogen.

does not change, indicating that the inner core is saturated. Prior to transportation, the free liquid is poured off, leaving only liquid nitrogen vapor to keep the contents cool, and thus its name, a ‘dry’ shipper. Dry shippers come in a variety of sizes and life spans for keeping the contents cool, ranging from 7 days to 21 days. They are fragile and are normally transported in a strong case with foam protection. If the vacuum is broken, the shipper will start to warm up and the contents inside will thaw. All patients should therefore be advised by the IVF clinic of the risks associated with the dry shipper, and indeed also general transport risks with couriers, etc., before proceeding with the transportation. The dry shippers are exempt from International Air Transport Association (IATA) packaging instruction 202, and are therefore not classed as dangerous goods because there is no free liquid as such. This exemption permits the shipper to be carried by normal courier companies and even be taken on airplanes. International transportation involves correct customs documentation to be completed for both the country of entry and the country from which the shipper is leaving, even if the shipper is empty. This should state that the dry shipper is not dangerous goods, and indicate how many straws or ampules of noninfectious human tissue are in the shipper and also the names and contact details of the participating clinics. Embryos are classified under the category of human tissues and fluids and are noninfectious, and therefore quarantine permits are not required. However, it

Fig 22.2

‘Dry shipper’ for transport of embryos.

is important that, with every vessel, there is a description provided by the IVF clinic of what is in the shipper, e.g. noninfectious human tissue, so as not to be held up by quarantine to delay the transit time. Some states or cities require written approval by the governing reproductive authority before movements of embryos or sperm can take place, e.g. The Infertility Authority of the state of Victoria, Australia. The clinic should obtain written consent from the patients stating that they would like to transport their gametes away from the clinic to a nominated laboratory. When any embryos or sperm either leave or arrive at an IVF clinic, updating the records of where they were or will be stored must be accurately maintained, and all appropriate information regarding the gametes exchanged between clinics. It is also useful to find which freezing method was used and which thaw method is recommended by the clinic where the gametes were frozen.

Risk of infection The storage of ampules or straws in liquid nitrogen with material from other patients must carry some risk of the transfer of infectious agents because (1) liquid nitrogen freezers are not sterile and will eventually become contaminated with potential pathogens, albeit at a low level,21 (2) serologic testing of IVF patients will not always detect the presence of current infection because of the lag for seroconversion in some diseases, and (3) many microorganisms can survive freezing and

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thawing.22 Whereas there has been one recorded case of the transmission of hepatitis B during the storage of bone marrow due to the leakage of a storage bag,23 there does not seem to have been any report of the transfer of infection with embryos, and so the risk would appear to be infinitesimally small. The storage of embryos from patients that have a known infection is another matter. Many laboratories opt to reserve storage tanks exclusively for those infected with a particular disease, e.g. hepatitis B, but patients should be aware that their embryos will be kept with the embryos of other infected people. If patients are unduly concerned, then they perhaps might consider the purchase of a small vessel for their own exclusive use. The availability and use of highsecurity straws24 may well reduce the risk of crossinfection further.

Summary The storage of human embryos is now often governed by local legislation, or has the potential for legal proceedings should something go wrong. Appropriately qualified staff, accurate records, and a clearly delineated protocol for managing the embryo bank should therefore be in place within the laboratory. The completion of consent forms should be used as a safeguard to ensure that the wishes of clients (the husband, wife, or donor) are known and adhered to wherever possible. The strategy for timing of the thaw of embryos should be clearly laid out in the laboratory manual for the various kinds of replacement cycles, and an approved method of embryo disposal should exist for those patients not wishing to keep or donate their embryos.

References 1. Beier HM, Beckman JO. German Embryo Protection Act (October 24, 1990): Gesetz zum Schutz von Embryonen (Embryonenshutzgesetz-EschG). Hum Reprod 1991; 6: 605–6. 2. Yovich JL, Matson PL. Legislation on the practice of assisted reproduction in Western Australia. J In Vitro Fert Embryo Transf 1996; 13: 197–200. 3. Wise J. Storage period ends for 4000 embryos. Br Med J 1996; 313: 189. 4. Edwards RG, Beard HK. UK law dictated the destruction of 3000 cryopreserved human embryos. Hum Reprod 1997; 12: 3–5. 5. Deech R. A reply from the Chairman of the HFEA. Hum Reprod 1997; 12: 5–6. 6. Schafer D, Kettner M. Moral concern over cryopreserved human embryos: too much or too little? Hum Reprod 1997; 12: 10–11. 7. Dickey RP, Krentel JB. Storage of sperm and embryos. Couples having IVF should be asked their wishes about spare embryos before egg retrieval. Br Med J 1996; 313: 1078–9.

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8. Smith L. Conman is jailed for IVF fraud. The Times 2003; 16 January: 3. 9. Fuscaldo G. Gamete donation: when does consent become irrevocable? Hum Reprod 2000; 15: 515–19. 10. Mandelbaum J, Junca AM, Plachot M, Cohen J, SalatBaroux J. Timing of embryo transfer and success of pregnancy in the human. Reprod Nutr Dev 1988; 28: 1763–71. 11. Van der Elst J, Van den Abbeel E, Vitrier S, et al. Selective transfer of cryopreserved human embryos with further cleavage after thawing increases delivery and implantation rates. Hum Reprod 1997; 12: 1513–21. 12. Ziebe S, Bech B, Petersen K, et al. Resumption of mitosis during post-thaw culture: a key parameter in selecting the right embryos for transfer. Hum Reprod 1998; 13: 178–81. 13. Van den Abbeel E, Camus M, Van Waesburghe L, Devroey P, Van Steirteghem AC. Viability of partially damaged human embryos after cryopreservation. Hum Reprod 1997; 12: 2006–10. 14. Horne G, Crithclow JD, Newman MC, et al. A prospective evaluation of cryopreservation strategies in a two-embryo transfer programme. Hum Reprod 1997; 12: 542–7. 15. Cohen J, Inge KL, Wiker SR, et al. Duration of storage of cryopreserved human embryos. J In Vitro Fert Embryo Transf 1988; 5: 301–3. 16. Darlington N, Matson P. The fate of cryopreserved human embryos approaching their legal limit of storage within a West Australian in vitro fertilization clinic. Hum Reprod 1999; 14: 2343–4. 17. Brzyski RG. Efficacy of postal communication with patients who have cryopreserved pre-embryos. Fertil Steril 1998; 70: 949–51. 18. Sehnert B, Chetkowski RJ. Secondary donation of frozen embryos is more common after pregnancy initiation with donated eggs than after in vitro fertilization–embryo transfer and gamete intrafallopian transfer. Fertil Steril 1998; 69: 350–2. 19. Lornage J, Chorier H, Boulieu D, Mathieu C, Czyba JC. Six year follow-up of cryopreserved human embryos. Hum Reprod 1995; 10: 2610–16. 20. Hounshell CV, Chetkowski RJ. Donation of frozen embryos after in vitro fertilization is uncommon. Fertil Steril 1996; 66: 837–8. 21. Fountain D, Ralston M, Higgins N, et al. Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components. Transfusion 1997; 37: 585–91. 22. Leiva JL, Peterson EM, Wetkowski M, da la Maza LM, Stone SC. Microorganisms in semen used for artificial insemination. Obstet Gynecol 1985; 65: 669–72. 23. Tedder RS, Zuckerman MA, Goldstone AH, et al. Hepatitis B transmission from contaminated cryopreservation tank. Lancet 1995; 346: 137–40. 24. Letur-Konirsch H, Devaux A, Collin G et al. Viral risk and straw for cryopreservation. A preliminary experimental study with HIV1. Presented at the 11th World Congress on In Vitro Fertilization and Human Reproductive Genetics, Sydney, May 1999.

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23 Cryopreservation and storage of spermatozoa Eileen A McLaughlin, Allan A Pacey

Overview Human semen cryopreservation has a long history that stretches back over 200 years to the first recorded experiments involving cooling followed by successful rewarming of spermatozoa in snow.1 Despite this early success, it was not until the fortuitous discovery of glycerol as a cryoprotectant,2 and the subsequent live birth of a calf3 in the early 1950s, that cryopreservation of human semen for assisted reproduction became a realistic option. The ability to store human semen greatly improved the flexibility of donor insemination treatment, resulting in the first live human births in 19534 and donor insemination (DI) became a mainstay of fertility treatment for the next 40 years.5 More recently, sperm cryopreservation has become an essential part of fertility preservation for young men who face the possibility of sterility as a consequence of the treatments given to combat cancer.6 As cryopreservation of human semen results in a significant loss of spermatozoal motility and viability,7–9 with considerable variation between ejaculates of different individuals, only semen from a highly selected population of men are suitable as donors for DI.10,11 Reasons for the differences between individuals in cryosurvival rates are only just beginning to be elucidated,11–13 but the ability to predict post-thaw survival remains limited.14 For men storing their own sperm prior to cancer treatment, this means that they may face complex fertility treatments, such as intracytoplasmic sperm injection (ICSI), if their fertility does not recover and their frozen samples were initially poor or did not freeze well.6 With the availability of fresh semen, coupled with the fact that some human spermatozoa survive the freeze– thawing process tolerably well,15 little pressure to optimize semen cryopreservation protocols existed until the mid-1980s. Following the infection of four recipients with human immunodeficiency virus (HIV) after insemination with semen from a seropositive donor,3 the use of quarantined cryopreserved semen became mandatory in donor insemination.16–18 A small number of studies suggested that, whereas cryopreserved semen was equally fertile to fresh semen when used for in vitro fertilization (IVF),19,20 a significant decrease in conception rates was observed in vivo.21 This was linked largely to the reduction in the number of

motile sperm inseminated,22–24 and confirmed by analysis of donor fecundity related to numbers of motile sperm per straw post-thaw.25 Cryobiologists and reproductive biologists began to question the largely empirical approach taken towards semen cryopreservation and artificial insemination, and during the past decade a number of specific studies on the effects of freezing and thawing on human spermatozoa have been undertaken.26 These culminated with the investigation of semen preparation methods, complex cryoprotectants, novel forms of packaging, and the use of programmable freezing machines with variable freezing rates as methods of optimizing the recovery of motile morphologically normal spermatozoa post-thaw. Biosecurity has become a major issue, with surveys of clinical practice suggesting that a wide range of protocols are in operation requiring significant standardization across a variety of laboratory practices to ensure the safe cryopreservation of semen for the large cohort of patients currently benefiting from fertility preservation and assisted reproduction.27,28

Methods Semen preparation pre-freeze and post-thaw Traditionally, whole semen has been diluted with an appropriate volume of cryoprotectant prior to packaging and freezing. This method is largely applicable to goodquality samples used for intracervical insemination (ICI).29 As ICI pregnancy rates have been consistently lower than natural fecundity, some medical practitioners in their quest to improve conception rates have opted for prepared semen, suitable for intrauterine insemination (IUI) immediately post-thaw.30 This involves the separation of the spermatozoa from seminal plasma, either by a simple washing technique or the use of density gradients such as Isolate or PureSperm,31 and resuspension of the sperm in a suitable culture media such as Ham’s F10.2,32,33 In general, density gradient semen preparation improves both the concentration and the motility characteristics of spermatozoa available for insemination,34 with several studies suggesting that donor insemination pregnancy rates have benefited.30 Separation of goodquality sperm from those exhibiting defective characteristics such as that associated with peroxidative damage,

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induced by factors including xenobiotic exposure, antioxidant deficiency, leukocytospermia, or aberrant free radical generation by the spermatozoa themselves,35,36 may contribute substantially to the improved fertility rates achieved. Further selection of functional spermatozoa can be accomplished by selectively removing apoptotic sperm cells displaying externalized phosphatidylserine using annexin V coated magnetic beads, thus enriching for fertile sperm.37 While seminal plasma seems to confer a beneficial effect during cryopreservation,38 studies comparing the fertilizing potential of prepared spermatozoa have not demonstrated an impaired ability of the thawed sperm to bind to homologous zona pellucida39 or to fertilize after ICSI.40 In addition, advancements in oncology treatment with enhanced post-therapy long-term survival rates have resulted in an increased demand for the banking of sperm in order to preserve the reproductive potential of young male cancer patients.41,42 Epidemiological estimates suggest that by the early 21st century, 1 in 750 people in developed nations will have survived a childhood cancer.43 As these men will often have impaired semen characteristics and will require assisted conception treatment to achieve a pregnancy,43 concentration of their few viable spermatozoa into a small volume may be warranted prior to cryopreservation and assisted fertilization.6 Similarly, the provision of cryostorage of spermatozoa prior to chemotherapy and/or radiotherapy for men with testicular cancer or Hodgkin’s lymphoma is now a recognized treatment norm,44 and offers a significant psychological boost to men diagnosed with a potentially life-threatening condition.45 The advent of a consistently successful assisted fertilization technique, i.e ICSI,46 allows men with severe oligozoospermia or obstructive azoospermia to achieve pregnancies in vitro. In order to minimize unnecessary medical treatment of the female partner (such as superovulation and oocyte recovery) and to maximize the number of cycles of ICSI treatment, elective cryopreservation of the small numbers of surgically retrieved spermatozoa has become a recognized therapeutic option for these couples.47 Latterly, the recognition that men with certain chromosomal and genetics disorders, such as Y chromosome microdeletions and Klinefelter syndrome, have a constantly reducing reservoir of spermatozoa, due to a progressive decline in spermatogenesis, has given rise to a new and somewhat clinically problematic group of patients now benefiting from testicular and epidiymal sperm cryostorage and ICSI.48

Cryoprotectants and extenders Since the discovery of the cryoprotective characteristics of glycerol,2 a substantial number of other chemicals have been shown to have cryoprotective properties.49. These have been divided into two classes: •

•

those that function as permeating cryoprotectants, such as dimethyl sulfoxide, propylene glycol, and glycerol nonpermeating cryoprotectants, e.g. sucrose, raffinose, and glycine.

Glycerol has remained the cryoprotectant of choice for preservation of spermatozoa of most species,50 despite gathering evidence that the optimal cryoprotectant for human spermatozoa is, in fact, ethylene glycol.51 As any change in cryoprotectant will have to be evaluated for consequences on fertility and embryo development, many reproductive medicine laboratories continue to use glycerol, despite the known toxic effects of this compound.52,53 If glycerol is the only cryoprotectant added, then historically a 5–10% v/v final concentration has been used,54,55 with 7.5% considered optimal,56 although one study with higher concentrations (12–16%) had the best post-thaw motility rates.57 In order to improve cryosurvival rates, more complex diluents containing other mainly nonpermeable cryoprotective agents, such as glycine, zwitterions, citrate, and egg yolk, were developed. Among the earliest and bestknown extenders for human semen is glycerol–egg yolk– citrate (GEYC).56 Modifications58–60 are still used today, although evidence based on post-thaw sperm motility rates suggests that GEYC and its derivatives are only marginally better than glycerol alone.61,62 During the early 1980s, influenced by changes in animal semen programs, two other complex cryoprotective diluents, both containing organic buffers, were introduced. The first, termed human sperm-preserving medium (HSPM),59 is a modified Tyrode’s medium containing glycerol (5–7.5% v/v final volume) as well as sucrose, glucose, and glycine as cryoprotective agents, human serum albumin, and HEPES (N-(2-hydroxyethyl) piperazine-N′ (2-ethanesulfonic acid). In the original studies,59 HSPM was comparable with GEYC at maintaining post-thaw spermatozoal motility, with a slightly higher (although not statistically significant) pregnancy rate obtained after intracervical insemination. HSPM is available commercially as SFM (Medicult, Jyllinge, Denmark). The second complex cryoprotective buffer is a zwitterion buffer system termed TESTCY, developed by Jeyendran and colleagues.63 This buffer contains TES (N-tris-(hydroxymethyl)-methyl-2-aminoethanesulfonic acid), TRIS (tris-(hydroxymethyl)-aminomethane), sodium citrate, and egg yolk but no glycerol, and in the initial report it proved superior to glycerol alone as a cryoprotectant.63 This remarkable result was due largely to the very rapid freezing protocol employed and was impossible to duplicate using standard cooling methods.64 TESTCY (now containing 12% glycerol) gave satisfactory cryosurvival rates and is also available commercially (Irvine Scientific, Santa Ana, CA, USA). Studies have compared the recovery of motility post-thaw obtained with the above three main cryoprotective extenders and some of their derivatives.14,64–66 Results are conflicting, with no obviously superior candidate emerging, but this is probably a reflection of the various cooling and thawing rates employed by the different research groups, making comparison difficult, and the inherent variability between individuals in their spermatozoa to withstand the cellular stresses of cryopreservation.

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Cryopreservation and storage of spermatozoa As assisted reproduction enters a new phase of governmental regulation, such as the recent implementation of the European Union Tissues and Cells Directive,67,68 it is now necessary in Europe at least to adopt protocols employing only media and supplements, including cryoprotectants, that conform to pharmaceutical standards.69,70 This is in addition to minimum standards of air quality during the processing of standards, as well as the requirement that sperm banks have total quality management systems in place and have full traceability of all aspects of freezing and storage and record keeping for 30 years.67 After dilution with extender, semen should be packaged and cooled immediately, as evidence suggests that exposure prior to freezing of human spermatozoa to cryoprotectant should be less than 10 minutes in order to have optimal cryosurvival rates.71

Packaging Several forms of packaging have been routinely used by clinical scientists in human semen cryopreservation: (1) cryovials or ampoules;72 (2) straws;5 and (3) 1.0 ml syringes.73 There are advantages and disadvantages to all types of packaging, and cryobiologists need to view these in light of the need to maintain biosecurity within liquid nitrogen tanks for extended periods of time. Cryovials are easy to fill aseptically and hold around 1.0 ml of semen plus cryoprotectant. Storage on canes in goblets within liquid nitrogen tanks is bulky and inefficient, but feasible if only small amounts of semen are to be stored. Placing cryovials in drawers in racking systems is more efficient, but prone to induce fluctuation in storage temperature during retrieval. Screw-top vials do not maintain their seals, and leakage of liquid nitrogen into containers is common, with consequent risk of rupture during thawing. The manufacturers recommend that a secondary skin is used, but in practice this is difficult to employ without compromising the integrity of the gametes.74 One option is to store semen cryovials in liquid nitrogen vapour phase to avoid contamination as a result of leakage of vial contents.75 The original plastic straws or ‘paillettes’ were made of either polyvinyl chloride (PVC) or polyethylene terephthalate glycol (PETG) and have largely been replaced by more robust and secure 0.3/0.5-ml straws made from ionomeric resin (CBS High Security straws). These CBS straws require the use of a vacuum pump and filling nozzle to aseptically aspirate the semen cryoprotectant mixture. Straws are available in a variety of colors suitable for the easy identification of individuals, and many thousands can be stored in plastic goblets in canisters within liquid nitrogen vessels.76 Syringes, which are also difficult to fill aseptically and are impossible to seal safely, have been used in the past but have largely fallen out of favor. The one advantage is that they are ready for insemination, but this is heavily outweighed by the excessive amount of space needed to store multiple ejaculates and the significant biocontainment risk.76 ICSI is now routinely available in most reproductive medicine units and is offered to an increasingly large

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group of men with poor-quality ejaculates. Latterly, several novel methods of cryopreservation packaging have been explored for the freezing and storage of very small numbers of spermatozoa. These have included minidroplets,77,78 mouse and human zona pellucidae,79,80 and cryoloops,81 and even the spherical algae Volvox globulator.82 All of these methods have been designed for use in conjunction with testicular or epididymal sperm retrieval, to maximize the number of IVF attempts possible from a single surgical procedure.

Cooling and freezing Many laboratories continue to use the simple rapid method of suspending straws or ampules in the uncirculated liquid nitrogen vapor phase for a set time period, before plunging the straws into liquid nitrogen for longterm storage. This method requires no specialized equipment and, although not ideal, can give satisfactory cryosurvival rates.83–85 Problems include nonuniform cooling rates, both within and between aliquots of the same ejaculate,86 and difficulties in maintaining reproducible freezing conditions. Programmable freezers which circulate liquid nitrogen vapor at a controlled rate give much more reproducible cooling curves.86–88 The more sophisticated computer-controlled freezing machines allow for several different freezing rates during one cooling curve and holding temperatures to permit manual seeding.8 In-depth studies have shown that sperm cells do not behave in accordance with experimental predictions from observations of somatic cells and that application of traditional linear freezing protocols to these highly differentiated cells is inappropriate.89 Data from ‘controlled concentration’ or nonlinear freezing protocols in novel freezing machines suggest that significant improvements in post-thaw motility, vitality, and fertility may be achievable.89,90

Vitrification One strategy for the management of males with severe oligozoospermia is the use of cryoprotectant-free methods through vitrification.91,92 This has resulted in the successful recovery of motile spermatozoa, following fast freezing by plunging into liquid nitrogen and direct rewarming. This was achieved by loading prepared spermatozoa onto pre-cooled copper loops prior to plunging into liquid nitrogen. After storage, the spermatozoa were thawed by plunging the loops into media at 37°C under intense agitation prior to processing and evaluation.91,92 More recently, one study suggests that despite the less effective cryoprotecting capacity of sucrose, in conjunction with vitrification of spermatozoa in microtubes, is a safer method and has advantages when applied to severely oligozoospermic samples.93

Thawing The cooling rate determines the optimum thawing rate.94 In practice this means a slow cooling rate (1°C/min)

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requires a similar 1°C/min thawing rate, only achievable using a computer-controlled freezer. As most clinical situations require a simpler protocol, a more rapid cooling rate (10°C/min) is compatible with a rapid thawing rate (400°C/min) achieved by removing straws from liquid nitrogen and placing them on the bench top at 22°C.94

Assessment of post-thaw fertility Traditionally, the success of cryopreservation is measured by the number of motile spermatozoa recovered post-thaw.62 However, other effects of cryopreservation such as ultrastructural damage to the plasma membrane and loss of acrosomal contents may contribute to a loss of fertility.95–98 It is recommended that additional tests of functional competence, such as cervical mucus penetration or zona free hamster oocyte fusion testing,99 are applied to assess the potential fertility of cryopreserved spermatozoa. Proteomic analysis of membrane and cytoskeleton components have raised the possibility that fertility loss is correlated with time in storage,100 reinforcing the need for practitioners to monitor storage temperatures carefully. Application of assessment of DNA damage using the SCSA, or sperm chromatin structure assay,101 indicates that cryopreservation also results in a subpopulation of frozen–thawed spermatozoa displaying enhanced loss of chromatin integrity,102 translating into a reduction in fertility post-thaw, possibly through the activation of caspase-derived apoptotic signaling in damaged spermatozoa.103 However, in spite of these observations, successful pregnancies have been reported after sperm have been in cryostorage for 21104 and 28105 years, respectively. During direct insemination of the semen cryoprotectant mixture in simple ICI, it is assumed that the sperm will adjust to the change in osmolality as they enter the female reproductive tract. However, in order to minimize osmotic shock, care is usually advised during the removal of the cryoprotectant. In particular, practitioners should opt for slow dilution with medium, pre-equilibrated to room temperature, prior to washing or density centrifugation.51

Results Minimal number of motile cells per straw The number of motile sperm is dependent on the intended use of the contents of the straw ranging from donor ICI to patient ICSI. In general most authors report an overall fecundity of ≤12% and prospective studies from the French CECOS group have confirmed that conception rates from ICI of frozen–thawed donor semen are related to the number of motile sperm per straw. If <4 × 106 motile sperm were inseminated, the pregnancy rates were 9.1% per cycle, and reached 17.2% when >8 × 106 motile sperm were inseminated in an 0.25-ml straw.25 Intracervical insemination of larger volumes of semen did not significantly improve pregnancy rates,

presumably as only a small volume of semen can be absorbed by the endocervical mucus,106 suggesting that concentration of motile spermatozoa in semen prior to cryopreservation might improve conception rates. The use of insemination devices such as the intracervical cap has not contributed to an improved success in routine donor insemination.107 A straw containing less than the accepted minimum of 4 × 106 motile sperm can be used for IUI, as this involves the removal of seminal plasma and concentration of sperm prior to insemination. Overall, donor insemination pregnancy rates are higher when IUI is compared with ICI,25,108 with pregnancy rates only dropping when <0.5 × 106 motile sperm were inseminated, similar to results obtained with washed fresh patient semen.109 The number of motile sperm per straw of cryopreserved donor semen does not correlate with either fertilization rates or pregnancy rates in IVF,20 reflecting the highly selected nature of the semen stored. However, as the quality of cryopreserved semen cryostored by patients before cancer treatment is often poor, the only therapeutic option may be assisted fertilization. In these cases straws containing very few motile cells are often sufficient to give acceptable fertilization rates using ICSI.40 As very few sperm are required for assisted conception, it is sensible to freeze a small number of sperm per straw (about 40 000) in order to maximize the number of fertilization attempts/patient.

Future directions and controversies Cross-contamination In 1995, a group of six cases of hepatitis B infection occurred in oncology patients after autologous transplantation of bone marrow or peripheral blood stem cells.110 By using molecular testing, it was found that four of these cases were linked to a single infected bone marrow sample stored in the same liquid nitrogen tank.108 Contamination was thought to have occurred via a cracked or leaking cryopreservation bag, as examination of the detritus extracted from the storage tank also detected the same hepatitis B viral DNA. Following this report, the British authorities issued guidelines implementing many changes in tissue and cell banking practice throughout the United Kingdom; this led to the development across Europe of compilation common standards that cover the cryopreservation of all human tissues intended for donation (including heterologous use), excluding blood.67,68 The EU Tissues and Cells Directive sets standards of quality and safety for the donation, procurement, testing, processing, preservation, storage, and distribution of human tissue and cells intended for human application. However, owing to the problems in applying these guidelines to reproductive tissues, many aspects of both gamete and embryo banking still require modification to ensure the safety of staff, recipients, and offspring.

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Liquid nitrogen contamination and sampling It is generally recognized that although liquid nitrogen is effectively sterile at the point of manufacture, there is a significant risk of microbial infection with environmental organisms during storage and distribution. 112 Guidelines issued by the EU Tissues and Cells Directive recommend that liquid nitrogen storage containers and cooling machines be subject to regular cleaning and disinfection. 112 Two problems exist: first, the lack of suitable chemical cleaning agents for use with gametes; and, secondly, the inaccessibility of some potentially contaminated parts of programmable freezing machines. The manufacturers, following consultation with reproductive biologists and cryobiologists, are addressing both these problems. In the interim, clinics may be advised to consider sampling liquid nitrogen from all storage vessels to identify those that may harbor pathogenic organisms.

Packaging and leakage Cracking and leakage from bone marrow cryopreservation bags has been documented, and the UK Department of Health has recommended that all primary packaging is robust and leak-free at storage temperature (−196°C). In addition, to avoid contamination of the cryopreserved cells, the guidelines state that all samples should be encased in a secondary container, double bagged, to prevent external organisms infecting cells or tissues post-thaw.113 Until recently, no leak-free system was available for the storage of human semen. Evidence from workers in the animal field is that semen straws filled and sealed in the traditional dip and wipe method are at significant risk of contaminating the liquid nitrogen storage vessels114 and, theoretically, other ejaculates in storage. This is demonstrated in Fig 23.1. Similarly conclusive evidence of the leakage of liquid nitrogen into screw-top cryovials, within 3 hours of placement into storage,74 is another possible route of cross-contamination. Both the Royal College of Pathologists115 and the manufacturers recommend that vials containing biological material be secondarily sealed using cryoflex tubing (CryoFlex Nalge Nunc International Corp.), before placing them in liquid nitrogen. An alternative form of straw for human semen storage (Fig 23.2), which takes into account the difficulties of aseptic loading and permanent sealing, has been developed in France (Cryo Bio System, IMV Technologies, L’Aigle, France). Preliminary results of microbiological testing, and field trials conducted by the French Ministry of Agriculture suggested that they may substantially reduce the risk of cross-infection in liquid nitrogen storage. Subsequently, the conversion of the entire French Federation CECOS in 2000 to the use of the CBS ‘High Security’ straw and their successful implementation in human semen cryobanking indicates that this is a feasible alternative to the more traditional methods of semen cryopreservation. This packaging

Figure 23.1 Semen diluted with GEYC being aspirated into straws using the traditional method.

system and its competitors have been extensively reviewed76 and recent studies using HIV as a model organism suggest that this is the optimal storage method for human semen.116

Screening of patients and donors As leakage of contents into liquid nitrogen following accidental damage to straws or vials remains a possibility, the guidelines also recommend that steps be taken to minimize the risk of placing potentially infective material in storage by screening patients and donors for major viral markers in advance. All men should therefore be screened for the presence of HIV 1 and 2 antibody, hepatitis B surface antigen (HbsAg), hepatitis C (HVC) antibodies, HTLV I and II antibodies, and syphilis by using the appropriate serological tests. Unscreened samples should not be stored with screened samples and any samples from infected patients should be stored separately in a dedicated container (see A Code of Practice for Tissue banks providing tissues of human origin for therapeutic purposes – http://www.dh.gov.uk/prod_consum_ dh/ groups/dh_digitalassets/@dh/@en/ documents/digitalasset/dh_4079053.pdf). More extreme examples of risk avoidance include the selection of donors thought to have been at risk of having contracted new variant Creutzfeld– Jacob disease (vCJD), to be excluded from semen collection and storage. While this is an ultra conservative strategy to avoid the theoretical risk of vCJD transmission, expert opinion is that this will have had minimal if any impact on recipient safety.117 Clinicians and scientists may find it more useful to direct their attention towards more prevalent semen-contaminating microorganisms such as Chlamydia, Mycoplasma, Candida, and Cytomegalovirus, and take precautions to limit the risk to co-stored samples.118 Clearly, such additional screening is required if samples are intended for donor insemination, and current best practice guidelines include those produced by the British Andrology Society18 and the Practice Committee of the American Society for Reproductive Medicine.16,28.

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Textbook of Assisted Reproductive Technologies Aspiration system

Identification

human semen stored in cryovials in vapor phase.74 Since storage temperatures can vary depending on the equipment employed, some authors have advocated the use of an internal inventory storage system, which is made from materials of high conductance, packed tightly together to maintain the coolest temperature required (−160°C) at the top of the container.27

Conclusions

Sealing plug Semen plus cryoprotectant

Air gap

Filling nozzle

Heat-sealed ends

Optimization of cryoprotective diluents and freezing protocols for human spermatozoa remains a challenge to the reproductive biologist. Determination of fundamental cryobiological characteristics has contributed to improved cryopreservation protocols in a number of other species.50,119,120 Latest developments in human semen cryopreservation include the introduction of new cryoprotective agents such as glutamine121 and pentoxifylline,122 investigation of novel linear cooling protocols,89 vitrification,123 and the assessment of postthaw sperm washing regimens124 to restore membrane lipid fluidity. An understanding of these processes will influence the design of the next generation of cryopreservation protocols for human spermatozoa. Of particular importance is the detection of sublethal damage such as DNA fragmentation,125 particularly as sperm from infertile males are more vulnerable to insult during cryopreservation.126 The major challenge today is the introduction of suitable modifications to ensure the safety of recipients and offspring127 while maintaining the efficacy of cryopreservation protocols and storage.

Summary

Semen plus cryoprotectant

Fig 23.2 CBS Straw. Robust straw made of ionomeric resin, aseptically filled, heat sealed, and containing identification rod.

Vapor phase storage While, in theory, vapor phase as an alternative to liquid nitrogen storage should minimize the risk of contamination with infective organisms, recent research detected bacterial pathogens within the vapor phase of storage tanks.112 Coupled with concerns regarding maintenance of all samples at a satisfactory temperature and the risk of partial thawing during sample retrieval, vapor phase storage has not received universal endorsement. However, Clarke has demonstrated good short-term survival of

It is now more than 50 years since the birth of the first offspring following insemination with cryopreserved semen. Whereas the use of cryopreserved donor semen to circumvent childlessness has declined, many couples continue to benefit from the combined use of cryopreservation of spermatozoa in conjunction with assisted fertilization technologies. As it is now feasible for men with severe male factor infertility to father offspring, many previously intractable cases, such as oncology patients, are now encouraged to store semen prior to sterilizing therapy in the knowledge that fatherhood is a viable prospect. Cryobiologists continue to investigate and unravel the complex mechanics of cryoprotectants and cryopreservation, and with the aid of molecular technologies identify those genetic factors that influence cryosurvival. Assisted reproductive technologies (ART) practitioners and regulatory authorities play a major role in the introduction of peer-reviewed guidelines and evidence-based practice, ensuring safety of recipients and healthy offspring.

Acknowledgments This work was supported by University of Newcastle, Australia (and the ARC Centre of Excellence in

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Cryopreservation and storage of spermatozoa Biotechnology and Development EAM) and the School of Medical and Biomedical Sciences. The University of Sheffield (AAP).

Appendix Dilution of semen with cryoprotective media and packaging It is recommended that handling of semen, cryoprotectant, and packaging should be conducted within a class II safety cabinet to ensure sterility and personal safety of staff. Scientific staff should wear appropriate protective clothing and gloves, and handle all semen samples as if potentially infective and avoid the use of sharps such as needles. Only one sample (clearly labeled) should be handled at any one time, to avoid the possibility of confusion, and key stages should be double-witnessed by a second trained individual. 1. Record patient details in laboratory register and assign unique identifier to ejaculate, e.g. sample number and color coding. 2. Ask patient/donor to collect semen into labeled sterile pot provided (preferably by masturbation) in a room close to the laboratory. 3. Allow semen to liquefy in incubator at 37°C and perform semen analysis96 as soon as possible, preferably within 1 hour of production 4. Allow preprepared cryoprotectant media to warm to room temperature. 5. Measure volume of semen to be cryopreserved using wide-necked sterile pipette and transfer into bottom of clean container – allow semen to cool to room temperature. 6. Add appropriate volume of cryoprotectant dropwise over 2–5-minute period with continuous gentle ‘swirling’ to ensure thorough mixing. 7. Aspirate diluted semen–cryoprotectant into CBS straw via sterile filling device until semen contacts cotton plug and polyvinyl sealing power, leaving a 1-cm air space at opposite (filling) end (Fig 23.2). Do not allow straw to come in contact with semen or sides of container. 8. Remove filling device and heat-seal end of straw using thermal sealer. Repeat steps 7 and 8 until sufficient straws are filled or entire sample is packaged. 9. Insert printed rod(s) labeled with patient/donor details and unique sample number into opposite end of straw and heat-seal with thermal sealer. 10. Transfer straws to liquid nitrogen vapor phase or programmable freezer as soon as possible.

Vapor phase freezing Suspend straws 5 cm above liquid nitrogen horizontally on metal platform in uncirculated liquid nitrogen vapor for 10–20 minutes. Ensure straws are evenly spread and not touching. After freezing, plunge into liquid nitrogen and transfer to labeled liquid nitrogen-filled visitube

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before transferring to goblet in liquid nitrogen storage tanks – record location of sample in laboratory records.

Programmable freezing Transfer straws into freezer racking system – to ensure uniform freezing rates, load ‘dummy’ straws containing cryoprotectant only into unfilled spaces in straw holders. Start programmable freezer cycle. Typical semen freezing protocol:86 1. 2. 3.

4. 5.

From room temperature (22°C) to −5°C at 3°C/minute. Hold at −5°C for 10 minutes. Manually seed after 3 minutes at −5°C using liquid nitrogen cooled forceps – touch straw within 1 cm of top of semen within straw, taking care not to remove remainder of straw from nitrogen vapor. From −5°C to −80°C at 10°C/minute. Hold at −80°C for 10 minutes.

After freezing, plunge straws into liquid nitrogen and transfer to labeled liquid nitrogen-filled visitube before transferring to goblet in liquid nitrogen storage tanks − record location of sample in laboratory records. Store at −196°C at all times, as partial thawing–rewarming will significantly impact on cell viability. Avoid contamination of liquid nitrogen with environmental organisms and protect straws from chemical and radiation exposure.

Thawing Identify location of straws in storage bank and confirm with laboratory records and patient/donor records: 1.

2.

3. 4.

Remove straws and thaw quickly by placing in 37°C water bath for 10 seconds, then transfer to bench top for 1 minute (or alternatively place on bench top at room temperature for 2–3 minutes). First wipe straws dry with paper tissue, then wipe semen end of straw with sterile alcohol swab and cut open with sterile scissors. Then wipe labeled end of straw with sterile swab and cut open with sterile scissors – remove labeled rod. Transfer straw to insemination device for intracervical insemination if required – alternatively, empty contents of straw into sterile container for post-thaw analysis and preparation.

References 1. Spallanzani L. II. Observationi e sperienze intorno ai vermicelli spermatica dell’homo e degli animali. Modena, 1776. 2. Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 1949; 164: 666. 3. Stewart GJ, Tyler JP, Cunningham AL, et al. Transmission of human T-cell lymphotropic virus

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Cryopreservation and storage of spermatozoa 38. Grizard G, Chevalier V, Griveau JF, Le Lannou D, Boucher D. Influence of seminal plasma on cryopreservation of human spermatozoa in a biological material-free medium: study of normal and lowquality semen. Int J Androl 1999; 22(3): 190–6. 39. Yogev L, Gamzu R, Paz G, et al. Pre-freezing sperm preparation does not impair thawed spermatozoa binding to the zona pellucida. Hum Reprod 1999; 14(1): 114–17. 40. Tournaye H, Merdad T, Silber S, et al. No differences in outcome after intracytoplasmic sperm injection with fresh or with frozen–thawed epididymal spermatozoa. Hum Reprod 1999; 14(1): 90–5. 41. Pfeifer SM, Coutifaris C. Reproductive technologies 1998: options available for the cancer patient. Med Pediatr Oncol 1999; 33(1): 34–40. 42. Tomlinson MJ, Pacey AA. Practical aspects of sperm banking for cancer patients. Hum Fertil 2003; 6(3): 100–5. 43. Wallace WH, Anderson RA, Irvine DS. Fertility preservation for young patients with cancer: who is at risk and what can be offered? Lancet Oncol 2005; 6(4): 209–18. 44. Schmidt KL, Carlsen E, Andersen AN. Fertility treatment in male cancer survivors. Int J Androl 2007; 30(4): 413–19. 45. Saito K, Suzuki K, Iwasaki A, Yumura Y, Kubota Y. Sperm cryopreservation before cancer chemotherapy helps in the emotional battle against cancer. Cancer 2005; 104(3): 521–4. 46. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992; 340(8810): 17–18. 47. Al-Hasani S, Demirel LC, Schopper B, et al. Pregnancies achieved after frozen–thawed pronuclear oocytes obtained by intracytoplasmic sperm injection with spermatozoa extracted from frozen– thawed testicular tissues from non-obstructive azoospermic men. Hum Reprod 1999; 14(8): 2031–5. 48. Krausz C, Forti G. Sperm cryopreservation in male infertility due to genetic disorders. Cell Tissue Bank 2006; 7(2): 105–12. 49. Karow AM, Jr. Cryoprotectants – a new class of drugs. J Pharmacol 1969; 21(4): 209–23. 50. Curry MR. Cryopreservation of semen from domestic livestock. Rev Reprod 2000; 5(1): 46–52. 51. Gilmore JA, Liu J, Gao DY, Critser JK. Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa. Hum Reprod 1997; 12(1): 112–18. 52. McLaughlin EA, Ford WC, Hull MG. The contribution of the toxicity of a glycerol–egg yolk–citrate cryopreservative to the decline in human sperm motility during cryopreservation. J Reprod Fertil 1992; 95(3): 749–54. 53. Gao DY, Ashworth E, Watson PF, et al. Hyperosmotic tolerance of human spermatozoa: separate effects of glycerol, sodium chloride, and sucrose on spermolysis. Biol Reprod 1993; 49(1): 112–23. 54. Sherman JK. Improved methods of preservation of human spermatozoa by freezing and freeze-drying. Fertil Steril 1963; 14: 49–64.

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55. Behrman SJ, Sawada Y. Heterologous and homologous inseminations with human semen frozen and stored in a liquid-nitrogen refrigerator. Fertil Steril 1966; 17(4): 457–66. 56. Behrman SJ, Ackerman DR. Freeze preservation of human sperm. Am J Obstet Gynecol, 1969; 103(5): 654–64. 57. Pilikian S, Czyba JC, Guerin JF. Effects of various concentrations of glycerol on post-thaw motility and velocity of human spermatozoa. Cryobiology 1982; 19(2): 147–53. 58. Richardson DW, Joyce D, Symonds EM. Frozen human semen: A Royal College of Obstetricians and Gynaecologists workshop on the cryobiology of human semen, and its role in artificial insemination by donor, March 22 and 23, 1979. The Hague; London: Nijhoff for The Royal College of Obstetricians and Gynaecologists, 1980. 59. Mahadevan M, Trounson AO. Effect of cryoprotective media and dilution methods on the preservation of human spermatozoa. Andrologia 1983; 15(4): 355–66. 60. Mayaux MJ, Schwartz D, Czyglik F, David G. Conception rate according to semen characteristics in a series of 15 364 insemination cycles: results of a multivariate analysis. Andrologia 1985; 17(1): 9–15. 61. Friberg J, Gemzell C. Inseminations of human sperm after freezing in liquid nitrogen vapors with glycerol or glycerol–egg-yolk–citrate as protective media. Am J Obstet Gynecol 1973; 116(3): 330–4. 62. Harrison RF, Sheppard BL. A comparative study in methods of cryoprotection for human semen. Cryobiology 1980; 17(1): 25–32. 63. Jeyendran RS, Van der Ven HH, Kennedy W, PerezPelaez M, Zaneveld LJ. Comparison of glycerol and a zwitterion buffer system as cryoprotective media for human spermatozoa. Effect on motility, penetration of zona-free hamster oocytes, and acrosin/ proacrosin. J Androl 1984; 5(1): 1–7. 64. Hammitt DG, Walker DL, Williamson RA. Concentration of glycerol required for optimal survival and in vitro fertilizing capacity of frozen sperm is dependent on cryopreservation medium. Fertil Steril 1988; 49(4): 680–7. 65. Peek JC, Gilchrist SJ, Kelso CM, Quinn PJ. Comparison of three cryoprotective solutions for human semen. Clin Reprod Fertil 1982; 1(4): 301–5. 66. Prins GS, Weidel L. A comparative study of buffer systems as cryoprotectants for human spermatozoa. Fertil Steril 1986; 46(1): 147–9. 67. European Union. Commission Directive 2004/ 23/EC of the European Parliament and of the Council of 31 March 2004 on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells. Official Journal of the European Union 2004; 47 (L102): 48–58. 68. European Union. Commission Directive 2006/ 17/EC implementing Directive 2004/23/EC of the European Parliament and of the Council as regards certain technical requirements for the donation, procurement and testing of human tissues and cells. Official Journal of the European Union 2006; L38/40: 9.2.2006.

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69. Rules and Guidance for Pharmaceutical Manufacturers and Distributors 2007. London: Pharmaceutical Press, 2007. 70. Auterhoff G. EC Guide to Good Manufacturing Practice for Medicinal Products. Aulendorf: Editio Cantor Verlag, 1993. 71. Fink K, Zech H. [Effect of incubation time in deep freezing human sperm]. Wien Klin Wochenschr 1991; 103(23): 707–9. 72. Graham EF, Crabo BG, Pace MM. Current status of semen preservation in the ram, boar and stallion. J Anim Sci 1978; 47 (Suppl 2): 80–119. 73. Kremer J, Dijkhuis JR, Jager S. A simplified method for freezing and storage of human semen. Fertil Steril 1987; 47(5): 838–42. 74. Clarke GN. Sperm cryopreservation: is there a significant risk of cross-contamination? Hum Reprod 1999; 14(12): 2941–3. 75. Tomlinson M, Sakkas D. Is a review of standard procedures for cryopreservation needed? safe and effective cryopreservation – should sperm banks and fertility centres move toward storage in nitrogen vapour? Hum Reprod 2000; 15(12): 2460–3. 76. Mortimer D. Current and future concepts and practices in human sperm cryobanking. Reprod Biomed Online 2004; 9(2): 134–51. 77. Craft I, Tsirigotis M. Simplified recovery, preparation and cryopreservation of testicular spermatozoa. Hum Reprod 1995; 10(7): 1623–6. 78. Gil-Salom M, Romero J, Rubio C, et al. Intracytoplasmic sperm injection with cryopreserved testicular spermatozoa. Mol Cell Endocrinol 2000; 169(1–2): 15–19. 79. Cohen J, Garrisi GJ, Congedo-Ferrara TA, et al. Cryopreservation of single human spermatozoa. Hum Reprod 1997; 12(5): 994–1001. 80. Hsieh Y, Tsai H, Chang C, Lo H. Cryopreservation of human spermatozoa within human or mouse empty zona pellucidae. Fertil Steril 2000; 73(4): 694–8. 81. Schuster TG, Keller LM, Dunn RL, Ohl DA, Smith GD. Ultra-rapid freezing of very low numbers of sperm using cryoloops. Hum Reprod 2003; 18(4): 788–95. 82. Just A, Gruber I, Wober M, et al. Novel method for the cryopreservation of testicular sperm and ejaculated spermatozoa from patients with severe oligospermia: a pilot study. Fertil Steril. 2004; 82(2): 445–7. 83 Thachil JV, Jewett MA. Preservation techniques for human semen. Fertil Steril 1981; 35(5): 546–8. 84. Wolf DP, Patton PE. Sperm cryopreservation: state of the art. J In Vitro Fert Embryo Transf 1989; 6(6): 325–7. 85. Verheyen G, Pletincx I, Van Steirteghem A. Effect of freezing method, thawing temperature and postthaw dilution/washing on motility (CASA) and morphology characteristics of high-quality human sperm. Hum Reprod 1993; 8(10): 1678–84. 86. McLaughlin EA, Ford WC, Hull MG. A comparison of the freezing of human semen in the uncirculated vapour above liquid nitrogen and in a commercial semi-programmable freezer. Hum Reprod 1990; 5(6): 724–8. 87. Czyba JC, Pinatel MC, Guerin JF. Preservation and storage of human sperm. Acta Medica Polona 1978; 19(1–2): 133–46.

88. Serafini P, Marrs RP. Computerized staged-freezing technique improves sperm survival and preserves penetration of zona-free hamster ova. Fertil Steril 1986; 45(6): 854–8. 89. Morris GJ, Acton E, Avery S. A novel approach to sperm cryopreservation. Hum Reprod 1999; 14(4): 1013–21. 90. Morris GJ. A new development in the cryopreservation of sperm. Hum Fertil 2002; 5(1): 23–9. 91. Isachenko E, Isachenko V, Katkov II, et al. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryoprotectant-free vitrification. Hum Reprod 2004; 9(4): 932–9. 92. Isachenko V, Isachenko E, Katkov II, et al. Cryoprotectant-free cryopreservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol Reprod 2004; 71(4): 1167–73. 93. Hossain AM, Osuamkpe CO. Sole use of sucrose in human sperm cryopreservation. Arch Androl 2007; 53(2): 99–103. 94. Henry MA, Noiles EE, Gao D, Mazur P, Critser JK. Cryopreservation of human spermatozoa. IV. The effects of cooling rate and warming rate on the maintenance of motility, plasma membrane integrity, and mitochondrial function. Fertil Steril 1993; 60(5): 911–18. 95. Mahadevan MM, Trounson AO. Relationship of fine structure of sperm head to fertility of frozen human semen. Fertil Steril 1984; 41(2): 287–93. 96. McLaughlin EA, Ford WC, Hull MG. Effects of cryopreservation on the human sperm acrosome and its response to A23187. J Reprod Fertil 1993; 99(1): 71–6. 97. McLaughlin EA, Ford WC, Hull MG. Adenosine triphosphate and motility characteristics of fresh and cryopreserved human spermatozoa. Int J Androl 1994; 17(1): 19–23. 98. McLaughlin EA, Ford WC. Effects of cryopreservation on the intracellular calcium concentration of human spermatozoa and its response to progesterone. Mol Reprod Dev 1994; 37(2): 241–6. 99. World Health Organization WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction. 4th edn. Cambridge, UK: Cambridge University Press, 1999. 100. Desrosiers P, Legare C, Leclerc P, Sullivan R. Membranous and structural damage that occur during cryopreservation of human sperm may be timerelated events. Fertil Steril 2006; 85(6): 1744–52. 101. Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J Androl 2002; 23(1): 25–43. 102. Gandini L, Lombardo F, Lenzi A, Spano M, Dondero F. Cryopreservation and sperm DNA integrity. Cell Tissue Bank 2006; 7(2): 91–8. 103. Wundrich K, Paasch U, Leicht M, Glander HJ. Activation of caspases in human spermatozoa during cryopreservation – an immunoblot study. Cell Tissue Bank 2006; 7(2): 81–90. 104. Horne G, Atkinson AD, Pease EH, et al. Live birth with sperm cryopreserved for 21 years prior to cancer treatment: case report. Hum Reprod 2004; 19(6): 1448–9.

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Cryopreservation and storage of spermatozoa 105. Feldschuh J, Brassel J, Durso N, Levine A. Successful sperm storage for 28 years. Fertil Steril 2005; 84(4): 1017. 106. Corrigan E, McLaughlin EA, Coulson C, Ford WC, Hull MG. The effect of halving the standard dose of cryopreserved semen for donor insemination: a controlled study of conception rates. Hum Reprod 1994; 9(2): 330–3. 107. Coulson C, McLaughlin EA, Harris S, Ford WC, Hull MG. Randomized controlled trial of cervical cap with intracervical reservoir versus standard intracervical injection to inseminate cryopreserved donor semen. Hum Reprod 1996; 11(1): 84–7. 108. Hurd WW, Randolph JF Jr, Ansbacher R, et al. Comparison of intracervical, intrauterine, and intratubal techniques for donor insemination. Fertil Steril 1993; 59(2): 339–42. 109. Campana A, Sakkas D, Stalberg A, et al. Intrauterine insemination: evaluation of the results according to the woman’s age, sperm quality, total sperm count per insemination and life table analysis. Hum Reprod 1996; 11(4): 732–6. 110. Tedder RS, Zuckerman MA, Goldstone AH, et al. Hepatitis B transmission from contaminated cryopreservation tank. Lancet 1995; 346(8968): 137–40. 111. Hawkins AE, Zuckerman MA, Briggs M, et al. Hepatitis B nucleotide sequence analysis: linking an outbreak of acute hepatitis B to contamination of a cryopreservation tank. J Virol Meth 1996; 60(1): 81–8. 112. Fountain D, Ralston M, Higgins N, et al. Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components. Transfusion 1997; 37(6): 585–91. 113. Department of Health. Guidance notes on the processing, storage and issue of bone marrow and blood stem cells. HMSO, London: 1997. 114. Russell PH, Lyaruu VH, Millar JD, Curry MR, Watson PF. The potential transmission of infectious agents by semen packaging during storage for artificial insemination. Anim Reprod Sci 1997; 47(4): 337–42. 115. Working Party of Royal College of Pathologists. HIV and the practice of pathology. London: Royal College of Pathologists, 1995. 116. Letur-Konirsch H, Collin G, Sifer C, et al. Safety of cryopreservation straws for human gametes or

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embryos: a study with human immunodeficiency virus-1 under cryopreservation conditions. Hum Reprod 2003; 18(1): 140–4. Mortimer D, Barratt CL. Is there a real risk of transmitting variant Creutzfeldt–Jakob disease by donor sperm insemination? Reprod Biomed Online 2006; 13(6): 778–90. Mazzilli F, Delfino M, Imbrogno N, Elia J, Dondero F. Survival of microorganisms in cryostorage of human sperm. Cell Tissue Bank 2006; 7(2): 75–9. Phelps MJ, Liu J, Benson JD, et al. Effects of Percoll separation, cryoprotective agents, and temperature on plasma membrane permeability characteristics of murine spermatozoa and their relevance to cryopreservation. Biol Reprod 1999; 61(4): 1031–41. Guthrie HD, Liu J, Critser JK. Osmotic tolerance limits and effects of cryoprotectants on motility of bovine spermatozoa. Biol Reprod 2002; 67(6): 1811–16. Renard P, Grizard G, Griveau JF, et al. Improvement of motility and fertilization potential of postthaw human sperm using glutamine. Cryobiology 1996; 33(3): 311–19. Esteves SC, Sharma RK, Thomas AJ Jr, Agarwal A. Cryopreservation of human spermatozoa with pentoxifylline improves the post-thaw agonist-induced acrosome reaction rate. Hum Reprod 1998; 13(12): 3384–9. Nawroth F, Isachenko V, Dessole S, et al. Vitrification of human spermatozoa without cryoprotectants. Cryo Lett 2002; 23(2): 93–102. James PS, Wolfe CA, Mackie A, et al. Lipid dynamics in the plasma membrane of fresh and cryopreserved human spermatozoa. Hum Reprod 1999; 14(7): 1827–32. Linfor JJ, Meyers SA. Detection of DNA damage in response to cooling injury in equine spermatozoa using single-cell gel electrophoresis. J Andrology 2002; 23(1): 107–13. Donnelly ET, Steele EK, McClure N, Lewis SE. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum Reprod 2001; 16(6): 1191–9. Avery SM, McLaughlin EA, Dawson KJ. Safe cryopreservation of sperm and embryos. Hum Fertil 1998; 1(1): 84–6.

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24 Handling and cryopreservation of testicular sperm Joseph P Alukal, Dolores J Lamb, Larry I Lipshultz

The treatment of male infertility presents many unique challenges. Amongst these are the dilemmas that arise from needing to coordinate treatments for both male and female patients. These logistical issues, such as the need to time testicular sperm extraction (TESE) to coincide with oocyte retrieval, can end up posing as much of a problem to practitioners as almost any cause of male or female infertility. Of course, the real impact of this dilemma is the fact that many patients with male factor infertility will have unsuccessful attempts at sperm extraction, thereby wasting the efforts of their female partner, who has by this point gone through ovarian hyperstimulation. The financial and emotional cost of this outcome can be devastating. Amongst the tools available to practitioners to combat this dilemma, cryopreservation of testicular sperm may be the most useful. Recovery of viable spermatozoa after freeze–thawing of testicular spermatozoa (obtained via percutaneous extraction) was first described by Craft and Tsirigotis in 1995.1 Subsequently, Romero et al demonstrated successful fertilization using cryopreserved spermatozoa obtained via testis biopsy; intracytoplasmic sperm injection (ICSI) performed using testicular spermatozoa at the time of the biopsy was unsuccessful.2 However, repeat attempts at ICSI using excess tissue that had been cryopreserved did result in fertilization. Importantly, Romero et al described this procedure in only two patients, both of whom were azoospermic, and pregnancy did not result in either case. The widespread application of cryopreservation to testicular spermatozoa is the result of the work of Oates and colleagues.3 In 1997, they published their results from a series of 10 patients with nonobstructive azoospermia (NOA) who underwent TESE with planned cryopreservation of testicular tissue. With subsequent thawing and usage for ICSI, their fertilization rate was 48% and clinical pregnancies with live births did occur. An acceptable fertilization rate, comparable to those found in routine cycles of in vitro fertilization (IVF)–ICSI using freshly obtained testis spermatozoa from patients with NOA, was consistent

with the hypothesis that freeze–thawing of testis tissue was not injurious to testicular spermatozoa in any meaningful way. Cryopreservation of testicular spermatozoa remains a commonly used and vital technique in assisted reproduction. There are several reasons for this, many of which relate to the unique treatment dilemmas posed by patients with NOA. First, the chance of an unsuccessful biopsy in a patient with NOA is approximately 30%.4 Obviously, unsuccessful biopsy makes the effort undertaken by the couple to coordinate ovarian hyperstimulation and oocyte retrieval completely wasted. However, there is no way to predict the likelihood of this occurrence. Cryopreservation allows the couple to time oocyte retrieval to their convenience and only in the event of successful testis sperm extraction. Second, without cryopreservation, the couple is forced to undergo repeated testis biopsies for each desired cycle of IVF–ICSI. This assumes that couples will need repeat cycles due to (1) failure of attempted cycles and (2) desire for multiple children. Both these occurrences are common; if patients with NOA were required to undergo fresh testis biopsy every time they planned to undergo an IVF–ICSI cycle, there would be a far higher likelihood of failure at each cycle (again a 30% risk of unsuccessful biopsy). In addition, there is significant injury to the testis incurred with each biopsy.5 This injury is compounded by the fact that both androgen production and spermatogenesis are often already impaired in the patient with NOA. Taken together, these reasons help illustrate why cryopreservation of testicular sperm is so crucially important to patients with NOA. On the other hand, there remain serious limitations to this technology as it currently exists. As many as 50% of viable sperm may be lost with each freeze–thaw cycle in experienced hands;6 in patients with severe oligospermia or azoospermia, this can result in an unacceptable rate of loss when viable testicular spermatozoa are rare in the first place. Continued research into cryopreservative techniques, allowing for better preservation of single digit numbers of cells, is yielding promising results.

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This chapter will serve as an update on technologies available for cryopreservation of testicular sperm. We will also outline handling techniques that are crucial to increasing the likelihood of successful sperm preservation after freeze–thaw. Finally, we outline our own technique for cryopreservation of testicular sperm, in the hope of providing a simple set of directions for anyone wishing to perform this technique for their patients.

Cryopreservation As is expected, spermatozoa are not different from other living cells in that they do not tolerate massive changes in temperature well. Much of the physiology of cryopreservation of testis spermatozoa derives from our understanding of cryopreservation of ejaculated spermatozoa, first used to achieve pregnancy by Bunge and Sherman in 1953.7 Freezing results in dehydration, ionic concentration, and loss of plasma membrane integrity in donor sperm; as a result, approximately 50% of sperm die during a single freeze–thaw cycle. Similar losses are encountered with a single freeze– thaw cycle performed on testicular spermatozoa.6,8–10 This is problematic given the low numbers of spermatozoa that are typically returned from testis biopsies on patients with NOA. Inquiry into the specific causes of lethal injury to living cells undergoing cryopreservation identified several correctable factors that minimize the likelihood of success. These include avoidance of phase transition and resultant membrane damage, optimization of cooling and warming rates, and prevention of ice formation. Finally, the usage of cryoprotectants represents an additional means of preventing lethal injury. Phase transition describes the thermodynamic event in which a compound is transformed from one phase to another; this event declares itself through an abrupt change in the physical properties of the compound after only a small change in temperature (e.g. liquid water to ice at 0°C). Numerous animal and human studies have demonstrated that by preventing the phase transition of the surrounding media, lethal injury to sperm is prevented. Sawada first demonstrated plasma membrane damage and cell death in human spermatozoa as a result of phase transition;8 subsequent work by Leibo in 1977 demonstrated optimal freezing characteristics for both sperm and embryos.11 This work focused on the notion that slow cooling rates allow for extracellular ice formation; this phase transition dehydrates cells, thereby resulting in hyperosmolarity of the intracellular environment and cell death. Conversely, rapid cooling rates resulted in intracellular ice formation, which was also lethal. More recent work by Jeyendren demonstrated that the membrane damage Sawada observed during freezing may specifically be due to lipid peroxidation in the setting of phase transition.12 Consequently, the media within which spermatozoa are kept and the rate at which this media are

cooled is vitally important. Mazur and Schmidt elaborated on this balance in yeast, showing clearly that rapid cooling rates prevented extracellular ice formation and increased cell survival up until a rate was reached that allowed for intracellular ice formation.13 This optimal cooling rate varied from cell type to cell type.9 In human ejaculated sperm and testicular spermatozoa, optimal cooling is achieved with a stepwise cooling of −10°C/min to −80°C. The sample is maintained at this temperature for 20 minutes and then stored permanently in liquid nitrogen at a temperature of −196°C.14 Warming rates are also important to cell survival; rapid warming prevents aggregation of extracellular ice crystals.9 Finally, cryoprotectants act to protect cells from the abrupt fluid shifts involved in freezing. They can be grouped into two categories. Intracellular cytoprotectants permeate the plasma membrane of cells and prevent intracellular solute toxicity; conversely, extracellular cryoprotectants act to prevent ice formation. Commonly used intracellular cryoprotectants include glycerol and polyethylene glycol; extracellular cytoprotectants include dextran, glucose, and sucrose. The cryoprotectant most commonly used for the purpose of freezing sperm and/or spermatozoa is glycerol; many cryobanks employ egg-yolk buffer containing glycerol as media for freezing specimens.15

Processing of specimens Methods for processing testis tissue are also vitally important to the eventual success of cryopreservation. The initial method described by Oates involves mechanical homogenization followed by repeated aspiration through a 16-gauge hypodermic needle; the resultant specimen is stored with media in polypropylene tubes.3 Non-mechanical methods for processing include ones such as that described by Salzbrun.16 This enzymatic digestion protocol uses type IV collagenase, trypsin, and trypsin inhibitor. Obviously, both methodologies introduce the risk of extensive cellular injury. The mechanical protocols can introduce cellular injury through shearing force. Enzymatic digestion results in unintentional digestion of healthy spermatozoa. Regardless of the methodology used, minimal processing of tissue results in maximized live-cell yield after freeze–thaw (RD Oates, pers comm). Much of the difficulty in processing testicular specimens derives from the paucity of spermatozoa in specimens obtained from patients with NOA. Identification of sperm within specimens is labor-intensive, often resulting in only tens or hundreds of cells appropriate for freezing. Unfortunately, standard techniques for freezing ejaculated sperm and/or testicular spermatozoa have been shown to yield poor results when applied to specimens with a minimal number of sperm.17 As a result, significant effort has been made to develop techniques that are appropriate for poor sperm density specimens.

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The most intriguing of these was described by Cohen in 1991;18 this method employs empty hamster oocytes as a storage vessel for individual sperm. After removal of the ooplasm via microdissection technique, individual sperm are inserted in the zona pellucida and then frozen. Excellent results with regard to eventual thawing, viability of sperm, and fertilization have been obtained. These results were confirmed by Borini in 2000 using sperm obtained via testicular sperm aspiration (TESA).19 Despite this, due to concerns of trans-species migration of viral infection from hamsters, this technique is not employed for clinical purposes, and no human pregnancy as a result of this technique has been described. Other methodologies for the storage of individual or low numbers of sperm include freezing of small aliquots on nylon microloops, as described by Schuster.20 This technique, which was described in 2003, was offered as a simple alternative to the empty zona pellucida technique. This experiment considered sperm motility and viability after freeze–thaw with the microloop technique; in addition, four separate cryoprotective solutions were used in an effort to ascertain whether or not a difference existed with regard to success. Ultimately, viability was poor with each solution used. Despite this, research into this technique continues, as the opportunity to individually store and then use single digit numbers of sperm would represent a significant advance in cryopreservation. The opportunity to avoid multiple freeze–thaw cycles, which are injurious to sperm, represents a meaningful step forward, especially when dealing with samples possessing minimal sperm.21 Research into other methodologies for addressing this challenge continues. In 2004, Just and colleagues reported successful freeze–thaw of human spermatozoa using the algae species Volvox globator as a vehicle.22 More fascinating developments in this field are sure to follow.

Technique for testicular sperm extraction and cryopreservation Diagnostic biopsy and testicular sperm extraction is performed at our institution using an open-window technique.23 After administration of IV conscious sedation and local anesthesia, a transverse scrotal incision is made through the scrotal skin, dartos, and tunica vaginalis. The tunica albuginea is then incised, and seminiferous tubules are extruded from the testis. These tubules are sharply dissected from the testis using Iris scissors, and then a wet preparation is prepared using modified sperm-washing medium (Irvine Scientific). A portion of the tissue is set aside in Bouin’s fixative for formal histologic examination. The wet preparation is then examined using phase contrast microscopy; if mature spermatozoa are identified, then additional tubules are harvested via the same incision and placed in 1 ml of test-yolk buffer (TYB, Irvine

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Scientific). If mature spermatozoa are not seen, additional biopsies are taken first from the ipsilateral testis and then the contralateral testis, until sperm are found. The testis tissue is then mechanically dispersed under an inverted dissecting microscope; this is performed in a cell culture dish (BD Falcon) using fine jeweler’s forceps. Fluid from the homogenized tissue is then examined for spermatozoa. Once sperm are found, the homogenate is adjusted to a total of 5 ml using equal parts of TYB with 20% egg yolk and 12% glycerol. The final volume is then partitioned into 1 ml aliquots and stored in sterile polypropylene vials. These are then frozen in liquid nitrogen using the above-described protocol (stepwise cooling to −80°C in liquid nitrogen vapor followed by immersion in liquid nitrogen at −196°C).

Conclusion Cryopreservation of testicular spermatozoa remains crucially important to treatment of patients with male infertility; along with IVF–ICSI, this technique allows for reasonable hope of live pregnancy for patients with NOA, while at the same time minimizing morbidity and unnecessary procedures. Future directions for inquiry include safe and effective means for cryopreservation of specimens with limited numbers of spermatozoa, and minimization of cell death with multiple freeze–thaw cycles. As these and other advances in cryopreservation are made, they will continue to directly benefit patients with severe male factor infertility.

References 1. Craft I, Tsirigotis M. Simplified recovery, preparation and cryopreservation of testicular spermatozoa. Hum Reprod 1995; 10(7): 1623–6. 2. Romero J, Remohi J, Minguez Y, et al. Fertilization after intracytoplasmic sperm injection with cryopreserved testicular spermatozoa. Fertil Steril 1996; 65(4): 877–9. 3. Oates RD, Mulhall J, Burgess C, Cunningham D, Carson R. Fertilization and pregnancy using intentionally cryopreserved testicular tissue as the sperm source for intracytoplasmic sperm injection in 10 men with non-obstructive azoospermia. Hum Reprod 1997; 12(4): 734–9. 4. Mulhall JP, Burgess CM, Cunnigham D, et al. Presence of mature sperm in testicular parenchyma of men with nonobstructive azoospermia: prevalence and predictive factors. Urology 1997; 49(1): 91–5. 5. Schlegel PN, Su LM. Physiological consequences of testicular sperm extraction. Hum Reprod 1997; 12(8): 1688–92. 6. Pegg DE. The history and principles of cryopreservation. Semin Reprod Med 2002; 20(1): 5–13. 7. Bunge RG, Sherman JK. Fertilizing capacity of frozen human spermatozoa. Nature 1953; 172(4382): 767–8. 8. Sawada Y, Ackerman D, Behrman SJ. Motility and respiration of human spermatozoa after cooling to various low temperatures. Fertil Steril 1967; 18(6): 775–81.

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9. Leibo SP, Mazur P. The role of cooling rates in lowtemperature preservation. Cryobiology 1971; 8(5): 447–52. 10. Mazur P, Leibo SP, Chu EH. A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells. Exp Cell Res 1972; 71(2): 345–55. 11. Leibo SP. Fundamental cryobiology of mouse ova and embryos. Ciba Found Symp 1977; 52: 69–96. 12. Jeyendran RS, Van der Ven HH, Kennedy W, PerezPelaez M, Zanereld LJ. Comparison of glycerol and a zwitter ion buffer system as cryoprotective media for human spermatozoa. Effect on motility, penetration of zona-free hamster oocytes, and acrosin/proacrosin. J Androl 1984; 5(1): 1–7. 13. Mazur P, Schmidt JJ. Interactions of cooling velocity, temperature, and warming velocity on the survival of frozen and thawed yeast. Cryobiology 1968; 5(1): 1–17. 14. Anger JT, Gilbert BR, Goldstein M. Cryopreservation of sperm: indications, methods and results. J Urol 2003; 170(4 Pt 1): 1079–84. 15. Gilmore JA, Liu J, Gao DY, Critser JK. Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa. Hum Reprod 1997; 12(1): 112–18. 16. Salzbrunn A, Benson DM, Holstein AF, Schulze W. A new concept for the extraction of testicular

17.

18.

19.

20.

21.

22.

23.

spermatozoa as a tool for assisted fertilization (ICSI). Hum Reprod 1996; 11(4): 752–5. Hewitt J, Cohen J, Mathew T, Rowland G. Cryopreservation of semen in patients with malignant disease: role of in-vitro fertilisation. Lancet 1985; 2(8452): 446–7. Cohen J, Garrisi GJ, Congedo-Ferrara, et al. Cryopreservation of single human spermatozoa. Hum Reprod 1997; 12(5): 994–1001. Borini A, Sereni E, Bonu, Flamigni C. Freezing a few testicular spermatozoa retrieved by TESA. Mol Cell Endocrinol 2000; 169(1–2): 27–32. Schuster TG, Keller LM, Dunn RL, Ohl DA, Smith GD. Ultra-rapid freezing of very low numbers of sperm using cryoloops. Hum Reprod 2003; 18(4): 788–95. Rofeim O, Brown TA, Gilbert BR. Effects of serial thaw–refreeze cycles on human sperm motility and viability. Fertil Steril 2001; 75(6): 1242–3. Just A, Gruber I, Wöber M, et al. Novel method for the cryopreservation of testicular sperm and ejaculated spermatozoa from patients with severe oligospermia: a pilot study. Fertil Steril 2004; 82(2): 445–7. Coburn M, Wheeler T, Lipshultz LI. Testicular biopsy. Its use and limitations. Urol Clin North Am 1987; 14(3): 551–61.

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25 Ovarian tissue cryopreservation and other fertility preservation strategies Erkan Buyuk, Ozgur Oktem, Murat Sonmezer, Kutluk H Oktay

Overview Studies performed on laboratory animals have proved the feasibility of cryopreservation and transplantation of ovarian tissue.1,2 In this chapter, we discuss animal and human ovarian xenograft models, and we present our preliminary findings on the application of this technology in humans. We also illustrate practical points relating to ovarian tissue cryopreservation. Other fertility preservation procedures are also discussed.

Background Ovarian tissue banking relies on the principle of resistance of the primordial follicles to cryotoxicity.3 Because of their relatively inactive metabolism, absence of zona pellucida, and lack of metaphase spindle, primordial follicle oocytes are more durable than larger, growing follicles when exposed to extreme changes in ambient temperature.4 Cryoprotectants are still needed to preserve the viability of primordial follicles; a relatively smaller cell size makes it easier for cryoprotectants to penetrate. In larger follicles, it takes longer for cryoprotectants to diffuse and distribute evenly,5 although birth of healthy pups following cryopreservation–thawing of preantral follicles followed by in vivo maturation has been demonstrated.6 Comparison of the ‘pros and cons’ of freezing ovarian follicles and oocytes at various stages is made in Table 25.1. Primordial follicles are embedded in the fibrous cortical stroma of the ovary and therefore cannot be retrieved by needle aspiration. To obtain primordial follicles for cryopreservation, one has to isolate them from the tissue by chemical and mechanical means. We have described a method of isolating primordial follicles with reasonable survival. This method is discussed in detail later in this chapter.3 The procedure is extremely meticulous and therefore currently impractical for clinical use. In addition, it has not been possible to grow isolated primordial follicles in vitro.

Consequently, we have concentrated our efforts on cryopreserving ovarian cortical pieces, with which there has been considerable success in achieving follicle development after grafting in animals.

Animal models of ovarian transplantation The sheep ovary provides a useful model for studying ovarian tissue cryopreservation and transplantation. It has a dense fibrous stroma and comparatively high primordial follicle density in the ovarian cortex, similar to the human ovary. In the first study by Gosden et al, frozen banked strips of ovarian cortical pieces were autotransplanted on the infundibulopelvic ligament.7 Each animal also had a fresh transplant on the opposite site serving as a control. Four months after the transplant, the first signs of ovulation were detected. Two pregnancies had occurred, one from a fresh and another from a frozen–thawed graft. In the second study, autotransplants were performed with frozen–thawed tissue in eight sheep, and the animals were monitored for up to 22 weeks.8 All the animals resumed cyclicity and showed hormone production. In that study, baseline follicle-stimulating hormone (FSH) concentrations were elevated, but luteal phase progesterone measurements were normal. However, serum inhibin A levels were found to be low in the luteal phase. Aubard et al compared the functions of fresh and frozen–thawed ovarian cortex transplanted heterotopically (under the skin of the abdomen) and orthotopically (to the uterine horn) in the sheep. Although preantral and antral follicle development were similar in both grafts and ovulation resumed in most of the ewes, none of the ewes grafted orthotopically became pregnant. Seven months later, their effort to develop blastocysts from oocytes collected from heterotopic and orthotopic grafts failed.9 In a later study, the same researchers showed spontaneous ovulation and blastocyst formation after in vitro fertilization (IVF) of oocytes obtained from heterotopic

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Table 25.1

Comparison of available options for banking follicles, oocytes, and ovarian tissue

Cell/tissue type

Size

Advantage

Disadvantage

Primordial follicle

30–50 µm

Preantral follicle

60–200 µm

Difficult to isolate Damage to basement membrane Culture not yet possible More differentiated ZP damage

Prophase I oocyte

80–100 µm

Least differentiated No ZP No cell spindle Easy isolation No cell spindle Culture possible No spindle Short in vitro maturation

Metaphase II oocyte

80–100 µm

Ovarian cortex

1 × 1-mm to 1 × 3-cm strips

Easy to obtain

Easy to obtain Preserves stroma Can restore fertility Preventive before cancer treatment

ZP damage Low IVM and fertilization Very few live births ZP damage Cell spindle damage Organelle damage Few live births Experimental Risk of reimplantation of the cancer cell

ZP, zona pellucida; IVM, in vitro maturation.

grafts in the sheep. This is the first evidence of normal embryo development in large mammals after IVF of oocytes obtained from heterotopic ovarian grafts.10 A limiting factor in transplantation of ovarian cortical strips is the significant follicle loss due to initial ischemia. Gonadotropin11 and vitamin E12 administration are among the approaches tested to reduce follicle loss, and to facilitate revascularization of ovarian grafts. In rodent and sheep models, transplantation of frozen–thawed intact ovaries with vascular anastomosis was partially successful.13–15 The main difficulty in cryopreserving an organ is to optimize cryopreservation for each of the components. While it may be possible to cryopreserve follicles with one protocol, the same protocol may not be optimal to freeze the vascular component. In fact, in one study, the majority of grafts were lost due to vascular failure. Wang et al reported a successful pregnancy in a rat after transplantation of ovaries, fallopian tubes, and the upper segment of the uterus en bloc, after storage in liquid nitrogen.16 They later compared the functions of fresh and cryopreserved intact adult rat ovaries transplanted using microvascular anastomosis, and showed that freshly isotransplanted ovaries survived and resumed follicle growth and secretion. Although ischemia for 24 h at 4°C did not disrupt ovarian function, the organs had fewer follicles. Four out of seven (57%) cryopreserved transplants survived for ≥60 days, and one pregnancy was established. However, the ovarian reserve was compromised, as shown by the presence of fewer follicles, higher FSH, and lower estradiol levels. These results suggest that a 1-h ischemic period was not detrimental, whereas 24-h ischemia impaired the function of the ovaries. In the same study, all of the ovarian allotransplants were rejected despite immunosuppression with cyclosporin A, which rules out ovarian donation

except for cases of close tissue match between donor and recipient.17 In another study of a sheep model, intact ovaries with vascular pedicles removed laparoscopically were first cryopreserved for 1 week, and then thawed and autotransplanted into the rectus abdominis muscle with microvascular anastomosis of the ovarian artery and vein to the branches of deep inferior epigastric vessels.14 Ovarian cortical strips were also obtained, cryopreserved, and autotransplanted without anastomosis, for comparison. Transplants were removed 8–10 days later, and only three of 11 grafts (27%) were found to be viable. The remaining grafts were necrotic, owing to the occlusion of anastomosed vessels. A higher success rate was described for intact rabbit ovary cryopreservation and transplantation.18 The smaller size of the rabbit ovaries increases the likelihood of success due to perfusion of the ovary from surrounding tissues beside the vascular supply. Intact ovarian cryopreservation and transplantation is even less likely to succeed in humans, as the human ovary is larger and more fibrous, making it more difficult to cryopreserve the entire organ efficiently. Moreover, it is nearly impossible to devise a cryopreservation protocol that will optimally preserve both ovarian tissue and blood vessels. Obtaining ovarian vessels without damage during surgery can be challenging, and reanastomosing, especially the ovarian vein, would be technically difficult. On the other hand, Bedaiwy et al demonstrated no difference between cryopreserved cortical strips and whole ovary in terms of primordial follicle counts, amount of necrosis, and apoptosis. Lack of comparison after transplantation limits the interpretation of their findings.19 A laparoscopic approach to whole human ovary cryopreservation was recently described by Jadoul et al.20 If whole ovary cryopreservation is to be

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Table 25.2 Antral follicle development and estradiol (E2) production after transplantation of human ovarian tissue in severe combined immune deficiency (SCID) mice

Graft 1 2 3 4

Number of antral follicles (mm diameter)

Serum E2 (pmol/l)

1 (5) 2 (3, 4) 0 1 (2.5, hemorrhagic)

2070 780 35 280

Uterine weight (mg) 212 (ballooned) 131 123 126

Vaginal introitus Patent Patent Patent Patent

Reproduced from reference 27.

carried out, care must be taken to remove the ovary with a large pedicle from the infundibulopelvic ligament, for easy anastomosis during future transplantation. Since the ischemic period between the removal of the tissue and cryopreservation is the most important factor in tissue viability, the period from laparoscopy to infusion of the whole ovary with the cryoprotectants should be minimized. Ovarian transplantation combined with in vitro maturation (IVM) is another alternative approach in animals. In a mouse study, frozen–thawed newborn ovaries were first transplanted under the kidney capsule of 10–12-week-old mice to allow growth from the primordial stage onward, and removed 14 days later. Follicles were then mechanically isolated and grown in vitro for 12 days; oocytes were fertilized in vitro and transferred to pseudopregnant mice, which gave birth to healthy pups.21 In a more recent study, preantral follicles were isolated from vitrified–thawed ovaries and subjected to in vitro growth and maturation. Healthy pups were obtained following in vitro fertilization of the eggs obtained from these follicles in various animal species, including mice and rabbits.22 Again, because of the significant differences between rodent and human ovaries, the feasibility of this two-step procedure in humans is highly questionable. Finally, the technique of cryopreservation may have impact on the viability of the cryopreserved–thawed tissue. Currently, there are two well-defined approaches: namely, slow freezing and vitrification. Although some studies showed superiority of vitrification to slow freeze, they were challenged by others. A third approach has recently been described, where less concentrated cryoprotectants are used and liquid nitrogen is directly applied to the ovarian tissue. Although this method has proved to be superior to conventional vitrification and slow freeze in terms of follicle numbers, pregnancy rates, and litter size, it needs to be confirmed by other studies.23

Xenografting using human ovarian tissue Severe combined immune deficiency (SCID) mice carry a genetic mutation, which results in T cell and B

cell immunodeficiency.24 This allows xenografts to revascularize and survive in these animals without being rejected. Gosden et al have adopted this model for human ovarian xenografting. In earlier studies, both marmoset and sheep ovarian tissue was transplanted under the kidney capsule, and follicles grew to the antral stage.25 In the first study with human tissue xenografts, Gosden et al cryopreserved ovarian cortical pieces using various cryoprotectants and grafted them into SCID mice.26 After 18 days, the grafts were removed, and primordial follicle counts were obtained. With the exception of glycerol, all cryoprotectants (propanediol, ethylene glycol, dimethylsulfoxide [DMSO]) performed well, and 44–84% of the follicles survived. On the basis of these successful results with shortterm xenografting, we performed two long-term studies in SCID mice, using human tissue.27,28 In the first study, 1-mm3 ovarian cortical pieces from a 17-yearold patient were grafted under the kidney capsules of hypogonadal SCID mice.27 During the last 6 weeks of the 17-week grafting period, one group of animals received FSH stimulation. Estradiol levels were measured at the end of 17 weeks, when animals were killed. In the FSH-treated group, antral follicles as large as 5.5 mm were found, estradiol levels peaked at >700 pg/ml, and the uteri showed clear signs of estrogenization (Table 25.2). The source of estrogen was obviously the xenografts, because the animals were oophorectomized. In the second study, we grafted frozen–thawed human ovarian tissue into SCID mice.28 Because these animals were not hypogonadal, no FSH was administered. Grafts were recovered 22 weeks later. Histologic examination showed that many follicles had initiated growth. Interestingly, compared with controls, a higher percentage of follicles had initiated growth (5.6 ± 2.4 vs 12.5 ± 1.9%; p <0.05), but a significant number of primordial follicles/amount of graft (75 ± 6.8) remained. Presumably, because no exogenous FSH was given, the follicles did not develop beyond the one- to two-layer stage. However, the development of an antral follicle without exogenous gonadotropin administration was demonstrated in cryopreserved human ovarian pieces xenotransplanted under the kidney capsule of SCID mice, indicating that circulating levels of FSH in

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Table 25.3

‘Oktay’ modification of human ovarian tissue cryopreservation protocol

Freezing 1. Equilibrate 1–3-mm thick, 3 × 10-mm strips of ovarian cortex for 30 minutes at 4°C in phenol-free HEPES-buffered medium (i.e. L-15) containing 1.5 mol/l DMSO (propanediol/ethylene glycol), 20% serum (autologous), and 0.1 mol/l sucrose. Place the vials on a tissueroller during incubation to assure even penetration of cryoprotectant. 2. Load the tissue in cryovials into an automated freezer, starting at 0°C, and cool at 2°C/min to −7°C. 3. Soak for 10 minutes before manual seeding. 4. Continue to cool at 0.3°C/min to −40°C. 5. Cool at the faster rate of 10°C/min to −140°C. 6. Transfer to liquid nitrogen Dewar for storage. Thawing 7. Thaw at room temperature for 30 seconds. 8. Then place in 37°C water bath for 2 minutes. 9. Wash tissues stepwise in media containing progressively lower concentrations of cryoprotectant with 20% serum plus 0.1 mol/l sucrose, gently agitate tissue for 5 minutes in each step (1.5 mol/l, 1.0 mol/l, 0.5 mol/l, 0 mol/l). 10. Perform the last wash with medium containing 20% serum only. 11. Transfer to the operating room for transplantation in fresh medium with serum, on ice. HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DMSO, dimethylsulfoxide.

Table 25.4

Protocol for primordial follicle isolation

1. Obtain 1–3-mm cortical biopsies from patients <35 years of age. 2. Mince the tissue in 2 × 2-mm pieces. 3. Incubate in disaggregation medium containing 1 mg/ml of collagenase type IA and 8 U/ml DNAase in L-15 for 2 hours at 37°C on a roller. 4. Wash the tissue with L-15 with 10% serum three times. 5. Isolate primordial follicles using 27-gauge insulin needles and siliconized mouth.

oophorectomized mice may be sufficent to induce follicle development up to the antral stage in human ovarian pieces.29 Similarly Weissman et al demonstrated antral follicle formation in human cryopreserved cortical pieces transplanted to a nonobese diabetic SCID mouse after FSH stimulation.30 The same group later reported antral follicle development and oocyte retrieval after intramuscular xenografting of human ovarian tissue to nude mice.31 Recently, a similar study documented ovulation and corpus luteum formation in cryopreserved human ovarian tissue xenotransplanted into SCID mice stimulated with pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) administration.32 Our studies as well as others have further strengthened the concept that cryopreservation of ovarian tissue may be the future method of choice for preserving unfertilized gametes. We have quantified the survival rates of primordial follicles by viability stains after cryopreservation.3 Ovarian cortical pieces 2 × 2 mm were cryopreserved using a slow-freeze protocol (Table 25.3). After thawing, the tissues were partly digested using collagenase type IA (Sigma Co., St Louis, MO, USA), followed by microdissection of primordial follicles (Table 25.4). These follicles were then incubated with viability stains. We found that about 70% of follicles had survived this process and were viable. Later electron microscopy

studies, however, showed that this digestion method might damage the basement membrane of the follicle, but the oocyte is rarely affected.33 It appears that the process of partial digestion and microdissection tends to disrupt the follicle basement membrane, and that is the reason why isolated follicles commonly disintegrate in culture.33 These findings create another argument in favor of ovarian tissue freezing as opposed to the cryopreservation of isolated follicles.

Clinical trials of human ovarian tissue cryopreservation and transplantation Cryopreservation and heterotopic transplantation of ovarian tissue Encouraged by these laboratory studies, we have established a human ovarian transplantation project.

Heterotopic subcutaneous grafting The first subcutaneous ovarian transplantation was performed in a 35-year-old patient with stage IIIB cervical carcinoma.34 She had been scheduled for radiosensitizing chemotherapy and radiotherapy, and was not eligible for ovarian cryopreservation since the research protocol at that time was confined to patients under the

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age of 35. The ovarian cortex was prepared into 16, 0.5 × 5-mm strips of 1–3 mm in thickness. The pieces were placed through a 1-cm transverse incision made over brachioradialis muscle into a subcutaneous fossa created 5 cm below the antecubital fossa above the fascia, using a pull-through technique (Fig 25.1). Three days later the patient received a course of radiosensitizing chemotherapy, and another after 4 weeks, and then external-beam radiation to the pelvic area followed by two sessions of brachytherapy with cesium. During external-beam radiotherapy, care was taken to shield and protect her transplanted arm from the radiation field. Six weeks after the operation, FSH and luteinizing hormone (LH) levels were in the postmenopausal range. Four weeks later she presented with a painless bulge at the site of ovarian transplantation. Ultrasonic examination revealed a 15-mm dominant follicle and another four antral follicles measuring 5–7 mm. Hormone replacement therapy (HRT) was discontinued, and FSH and LH values reached a nadir at between 120 and 227 days post-transplantation: 8.6 ± 0.4 mIU/ml and 12.8 ± 0.8 mIU/ml, respectively. The continual development of antral follicles was observed on ultrasonography on a monthly basis, but progesterone levels never reached postovulatory values. An attempt to recover an oocyte percutaneously on postoperative day 216 retrieved a fractured germinal vesicle (GV)-stage oocyte from a 11-mm follicle. Another attempt was made to recover oocytes after 11 days of controlled ovarian hyperstimulation with gonadotropin-releasing hormone antagonist, recombinant FSH, and human menopausal gonadotropins. At day 11, four follicles measuring 11.5–15.5 mm were visualized by ultrasonography. The peak estradiol levels prior to hCG administration were 3482 pg/ml from the right cubital fossa representing the ovarian vein, and 264 pg/ml from the right hand showing peripheral measurement. Three oocytes were recovered percutaneously 36 hours later. Two oocytes obtained from 15.5-mm follicles were postmature, one oocyte from a 14-mm follicle had completed metaphase I, and a 11.5-mm follicle did not yield an egg. After overnight maturation, the oocyte from the 14-mm follicle extruded the first polar body, reaching metaphase II. Intracytoplasmic sperm injection in this oocyte did not result in fertilization. The second patient was a 37-year-old woman who developed a recurrent benign serous cyst in her only ovary, and had had the other ovary removed owing to serous cystadenoma. She had frozen pelvis subsequent to multiple laparotomies for cystectomies performed on the only remaining ovary. In the last operation, her gynecologist decided to remove the ovary, and healthy ovarian tissue was harvested. The transplantation technique was similar to that in the first patient except the tissue was placed more medially in the forearm for esthetic reasons. The postoperative FSH value was 50.7 mIU/ml. Five months after transplantation the patient felt a growing lump

331

a

b

c

d

Fig 25.1 Technique of transplantation of ovarian cortical strips to forearm.

at the transplantation site, and 1 month later ultrasonography demonstrated a 7.5-mm follicle, and HRT was discontinued. The following month she menstruated spontaneously. On day 13 of that cycle, a 9-mm follicle was noted. Hormone measurements showed a mid-cycle surge (FSH, 40 mIU/ml; LH, 90 mIU/ml; estradiol, 254 pg/ml; and progesterone, 2.1 ng/ml). The patient menstruated 2 weeks later. Ten months after grafting, and 6–11 days after an LH surge (62 mIU/ml), progesterone ranged from 7 to 10.1 ng/ml, confirming spontaneous ovulation. Cycle day-2 FSH, LH, and estradiol levels showed normal ovarian reserve.34 These cases were the first reports of endocrine function and oocyte retrieval after autologous transplantation of ovarian cortical strips to the forearm.

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Orthotopic transplantation There have been two cases of orthotopic ovarian transplantation with frozen-banked ovarian tissue. In the first case of a 29-year-old patient, frozen banked tissue was thawed 8 months after its storage.35 Histologic analysis of vials of ovarian pieces to assess the quality of cryopreservation showed a few follicles with one to two layers of granulosa cells, with 50% preservation of stromal cellularity. Six-day culture of ovarian pieces in the presence of gonadotropin stimulation showed the production of increasing amounts of estradiol, progesterone, and testosterone. Ovarian pieces were thawed using stepwise cryoprotectant dilution as previously described4 and strung with a 6-0 delayed absorbable suture, and three strings of pieces were then attached (Fig 25.2) to an absorbable cellulose membrane (Surgicel®; Ethicon Sommerville, NJ). The grafts were sutured to a peritoneal pocket created in the left pelvic ovarian fossa, and to ovarian fossa caudal to the first36 (Figs 25.3–25.5) The patient was given FSH 150 IU/ml and aspirin 80 mg to enhance vascularization for 1 week, and then put on HRT in the form of transdermal estradiol and oral progesterone. Fifteen weeks later the ovarian grafts were stimulated with human menopausal gonadotropins. On day 11, a dominant follicle emerged and estrogen therapy was discontinued. After 24 days of stimulation, the follicle diameter reached 17 mm. To sustain growth, the dose of gonadotropins was increased to 675 IU/ml, and 10 000 IU of hCG was given. Ovulation was documented by a rise in progesterone level from 0.7 to 13 ng/ml, ultrasonographic demonstration of the corpus luteum, free fluid in the cul-de-sac, and endometrial thickening. The patient menstruated 16 days after hCG administration. Ovarian function cannot be confirmed beyond 9 months post-transplant. In the second case reported by Radford et al, a 36-yearold woman with stage IIIB Hodgkin’s lymphoma underwent a right oophorectomy with cryopreservation of cortical strips before high-dose CBV chemotherapy (cyclophosphamide, carmustine, and etoposide) owing to the third relapse of the disease.37 Two ovarian cortical strips were thawed and transplanted onto the left ovary and at the site of the right ovary 19 months after chemotherapy, when serum sex hormones were in the postmenopausal range. Her hot flushes resolved 7 months after transplantation. One month later, serum estradiol became detectable, and ultrasound showed a 2cm follicle on the right side and 10-mm thick endometrium. The serum progesterone level never reached the ovulatory range, and no ovulation was detected. Unfortunately, the patient’s hormone levels confirmed ovarian failure 9 months after transplantation.

Fertility outcomes The studies mentioned above demonstrated the return of hormonal function after ovarian cryopreservation–transplantation. Later studies showed that the

Fig 25.2 Retrograde loading of graft that was reconstructed by stringing ovarian tissue between two strips of Surgicel®. Reprinted by permission of the American Society of Reproductive Medicine (reference 36).

Fig 25.3 Placement of leading suture in the pelvic pocket and through the lower peritoneal edge. Reprinted by permission of the American Society of Reproductive Medicine (reference 36).

restoration of fertility with live births is also possible with this technique. We reported the first case of human embryo development up to 4-cell stage following cryopreservation and heterotopic transplantation of ovarian cortical strips.38 The patient was a breast cancer survivor who was cleared by her oncologist, after 5 years from diagnosis, for transplantation and possible ovulation induction. A series of ovulation induction cycles yielded 20 oocytes with 8 suitable for fertilization and one normal embryo development. Lee et al reported the live birth of a healthy female monkey following cryopreservation and heterotopic transplantation.39 The eggs harvested were in vitro

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Fig 25.4 Placement of base suture through the upper peritoneal edge. By pulling on this suture, the raft is flattened against the vascular pelvic wall. Reprinted by permission of the American Society of Reproductive Medicine (reference 36).

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mouse model may provide the basis for understanding the mechanism of return of fertility after BMT.42 Lee et al demonstrated that BMT after chemotherapyinduced ovarian failure may restore fertility, by repairing chemotherapy-induced damage to the germ cells or to the ovarian stroma. They also showed that all pups born after BMT-mediated ovarian recovery were of recipient origin. Thus, bone marrow transplants do not contribute to regeneration of mature oocytes.43 Finally, another human live birth was recently reported in a patient with history of Hodgkin’s disease.44 In this case, the ovarian tissue was harvested and cryopreserved following the first chemotherapy cycle, before the sterilizing chemotherapy. After 2 years during which ovarian failure was demonstrated, orthotopic transplantation under the capsule of the contralateral ovary was performed. Ovarian stimulation, in vitro fertilization, and transfer of a single embryo resulted in a live birth. Collectively, these studies demonstrated the feasibility of ovarian cryopreservation with later autotransplantation for the return of both hormonal function and fertility.

Clinical and laboratory tips for cryopreserving human ovarian tissue Even though there are a limited number of studies addressing the most optimum way of cryopreserving human ovarian tissue, we have, in our experience, found the following points to be useful.

Age of the patient

Fig 25.5 Closure of peritoneum with interrupted sutures. Note the placement of two grafts side by side. Reprinted by permission of the American Society of Reproductive Medicine (reference 36).

fertilized and embryos were transferred to a surrogate mother, leading to healthy live birth. These landmark studies were followed by a human live birth after orthotopic transplantation of cryopreserved ovarian cortical strips underneath the ovarian capsule.40 This study was criticized since the possibility of eggs coming from the original ovarian tisue could not be completely ruled out. Restoration of hormonal function and return of fertility was also described using a combined orthotopic–heterotopic approach in a patient with a history of Hodgkin’s disease and bone marrow transplantation (BMT).41 A recent study in a

The follicle density in frozen–thawed ovarian tissue from women >40 years of age tends to be extremely low. Therefore, we do not recommend ovarian tissue freezing in women older than the age of 40. Earlier studies suggested that >60% of primordial follicles are lost due to the initial ischemia before revascularization after transplantation, and the freeze–thaw procedure results in only an additional 7% follicle loss.8 These losses are better tolerated in younger patients with a larger follicle reserve. A 25-year-old should still have a reserve of several hundred thousand follicles, and even a 70% loss would leave a reserve comparable to or better than that of a 35-year-old. This point underscores the importance of age in ovarian tissue cryopreservation, and perhaps better chances are offered to women <30 years of age.

Tissue collection Tissue collection is best done during the early follicular phase to avoid large ovarian follicles or a corpus luteum, which may result in hypervascularity and anatomic distortion. Ovarian cortical tissue can easily

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Table 25.5

Classification of cancer types according to the risk of ovarian metastasis

Low risk

Intermediate risk

High risk

Wilms’ tumor NonHodgkin’s lymphoma Hodgkin’s lymphoma

Lobular breast cancer Cervical adenocarcinoma Stage IV invasive ductal breast cancer Colorectal cancer

Leukemia Neuroblastoma Stage IV breast cancer Genital rhabdomyosarcoma

Stage I–III invasive ductal breast cancer Nongenital rhabdomyosarcoma Osteogenic sarcoma Cervical squamous-cell carcinoma Ewing’s sarcoma

be collected via laparoscopy. This can be done using a laparoscopic punch biopsy device (CasMed, London, UK) or by oophorectomy.45 The tissues should be transported to the laboratory on ice, and preferably in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered medium. Primordial follicles have a higher tolerance to hypoxia when kept in serumsupplemented medium; therefore, delays as long as 4–6 hours do not have a significant effect on follicle survival.46

dye that stains the dead tissue since it is actively excreted by live cells) and lack of ultrastructural damage on electron microscopy. Staining of enzymatically isolated follicles by trypan blue may predict the longevity of the ovarian cortical tissue.47 One possible method of pre-transplant ovarian tissue viability assessment is human ovarian tissue xenografting in SCID mice.27

Optimal tissue size

Fertility preservation and ovarian cryopreservation were initially indicated for cancer patients receiving sterilizing chemotherapy. Today its indication is extended beyond cancer, and covers patients receiving gonadotoxic chemotherapy for other systemic illnesses and those undergoing oophorectomy for benign ovarian conditions and for prophylactic purposes. The following gives indications for fertility preservation and ovarian cryopreservation from which both cancer and noncancer patients may benefit.

The tissues should not be frozen in sections that are too small. This results in excessive follicle damage during slicing and creates tissue pieces unmanageable for transplantation. In the sheep, 0.5 × 0.5–1.5-cm pieces have resulted in long-term follicle growth,7,8 and this has also been our experience with human ovarian tissue. It is best to slice the cortex in thin (1–2 mm) but long strips (1 × 0.5 cm) (Table 25.3).

Choice of cryoprotectant There is no significant difference between propanediol, ethylene glycol, or DMSO. Glycerol should not be used because it offers very poor protection against cryotoxicity. However, it is important to incubate the tissue in the cryoprotectant at 4°C for 30 minutes. The incubation should be done on a tissue-roller to facilitate uniform penetration of the cryoprotectant. The tissues are frozen with the slow-freeze protocol shown in Table 25.3.

Indications for ovarian cryopreservation

Patients receiving chemo- and/or radiotherapy for cancer The most common indication for ovarian cryopreservation is women receiving chemo- and/or radiotherapy for cancer. Among the various childhood and adult cancers, those with a low-to-intermediate probability of ovarian metastasis are better candidates for the procedure (Table 25.5).

Breast cancer

Assessment of viability Although demonstration of hormonal function and follicle growth is the best and ultimate proof of ovarian tissue viability after cryopreservation, various other methods of viability assessment before transplantation have been suggested. Fauque et al recently showed high correlation between lack of trypan blue staining (vital

Breast cancer is the most frequent cancer seen in reproductive-age women. Nearly 15% of 182 000 cases of invasive breast cancer occur in women under the age of 45.48,49 It is estimated that there will be more than 182 460 new cases of invasive and 67 770 new cases of in situ breast cancer, and 40 480 deaths, in the year 2008.50 Nearly all of these patients are subject to the risk of premature ovarian failure due to the

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combination chemotherapy commonly employed postoperatively for these patients. Infiltrative ductal carcinoma, which is the almost exclusive histologic type in reproductive-age women, does not metastasize to the ovaries, in the absence of stage IV (metastatic) disease.

Cervical cancer Cervical squamous-cell carcinoma may occur as early as the second decade of life. Approximately 3000 of 12 800 cases of invasive cervical cancer occur in premenopausal women.49,51 Ovarian involvement in cervical squamous cancer is less than 1%, compared with 12% for adenocarcinoma of the cervix.52 Because of the higher risk of ovarian metastasis in cervical adenocarcinoma, ovarian cryopreservation is recommended only for women with squamous-cell carcinoma of the cervix.

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have failed, oophorectomy followed by cryopreservation may be indicated.

Autoimmune diseases The prevalence of systemic lupus erythematosus (SLE) is 15–51 per 100 000, and 80% of these cases occur in women during child-bearing age. As a result, it may affect one in 1000 women.62 For patients with lupus glomerulonephritis, a regimen of cyclophosphamide, one of the most gonadotoxic agents, is commonly employed. Ovarian cryopreservation may be considered before chemotherapy in these patients. As in the case of SLE, patients with multiple sclerosis (MS) may be subjected to cytototoxic chemotherapy (e.g., mitoxantrone, cyclophosphamide). On the other hand, several endocrine and sexual disturbances have been described in MS patients, potentially interfering with fertility. As a result, among others, ovarian cryopreservation has been suggested as a means of preserving fertility in MS patients.63

Childhood and juvenile cancers Leukemia (especially acute lymphocytic leukemia), neuroblastoma, Hodgkin’s lymphoma, osteosarcoma, Wilms’ tumor, and nonHodgkin’s lymphoma are the most common childhood and youth cancers. With improvements in chemotherapy, radiotherapy, and BMT, cure rates have increased from less than 30% to 70% over the past 40 years,53 and more than 4000 female children are exposed to sterilizing chemo/radiotherapy per annum.49

Patients undergoing bone marrow transplantation BMT, initially used for the treatment of leukemia, is now increasingly being used for other cancerous and noncancerous systemic illnesses, including aplastic anemia, autoimmune and immunodeficiency diseases, rheumatoid arthritis, sickle-cell anemia, lymphoma, and breast cancer.54–58 High-dose chemo- and radiotherapy used prior to BMT for the ablation of bone marrow cause ovarian failure in nearly all patients.59

Adjunctive oophorectomy Recurrent breast cancer Oophorectomy can sometimes be offered as an adjunctive treatment for recurrent estrogen receptorpositive cases of breast cancer that do not respond to tamoxifen therapy.60 Ovarian cryopreservation can be done at the time of surgery in these patients.

Endometriosis Endometriosis is one of the most common reproductive diseases, and affects 10% of reproductive-age women.61 In cases where medical therapy and conservative surgery

Benign ovarian tumors When oophorectomy is performed for benign ovarian tumors, a healthy piece of the ovary may be cryopreserved at that time for future transplantation. Heterotopic transplantation may be superior to orthotopic transplantation if recurrence of disease is a concern.

Prophylactic oophorectomy Of all patients with ovarian cancer, 20% have a family history of the disease and 8% carry BRCA-1 or -2 mutations. The lifetime risk of developing ovarian cancer is 63% and 27% for patients having BRCA-1 and -2 mutations, respectively.64 Prophylactic oophorectomy is recommended when child-bearing is completed, or by age 35. In theory, the cryopreservation of ovarian tissue in patients who want to delay child-bearing until after age 35 may decrease the lifetime risk of ovarian cancer. Heterotopic transplantation to the forearm may be chosen in these patients.

Other fertility-preserving strategies There are several other fertility-preservation strategies. Depending on the patient’s age, type of cancer treatment, and available time, different strategies may be chosen for different patients.

Embryo cryopreservation In vitro fertilization and embryo cryopreservation are standard and clinically established procedures, and, if the patient has a partner, and sufficient time prior to cancer treatment, IVF can be performed to store embryos for future use. An IVF cycle will take approximately 2 weeks to complete from the onset of menses, and this

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time may not be available to most cancer patients prior to treatment. In breast cancer, there is typically a 6-week hiatus between surgery and chemotherapy, which would be sufficient to perform ovarian stimulation and IVF. However, standard ovulation drugs are contraindicated in breast cancer because of the resultant high levels of estradiol during stimulation.

Tamoxifen Tamoxifen is a nonsteroidal triphenylethylene antiestrogen compound, which was originally developed as a contraceptive in the UK.65 Later, it was found to stimulate ovarian follicle growth, and began to be used as an ovulation-induction agent in Europe.66 In 1966, a related compound, clomiphene, became a commonly used ovulation-induction agent in the USA.67 Then, it was discovered that tamoxifen had a suppressive effect on breast cancer,68 and it became the drug of choice in the treatment of breast cancer.69 We have recently used tamoxifen to perform ovarian stimulation and IVF in breast cancer patients undergoing or with a history of cancer therapy, and have found that a higher number of embryos, compared with natural cycle IVF, can be obtained without increasing the cancer recurrence risk in these patients.70 Twelve patients with breast cancer were given 40–60 mg/day tamoxifen on the second or third day of their menstrual cycle (15 cycles) for 5 days, and had IVF (TamIVF) with either fresh embryo transfer (six cycles) or cryopreservation of embryos (nine cycles). TamIVF patients had significantly less cycle cancellation (1/15 vs 4/9), and a greater number of mature oocytes (1.6 ± 0.3 vs 0.7 ± 0.2) and embryos (1.6 ± 0.3 vs 0.6 ± 0.2) per cycle, compared with retrospective natural cycle IVF (NCIVF) patients. Recently, we reported a protocol combining continuous tamoxifen with low-dose FSH stimulation in breast cancer patients. This approach more than doubled the embryo yield in breast cancer patients undergoing IVF, compared with the tamoxifen-only protocol.71

Aromatase inhibitors Because tamoxifen is stimulatory on the endometrium, it cannot be used in endometrial cancer in a similar fashion. For these patients, aromatase inhibitors can be used for ovarian stimulation, IVF, and embryo cryopreservation prior to radical surgery.72 Aromatase enzyme is a member of the cytochrome P450 family, which converts androgens (mainly androstenedione and testosterone) to estrogens (estrone and estradiol). Aromatization is a rate-limiting step in estrogen formation. The enzyme is abundant in subcutaneous fat, liver, normal breast tissue, muscle, and brain. After the promising result with the firstgeneration inhibitor aminoglutethimide, second- and third-generation aromatase inhibitors have been developed. The third-generation inhibitors (anastrozole, letrozole, and exemestane) are more specific for the enzyme and have fewer side effects.

Letrozole is a potent reversible inhibitor of the enzyme aromatase, and has recently been tested in phase III studies as a chemotherapeutic agent in postmenopausal women with metastatic breast cancer.73 Letrozole suppresses the intratumoral and systemic production of estrone from androstenedione by inhibiting aromatase activity. Letrozole in a single daily dose of 2.5 mg has been shown to achieve optimal suppression of serum estrogen levels in postmenopausal women.74 When administered to cycling female rats, it causes more than an 80% reduction in estradiol levels and a marked increase in FSH and LH levels.75 In a doubleblind randomized controlled trial, Fisher et al evaluated the effect of the aromatase inhibitor letrozole on ovulatory function in comparison with clomiphene citrate (CC) and natural cycles in normal ovulatory women. The patients received either letrozole 2.5 mg/day or CC 50 mg/day on days 5–9. There was no difference in the endometrial thickness at mid-cycle in the natural-cycle group or the study groups, but the number of mature follicles at the LH surge was higher in both treatment groups than in the control group. Follicular profiles of LH and FSH were similar between groups in both the natural and medicated cycles. In the study groups, CC resulted in a significant increase in estradiol levels, while estradiol levels in letrozole-stimulated cycles were lower than in natural cycles.76 Following the tamoxifen study,70 we performed a prospective controlled study where we compared letrozole with tamoxifen in breast cancer patients.77 In this study, we stimulated patients either with tamoxifen alone, with tamoxifen + low-dose FSH, or with letrozole + low-dose FSH. We found that patients undergoing controlled ovarian stimulation with tamoxifen–FSH or letrozole–FSH had higher number of follicles, mature oocytes, and embryos than patients stimulated with tamoxifen alone. Although there was no statistically significant difference between tamoxifen–FSH and letrozole–FSH groups in terms of number of follicles, mature oocytes, and embryos, patients in the letrozole–FSH group had significantly lower peak estradiol levels than patients in the tamoxifen–FSH group. There was no difference in the peak estradiol levels between tamoxifen only and letrozole–FSH groups. As a result, the letrozole–FSH protocol may be more desirable than tamoxifen–FSH in breast cancer patients due to lower levels of estradiol achieved during the stimulation cycle. This is confirmed by our follow-up study where we demonstrated significantly reduced peak estradiol levels and gonadotropin requirements in breast cancer patients undergoing controlled ovarian hyperstimulation with letrozole and gonadotropins compared to tubal disease patients undergoing standard IVF treatment.78 Moreover, lower peak estradiol levels were achieved with letrozole, compared to anastrozole, another aromatase inhibitor.79 It should be noted that aromatase inhibitors block the conversion of androgens to both estrogens and genotoxic estrogen metabolites, which may still be

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Strategies for fertility preservation in cancer patients

Low probability of ovarian involvement

Oophoropexy

Chemotherapy delay feasible

Ovarian tissue cryopreservation

Oocyte/embryo cryopreservation

ER-positive tumor

Ovarian stimulation using TMX or AI

Chemotherapy delay not feasible

ER-negative tumor

Standard ovarian stimulation

In vitro maturation

High probability of ovarian involvement

Cryopreservation of ovarian tissue

Xenografting

Close tissue surveillance necessary

Tissue surveillance not necessary

Heterografting

Autografting

Fig 25.6 Approach to cancer patients for fertility preservation. TMX, tamoxifen; AI, aromatase inhibitor; ER, estrogen receptor.

capable of inducing breast cancer, whereas antiestrogens only inhibit binding of estrogen to its receptor, and thus its transcriptional activation. On the other hand, we recently reported that the recurrence risk of breast cancer is not substantially increased in patients stimulated with letrozole and gonadotropins within an average of two years of follow-up,80 although longerterm follow-up is necessary to demonstrate the safety of this approach. The potential of aromatase inhibitors to preserve fertility in breast cancer patients is currently under investigation by our group.

Oocyte cryopreservation IVF and embryo cryopreservation is not an option for single patients unless they choose to use donor sperm. For single patients, unfertilized oocytes can be cryopreserved instead. Initial success rates with frozen–thawed oocytes have been much lower than with cryopreserved embryos, however. Success rates did not exceed 3–4% per thawed oocyte in the initial reports.81 Recent improvements in oocyte cryopreservation techniques appear to have resulted in better success rates.82 Pregnancy rates up to 18% per embryo transfer with 8% implantation rates were reported recently, even though the majority of these patients were young with >10 oocyte yield, and were not cancer patients.83 We recently published the results of a meta-analysis covering the literature on the results of oocyte cryopreservation.84 We

analyzed the results of all reports from 1997 to 2005 with the patients undergoing IVF–ICSI (intracytoplasmic sperm injection) with cryopreserved cycles. We compared the live birth rate per thawed oocyte and embryo transfer with those of patients who underwent IVF with fresh oocytes during comparable time periods in the USA using the SART (Society of Assisted Reproductive Technology) data. We found that while success rates were significantly lower with slow freezing, with the vitrification methods they were similar to success rates with fresh oocytes. Based on these results, oocyte cryopreservation is clearly justified for preserving fertility when a medical indication exists. Although higher pregnancy rates are achieved with the vitrification method compared to slow freezing, selective reporting and transfer of higher number of embryos may at least be partially responsible for this difference. Furthermore, since there are fewer babies born after vitrification, the safety of this method needs to be assessed further. Tamoxifen and aromatase inhibitors may also be used in breast cancer patients for ovulation-inductionoocyte cryopreservation as described above for embryo cryopreservation.

Ovarian transposition Protection of the ovaries from radiation in patients who will receive pelvic or whole-abdomen irradiation is another important issue that should be considered.

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Ovarian cryopreservation transplantation

Patients receiving chemo/radiotherapy Breast cancer Cervical cancer

Adjunctive oophorectomy Prophylactic oophorectomy Benign ovarian tumors

Childhood…

Ovarian tissue banking

Orthotopic transplantation

Resumption of cyclicity

Heterotopic transplantation

Autograft

Xenograft

Follicle isolation

In vitro maturation

Egg retrieval

Conception

In vitro fertilization

Fig 25.7 Strategies in utilizing banked human ovarian tissue. Frozen–thawed tissue can be returned to the original pedicle, and pregnancy may be achieved naturally. Alternatively, the tissue may be grafted to a heterotopic site, either as an autograft (in the brachioradialis muscle) or xenograft (in severe combined immune deficiency (SCID) mouse). Follicles can also be isolated from the ovarian tissue and grown in vitro. In the last instance and in the case of heterotopic grafts, in vitro fertilization will be required to achieve pregnancy. BMT, bone marrow transplantation.

Although the transposition of the ovaries has been shown to reduce the risk of ovarian failure, the ovaries are still susceptible to the effects of scattered radiation and vascular compromise. Attempts at ovarian transposition give protection rates of 0–66% for Hodgkin’s lymphoma85–89 and 17–83% for cervical cancer.90,91 Thus, these unpredictable results indicate ovarian transposition to be an unreliable measure. On the other hand, it has been suggested that unilateral ovarian transposition in combination with the cryopreservation of the contralateral ovary may increase the chances for future fertility and hormonal function in selected patients.92 A flowchart describing the approach to cancer patients for fertility preservation is presented in Fig 25.6

Conclusions Ovarian tissue banking can offer hope to cancer patients who want to safeguard their fertility against sterilizing

chemotherapy and radiotherapy. Even though this technology is currently being tested for cancer patients only, in the future it may find other applications, such as preventing premature ovarian failure and delaying reproductive aging. There are other fertility-preservation procedures such as embryo and oocyte cryopreservation, and ovarian transposition. Breast cancer patients can be stimulated with tamoxifen for embryo cryopreservation. In endometrial and breast cancer patients, aromatase inhibitors can be used.

Eye to the future The procedure of ovarian transplantation is no longer a futuristic idea. In our last review of the subject, we proposed several theoretical approaches to transplant cryopreserved ovarian tissue in humans (Fig 25.7). At the time of writing the second edition of this chapter, multiple autologous orthotopic and heterotopic transplants

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had already resulted in the restoration of ovarian function; in a very recent primate study the first pregnancy from heterotopic transplantation was reported.39 Clinical studies are now under way to restore fertility. In the future, further progress in enhancing graft revascularization, whole ovarian cryopreservation, IVM, and xenografting may be expected. Several new ovarian stimulation protocols will be established for breast and endometrial cancer patients. Chemoprevention of chemo- and radiotherapy-induced gonadal damage via selective antiapoptotic agents is also being investigated,93 and may one day obviate the need for surgical techniques of fertility preservation.

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Ovarian tissue cryopreservation 70. Oktay K, Buyuk E, Davis O, et al. Fertility preservation in breast cancer patients: IVF and embryo cryopreservation after ovarian stimulation with tamoxifen. Hum Reprod 2003; 18: 90–5. 71. Oktay KH, Buyuk E, Davis OK, Veeck L, Rosenwaks Z. A prospective comparison of tamoxifen alone and tamoxifen–FSH combined protocol for IVF and fertility preservation in breast cancer patient. Fertil Steril 2003; 80 (Suppl 3): 63–4. 72. Oktay KH, Buyuk E, Rosenwaks Z. Novel use of an aromatase inhibitor for fertility preservation via embryo cryopreservation in endometrial cancer: a case report. Fertil Steril 2003; 80 (Suppl 3): 144. 73. Lamb HM, Adkins JC. Letrozole: a review of its use in postmenopausal women with advanced breast cancer. Drugs 1998; 56: 1125–40. 74. Dowsett, M, Jones A, Johnston SR, et al. In vivo measurement of aromatase inhibition by letrozole (CGS 20267) in postmenopausal women with breast cancer. Clin Cancer Res 1995; 1: 1511–15. 75. Sinha S, Kaseta J, Santner SJ, et al. Effect of CGS 20267 on ovarian aromatase and gonadotropin levels in the rat. Breast Cancer Res Treat 1998; 48: 45–51. 76. Fisher SA, Reid RL, VanVugt DA, Casper RF. A randomized double-blind comparison of the effects of clomiphene citrate and the aromatase inhibitor letrozole on ovulatory function in normal women. Fertil Steril 2002; 78: 280–5. 77. Oktay K, Buyuk E, Libertella N, Akar M, Rosenwaks Z. Fertility preservation in breast cancer patients: a prospective controlled comparison of ovarian stimulation with tamoxifen and letrozole for embryo cryopreservation. J Clin Oncol 2005; 23: 4347–53. 78. Oktay K, Hourvitz A, Sahin G, et al. Letrozole reduces estrogen and gonadotropin exposure in women with breast cancer undergoing ovarian stimulation before chemotherapy. J Clin Endocrinol Metab 2006; 91: 3885–90. 79. Azim AA, Costantini-Ferrando M, Lostritto K, Oktay K. Relative potencies of anastrozole and letrozole to suppress estradiol in breast cancer patients undergoing ovarian stimulation before in vitro fertilization. J Clin Endocrinol Metab 2007; 92: 2197–200. 80. Azim AA, Costantini-Ferrando M, Oktay K. Safety of fertility preservation by ovarian stimulation with letrozole and gonadotropins in patients with breast cancer: a prospective controlled study. J Clin Oncol 2008; 26: 2630–5.

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81. Oktay K, Kan MT, Rosenwaks Z. Recent progress in oocyte and ovarian tissue cryopreservation and transplantation. Curr Opin Obstet Gynecol 2001; 13: 263–8. 82. Porcu E, Fabbri R, Seracchioli R, et al. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril 1997; 68: 724–6. 83. Fabbri R, Porcu E, Marsella T, et al. Oocyte cryopreservation. Hum Reprod 1998; 13 (Suppl 4): 98–108. 84. Oktay K, Cil AP, Bang H. Efficiency of oocyte cryopreservation: a meta-analysis. Fertil Steril 2006; 86: 70–80. 85. Le Floch O, Donaldson S, Kaplan HS. Pregnancy following oophoropexy and total nodal irradiation in women with Hodgkin’s disease. Cancer 1976; 38: 2263–8. 86. Ray GR, Trueblood HW, Enright L, Kaplan HS, Nelson TS. Oophoropexy: a means of preserving ovarian function following pelvic megavoltage radiotherapy for Hodgkin’s disease. Radiology 1970; 96: 175–80. 87. Thomas PR, Winstanly D, Pechham MJ, et al. Reproductive and endocrine function of inpatients with Hodgkin’s disease: effects of oophoropexy and irradiation. Br J Cancer 1976; 33: 226–31. 88. Guglielmi R, Calzavena F, Pizzi GB. Ovarian function after pelvic lymph node irradiation. Eur J Gynaecol Oncol 1980; 2: 99–107. 89. Hunter MCH, Glees JP, Gazet JC. Oophoropexy and ovarian function in the treatment of Hodgkin’s disease. Clin Radiol 1980; 31: 21–6. 90. Anderson B, LaPolla J, Turner D, Chapman G, Buller R. Ovarian transposition in cervical cancer. Gynecol Oncol 1993; 49: 206–14. 91. Husseinzadeh N, Nahhas WA, Velkley DE, Whitney CW, Mortel R. The preservation of ovarian function in young women undergoing pelvic radiation therapy. Gynecol Oncol 1984; 18: 373–9. 92. Martin JR, Kodaman P, Oktay K, Taylor HS. Ovarian cryopreservation with transposition of a contralateral ovary: a combined approach for fertility preservation in women receiving pelvic radiation. Fertil Steril 2007; 87: 189e5–7. 93. Tilly JL, Kolesnick RN. Realizing the promise of apoptosis-based therapies: separating the living from the clinically undead. Cell Death Differ 2003; 10: 493–5.

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26 Severe male factor: genetic consequences and recommendations for genetic testing Inge Liebaers, André Van Steirteghem, Willy Lissens

Overview Infertility in the presence of a severe male factor such as oligoastenoteratospermia or azoospermia may be of genetic origin. This means that either the number or the structure of the chromosomes may be aberrant or a gene defect may be at stake. For two major reasons genetic investigations are indicated in case of male infertility. One reason is to understand more about the possible causes of azoospermia or oligoastenoteratospermia. Another reason is to be able to offer genetic counseling to the patient, his partner, and his family whenever indicated. The role of genetic counseling in case of infertility has, of course, increased since the advent of assisted reproductive technologies (ART) in general and certainly since the use of intracytoplasmic sperm injection (ICSI), offering the possibility to men with almost no spermatozoa to have children.1–4 In the clinic genetic investigations are usually performed when the azoo- or oligospermia is part of a more complex disease or syndrome. Based on the available data today a number of genetic tests should also be performed in case of infertility in an otherwise healthy male. In the majority of such cases it will today be sufficient to start with the analysis of the karyotype in peripheral lymphocytes, the search for the presence or absence of a Yq11 deletion on the long arm of the Y chromosome, and/or the analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) genes in couples in which the male partner has congenital bilateral absence of the vas deferens (CBAVD). More specific genetic investigations can be done if indicated.

Genetic causes of male infertility Chromosomal aberrations It has been known for over 40 years that the presence of an extra X chromosome in males, resulting in a 47,XXY karyotype, causes the Klinefelter syndrome with testicular atrophy and nonobstructive azoospermia as main features.5,6 Since then, many chromosomal studies have been performed in series of infertile

males and the conclusions drawn from these studies are that constitutional chromosomal aberrations increase as sperm counts decrease. From these studies it is also clear that the incidence of numerical sex chromosomal aberrations such as 47,XXY and 47,XYY is proportionally higher in males with azoospermia compared to males with oligospermia, whereas structural chromosomal aberrations of autosomes such as Robertsonian (Fig 26.1a) and reciprocal (Fig 26.1b) translocations are proportionally more frequent in oligospermic males (Table 26.1).7–9 In azoospermic males it is also possible to find a 46,XX karyotype. In roughly 80% of these Klinefelterlike males the SRY gene, normally located close to the pseudoautosomal region of the short arm of the Y chromosome is now, due to a crossing-over event during meiosis, present in that same region on the X chromosomes.10,11 The SRY gene, referring to the sex-determining region of the Y chromosome, has to be expressed to induce the sexual development of an embryo towards a male phenotype.12 In the remaining 20% of XX males, most probably other genes concerned in sexual development are involved. Spermatogenesis seems to be absent in these males, whereas in apparently nonmosaic Klinefelter patients a few spermatozoa can be found in testicular tissue. Such spermatozoa have been used in ICSI procedures, and healthy as well as XXY children have been born.13–21

Microdeletion of the long arm of the Y chromosome (Yq11) In general, a microdeletion of a DNA sequence in the euchromatic part of the long arm of the Y chromosome (Yq11) will not be seen on a conventional or a high-resolution banded karyotype. Even a molecular cytogenetic fluorescent in situ hybridization (FISH) analysis is not suitable to identify such a microdeletion. One needs molecular techniques such as the polymerase chain reaction (PCR) to find these microdeletions of up to several tens of kilobases.22 Nevertheless, the first azoospermic male patients in

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Fig 26.1a 45,XY,der(13;14)(q10;q10) karyotype from a phenotypic normal male with a Robertsonian translocation of chromosomes 13 and 14 through centromeric fusion.

Fig 26.1b 46,XY, t(11;22)(q24.3;q12) karyotype from phenotypic normal male with a balanced reciprocal translocation of chromosomes 11 and 22 with break points in 11q24.3 () and 22q12 ().

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Table 26.1

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Incidence of chromosomal aberrations in infertile oligospermic and azoospermic males compared to newborns Infertile males (n = 7876)

Oligozoospermia (n = 1701)

Azoospermia (n = 1151)

Newborns (n = 94465)

Autosomes Sex chromosomes

1.3% 3.8%

3.0% 1.6%

1.1% 12.6%

0.25% 0.14%

Total

5.1%

4.6%

13.7%

0.39%

Aberrations

Summarized from reference 7.

Table 26.2

Risk calculations for a CF child or a CBAVD child in a case of CBAVD. Male

Female

Risk

No testing:

8/10

×

1/25

×

1/4

= 1/125

Testing female carrier: no carrier:

8/10 8/10

× ×

1 1/150

× ×

1/4 1/4

= 1/5 = 1/750

CF/CF CF/CF CF/5T

× × ×

1 1/150 1

× × ×

1/2 1/2 1/4

= 1/2 = 1/300 = 1/4 (CF) 1/8 (CBAVD)

Testing male + female female carrier: female no carrier: female carrier:

If the CBAVD patient is not tested for CF mutations, his risk of having at least one CF mutation is 8/10; if his partner is not tested and Caucasian, her risk of being a carrier of one CF mutation is 1/25. A carrier has a risk of 1/2 to transmit the mutation. Two carriers have a risk of 1/4 to transmit their mutated gene at the same time. A CBAVD patient with two mutations will always transmit a mutated gene. Risks for CF can be calculated if none of the partners are tested, if only the female partner is tested, if both partners are tested. In high-risk situations, PGD can be offered.98–100

whom the probable role of a deletion in the Yq11 region was linked to their infertility were identified through conventional cytogenetic analysis. At that time the concept of the azoospermia factor (AZF) region was introduced.23 During the last decade the content and the structure of the Y chromosome, consisting of the gene-containing euchromatic parts (Yp and Yq11) and the polymorphic heterochromatic parts (Yq12) have been studied in more depth. The existence of a single AZF, corresponding to one gene, did not hold through. The development of new approaches, such as the use of sequence-tagged sites (STSs) as primers to amplify parts of the Yq11 region of interest, induced several studies in azoospermic and oligospermic males. Depending on the inclusion criteria and the applied technology, the incidence of microdeletions in these patients varied from 1 to 55%, with a mean of 7.6%. Again it is clear that the lower the sperm count the higher the incidence of deletions.9,22,24–27 Careful evaluation of nonoverlapping microdeletions facilitated subdivision of the AZF region into at least AZFa, AZFb, and AZFc. In most patients the deletion spans the AZFb and/or AZFc region while in only a small number the AZFa region is deleted.22 Most deletions occur by intrachromosomal homologous recombination between repeat sequences spread over

the Yq11 region.28,29 The repeat sequences are either palindromes consisting of inverted repeat arms, or intrachromosomal repetitive sequences. Several genes have been identified in the AZF regions and they are currently being studied to prove their role in spermatogenesis.24,30–33 It is of course clear that if these microdeletions cause the spermatogenic defect that leads to a low to very low sperm count present in the ejaculate or to only a few sperm cells in the testes, these microdeletions will, through the use of ICSI, be transmitted to sons who most probably will be infertile as well.34 However, ICSI children are still too young to evaluate their fertility or their sperm count. In a few cases, fertility has been described in AZFcdeleted fathers who transmitted the deletion to their now infertile sons.32,35,36 Age at investigation may play a role, as observed in one patient with an AZFc deletion being oligospermic and later on azoospermic.37

Congenital bilateral absence of the vas deferens and cystic fibrosis Men with CBAVD have obstructive azoospermia. Spermatogenesis is usually normal and sperm can be obtained through microsurgical epididymal sperm aspiration (MESA), testicular sperm extraction (TESE), percutaneous epididymal sperm aspiration (PESA) or

1:8000

1:10 000

1:25 000

1:50 000

Myotonic dystrophy

Kallmann syndrome

Primary ciliary dyskinesia or immotile cilia syndrome

Kennedy disease or spinal bulbar muscular atrophy

Male (gynecomastia) Muscular atrophy

Male phenotype

Oligo-/azoospermia T normal or  LH, FSH 

Astenozoospermia

Azoospermia T, FSH, LH  No response to GnRH test

Normo-/oligospermia LH, FSH normal or  T normal or 

Lab tests

X-linked ‘CAG’ expansion in androgen receptor gene

AR Dynein deficiency Genetic heterogeneity (?)

X-linked Abnormal neuronal migration Point mutation in KAL1 gene AR and AD forms exist as well !!

AD ‘CTG’ expansion in DMPK gene

Cause

ICSI or AID

ICSI

Hormonal substitution

ICSI PGD

Treatment

72

68–71

60–66

56–59

References

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Male phenotype Pubertal delay Anosmia

Male phenotype Myotonia

Clinic

9/11/2008

AD, autosomal dominant; AR, autosomal recessive; AID, artificial insemination with donor sperm.

Frequency

Disease

Other known genetic causes of male infertility

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Table 26.3

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epididymal or testicular fine needle aspiration (FNA). These sperm can be used to fertilize oocytes in vitro through ICSI.3,38 CBAVD is known to be present in 97– 99% of male cystic fibrosis (CF) patients. CF is a frequent and by now well-known autosomal recessive disease in the Caucasian population, with an incidence of approximately 1/2500. Patients now survive into their twenties and thirties and suffer from severe lung disease and pancreatic insufficiency. They are often too ill to reproduce, although improved survival into adulthood generates interest in reproduction.39,40 The CFTR gene, with its mutations responsible for the eventual malfunction of the cystic fibrosis transmembrane conductance regulator involved in chloride transport across epithelial membranes, was described more than 10 years ago.41–43 CBAVD has also been observed in 1–2% of apparently healthy infertile males and in 6–10% of men with obstructive azoospermia.44 When the CFTR gene was studied in these males, mutations or splice site variants in intron 8 (the 5T-variant) interfering with gene expression were found in 80% of them.45–49 In 20% of the CBAVD patients, no link could be found with aberrant CFTR expression nor with any other etiology. However, in these patients, CBAVD-associated urinary tract malformations were observed.50,51 When performing ICSI with sperm from CBAVD males carrying CFTR mutations, their partners have to be tested for mutations in the same gene since the carrier frequency of CF mutations may be as high as 1/25. If both partners carry CFTR mutations, the risk of having a child with cystic fibrosis is 1/4 or 25%, or even 1/2 or 50% (Table 26.2). However, since the incidence and the type of CFTR mutations vary with the ethnic origin as well as with the geographical region, counseling and approach to treatment will have to be adjusted. In high-risk situations, prenatal diagnosis or preimplantation genetic diagnosis is indicated (see later).

Other known genetic causes of male infertility These males all have a 46,XY normal karyotype. Most of the defects are monogenic and either the specific gene defect is known or a chromosomal locus is known or suggested.52 A number of these rather rare conditions, which may be encountered in a fertility clinic, have been summarized in Table 26.3. Myotonic dystrophy is a rather common autosomal dominant muscular dystrophy with an incidence of 1/8000. The presence of an expanded CTGtrinucleotide repeat in the DMPK gene interferes with its function.53–56 Symptoms can be very mild, such as cataract at an advanced age, or very severe, as is the case in the congenital often lethal form of the disease. Severity is related to the number of CTG repeats.57 In 60–80% of the male patients, testicular tubular atrophy will develop and cause oligoasthenoteratospermia. When such spermatozoa are used to fertilize oocytes, the risk of transmitting the disease, often in a more severe

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form due to further expansion of the trinucleotide repeat, is 1/2 or 50%. Prenatal diagnosis or preferentially preimplantation diagnosis should be offered.58,59 Kallmann syndrome is characterized by hypogonadotropic hypogonadism, due to impaired gonadotropin-releasing hormone (GnRH) secretion, and anosmia. X-linked as well as autosomal recessive and autosomal dominant inheritance exists. The X-linked form of the Kallmann syndrome (KAL1 gene) is the most frequent and the best known one.60 An autosomal dominant form of Kallmann syndrome is caused by mutations in the FGFR1 gene.61 A possible interaction between the gene products of the KAL1 and FGFR1 genes has been suggested as a possible explanation for the higher prevalence of Kallmann syndrome in males than in females.62,63 In addition, mutations in the genes encoding prokineticin-2 and prokineticin receptor-2 have been implicated in Kallmann syndrome.64 The presence of mutations in different genes of some individuals suggests, that at least in some patients, a possible digenic mode of inheritance of Kallmann syndrome exists.64,65 Hormonal treatment will stimulate spermatogenesis in patients with Kallmann syndrome.66 Genetic counseling is indicated (Fig 26.2b). Primary ciliary dyskinesia or the immotile cilia syndrome is an autosomal recessive disease presenting with chronic respiratory tract disease, rhinitis, and sinusitis due to immotile cilia. Male patients are usually infertile because of asthenospermia. If the above symptoms are associated with situs inversus, the condition is called the Kartagener syndrome.67 With the help of ICSI, men with this condition can reproduce. Genetic counseling is hampered because the possibility for genetic testing is still limited.68–71 However, if we accept the incidence of 1/25 000, the carrier frequency must be 1/80, which means that the risk of a man to have an affected child is 1/160 (1 × 1/80 × 1/2). Kennedy disease or spinal bulbar muscular atrophy (SBMA) is a neuromuscular disease causing muscular weakness that is associated with testicular atrophy and leads to oligo- or azoospermia. It is an Xlinked disease caused by an expanded (CAG) trinucleotide repeat in the transactivation domain of the androgen receptor gene.72 If treated with ICSI, again genetic counseling is indicated. Point mutations in the androgen receptor gene, resulting in androgen insensitivity through impaired binding of dihydrotestosterone to the receptor, will interfere with sexual development. The resulting syndrome is testicular feminization or androgen insensitivity syndrome, causing a female phenotype.73 The presenting problem here will not be male infertility. Patients with an autosomal recessive 5α-reductase deficiency, and therefore unable to synthesize dihydrotestosterone from testosterone, may theoretically present at the clinic with azoospermia and pseudohermaphroditism.74 Very rarely, patients with other genetic defects may consult at a male infertility clinic. Patients with

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MCA 45,XX,der(13;14)

OAT 45XXder(13;14)

45,XY,der(13;14)

46,XY

Fig 26.2a Segregation of a Robertsonian translocation der(13;14) in a family: its consequences, and recommendations. OAT (our proband ) presents with infertility due to oligoastenoteratospermia. His sister had two miscarriages (
); his brother has two healthy children. His mother had two miscarriages (
), lost a brother born with multiple congenital anomalies (MCA), and has a healthy brother without children. This story is suggestive of a chromosomal translocation. The karyotype of OAT points indeed to a Robertson translocation der(13;14) (Fig 26.1a). His mother and his sister have the same translocation explaining the recurrent miscarriages (
). These miscarriages are most probably resulting from a trisomy 14, or a monosomy 13 or 14. The brother of OAT has a normal karyotype, which is perfectly possible. The MCA brother of the mother died and had most probably a trisomy 13. OAT should be informed about all the above possible risks in case of pregnancy. In case of ICSI, a preimplantation genetic diagnosis or a prenatal diagnosis should be offered.

KAL

KAL

Fig 26.2b X-linked Kallmann syndrome in a family: its consequences, and recommendations. KAL (our proband ) has Kallmann syndrome. The family history fits with an X-linked transmission since the brother of the mother of KAL has the same disease. This means that the mother of KAL must be a carrier() Her daughter, the sister of our proband, has therefore a 1/2 risk of being a carrier and a 1/4 risk of having an affected son. Preimplantation or prenatal diagnosis should be discussed. If the wife of KAL becomes pregnant, boys will be healthy and fertile (because they inherit the Y chromosome of their father), while girls will always be carriers.

Noonan syndrome may present with oligo- or azoospermia as a result of cryptorchidism. The diagnosis is so far based on other symptoms (including a small stature, a rather typical facial dysmorphism, and heart disease). Defects in a gene on chromosome 12q24.1, PTPN11, are responsible for approximately 50% of patients with Noonan syndrome.75 Another three genes involved in Noonan syndrome have been identified; more genes are probably involved. The autosomal dominant inheritance requires genetic counseling.76,77 Other possible patients may be affected by the Aarskog–Scott syndrome with acrosomal sperm defects,78 the Beckwith–Wiedemann syndrome with cryptorchidism79 and adrenomyeloneuropathy with oligo- or azoospermia.80 Syndromes such as the Bardet–Biedl syndrome and the Prader–Willi syndrome, both presenting with hypogonadism, are associated with other major symptoms including mental retardation, which limit procreation.81,82 It is noteworthy that the Prader–Willi syndrome is an imprinting syndrome resulting from the absence of the expression of the paternal alleles in the 15q11-q13 imprinted region.83,84 Other causes of male infertility include a deficiency in enzymes involved in testosterone

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synthesis,74 luteinizing hormone-, and luteinizing hormone receptor deficiencies.85,86 Defects in energy production by the mitochondria have been implicated in male infertility. Mitochondria are the main source of energy production for the cells through the process of oxidative phosphorylation (OXPHOS). The synthesis of ATP occurs through the action of five enzyme complexes that are encoded by nuclear genes and partly by the small mitochondrial genome that is exclusively maternally inherited. Mitochondrial diseases usually evolve as multisystem disorders mainly affecting the central nervous system and muscle. In addition, these defects in the respiratory function are believed to cause a decline in sperm motility because of ATP depletion that is necessary for flagellar propulsion of the spermatozoa.87,88 Reduced sperm motility and resulting male infertility have been well documented in several patients with mitochondrial encephalopathies caused by mitochondrial tRNA point mutations or multiple mtDNA deletions.89–91 Moreover, several research groups have demonstrated the presence of an almost 5 kb deletion in sperm with diminished motility in otherwise healthy males.92–95

Consequences and recommendations in the clinic Genetic evaluation of infertile males before ART Not only a personal history should be taken. Also, a detailed pedigree should be drawn and completed for miscarriages or children (also deceased) with multiple congenital malformations in first- or second-degree relatives. It is also important to know about infertility in sibs or other family members. This information may suggest a possible chromosomal aberration such as a translocation or a monogenic disease like Kallmann syndrome or cystic fibrosis (Fig 26.2a; Table 26.2). A thorough inquiry of the proband and his partner may also pinpoint other hereditary diseases not necessarily causing infertility but causing morbidity or being lethal to offspring. A complete clinical examination of the proband and his partner is useful to establish a clinical diagnosis of a disease or a syndrome associated with infertility such as Klinefelter syndrome or CFlinked CBAVD. This examination may also reveal other possible hereditary diseases not identified as such before, which means that the couple does not yet know and should be counseled before treatment starts. Complementary tests, mainly laboratory investigation, will allow confirmation of a clinical diagnosis. In the case of male infertility, the history, clinical examination, a semen analysis, and hormonal tests are sufficient to characterize most of the patients as being: 1. 2.

Infertile in association with other physical or mental problems. Infertile, but otherwise healthy; these patients can be subdivided into oligospermic or eventually

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oligoasthenoteratospermic males and into obstructive or nonobstructive azoospermic males. Genetic investigations will help to refine the diagnosis and to counsel accordingly. The above information will help to select the additional tests to be performed. In most cases of male infertility due to severe OAT or nonobstructive azoospermia, a peripheral karyotype should be performed, even if the family history is not suggestive of a chromosomal disorder.7–9 In the same patients AZFa,b,c deletions on Yq11 should be looked for in peripheral blood. With this genetic test, attention should be paid to the techniques used to confirm the presence of a Yq11 deletion. To avoid erroneous results, laboratories can now participate in quality control studies.96 The possibility of fertility treatment in couples in whom the male has an AZF deletion is strongly dependent on the type of deletion present.97 In men with CBAVD without other anomalies of the urogenital tract, mutations in the CFTR gene should be looked for in the patient and, even more important in his partner. At present, it is possible to identify 85– 90% of carriers in the Caucasian population by using a laboratory kit detecting 19–35 of the most common mutations (ex: INNO-LiPATM CFTR19 and CFTR 17+Tn, Innogenetics, Ghent, Belgium). Depending on whether CFTR mutations have been identified in the male patient and/or his female partner, the risk of conceiving a child with cystic fibrosis can be calculated (Table 26.2). These figures, together with the type of mutations, allow prenatal diagnosis or preimplantation genetic diagnosis to be offered.40,99,100 Other, more specific tests should be performed if other diseases such as Kennedy disease, Kallmann syndrome, myotonic dystrophy, the immotile cilia syndrome, or other syndromes or diseases are suspected. In these cases it is again not only important to establish a correct diagnosis to treat correctly but also to counsel the proband and his family concerning recurrence risks and prenatal or preimplantation diagnosis.

Genetic testing during ART for severe male infertility Genetic tests, which can be performed during ART, refer to preconceptional or preimplantation genetic diagnosis. These procedures refer to the genetic analysis (PGD) of one or two polar bodies before fertilization or to the analysis of one or two blastomeres of the 8- to 10-cell embryo in vitro.101,102 The aim is to avoid the birth of a child with a genetic disease. PGD makes conventional prenatal diagnosis, eventually followed by termination of pregnancy, obsolete. PGD is a complex procedure because of the ‘single cell’ genetic diagnosis. It was developed and first applied in the clinic more than 15 years ago.103 In reference to this chapter, most of the PGDs performed were for cystic fibrosis, myotonic dystrophy, Huntington’s disease, and

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Duchenne’s muscular dystrophy, but many others have been performed for either infertile or fertile couples.104–106 For chromosomal aberrations, most PGDs have been done for reciprocal and Robertsonian translocations.104–111 In general, the take-home baby rate is of the same order of magnitude of 20–25%, as in ICSI cycles in general.3,4 A number of PGDs have been performed for Klinefelter patients in whom spermatozoa found in the testes were used to fertilize oocytes.13–18,112–115

Genetic evaluation of pregnancies and children conceived through ICSI because of severe male infertility Follow-up studies of pregnancies established and children born after the use of ICSI were initiated as soon as this new procedure was applied in the clinic. From these, still ongoing, studies it became clear that the number of major malformations was comparable to the number of major malformations in IVF children and possibly slightly higher than in naturally conceived children. Preliminary results on the psychomotoric development of these children are also reassuring.3,4,116–125 The ‘de novo’ chromosomal aberrations found at prenatal diagnosis indicate that numerical sex chromosomal anomalies are slightly increased when compared to a large newborn population. If the incidence in the newborn is 0.2%, the incidence in ICSI children is 0.8%. This is a four-fold increase, but of course the overall incidence remains low (<1%). Apart from sex chromosome anomalies, also de novo balanced translocations have been observed.3,4,126 These aberrations occurring in children from men with a normal peripheral karyotype could be related to chromosomal anomalies being present in their sperm, but not in their lymphocytes.127–130

Controversies

Who to test? Among those clinicians who are convinced that genetic tests are useful and among the geneticists performing the tests, the main ongoing discussion is in which infertile male patients karyotypes and Yq deletion tests should be performed. With time, many do now agree on performing these genetic tests if the sperm count is below 1 or 5 × 106 spermatozoa/ml, although chromosomal aberrations as well as Yq deletions have been found in patients with more than 5 × 106 spermatozoa/ml, although to a lesser extent.4,25,133 Based on a few reports, one can also wonder whether karyotypes of the female partners should be performed.134–137 One reason, of course, to limit the patient population to be tested is that most of these tests are still cumbersome and costly. Prenatal diagnosis through chorionic villus sampling or amniocentesis after ICSI should be discussed with the couple in view of the known increase in sex chromosomal aberrations in the offspring.3,138

Genetic testing versus genetic screening Genetic screening is different from genetic testing. A screening test is offered to a ‘healthy’ population. In that case, the persons who are tested have no particular problem but they may be interested to know whether they are carriers of a particular gene mutation in order to take preventive measures. Examples are screening programs for cystic fibrosis, Tay–Sachs disease, and other diseases in certain high-risk populations.139–141 Couples may want to know before having children, since if both partners are carriers of such an autosomal recessive gene the risk of having an affected child is 1/4. Such screening programs are not specific to infertile patients. However, a fertile couple with a 25% recurrence risk may choose to have prenatal diagnosis to prevent the birth of an affected child, while if the couple is infertile and can be helped with IVF they may choose to have preimplantation genetic diagnosis.142

To test or not to test Some clinicians claim that now that ICSI is available to alleviate male infertility, it is sufficient to know whether these patients are oligospermic or azoospermic. Oligospermic and obstructive azoospermic males can be treated immediately and often successfully even if repetitive in vitro fertilization (IVF) cycles are necessary.131 Even in case of nonobstructive azoospermia, repeated testicular sperm extraction leads to a high sperm recovery rate that allows ICSI.132 It is probably true that in the majority of the cases a healthy although maybe infertile child will be born. Nevertheless in a number of cases, e.g. in the case of a chromosomal translocation, the treatment will fail and be repeated endlessly or recurrent miscarriages will occur. Furthermore, a few CF children will be born, and probably a few other children with genetic disease which could have been avoided. Another option could be not to use ICSI further and leave decisions to nature.

Preimplantation genetic diagnosis for aneuploidy screening PGD-aneuploidy screening, a novel approach to select the ‘better’ embryos for transfer after IVF/ICSI is at present offered to selected groups of patients. Here the embryos are biopsied, and a variable number of chromosomes, usually 13, 16, 18, 21, 22, X, and Y are enumerated using specific fluorescent in situ hybridization probes. Embryos diploid for the chromosomes tested are then transferred without (of course) having information on the other chromosomes. The observations reported so far are that in women over 37 years old, the IVF success rate increases,143 the rate of miscarriage decreases,144 and the implantation rate per embryo increases.145–147 Recently, some studies have failed to show a benefit of PGD for aneuploidy screening in selected populations needing ART.146,147 Therefore,

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more data are needed to evaluate the benefit of this aneuploidy screening performed on IVF embryos. 6.

Is ICSI in case of severe male infertility safe? Although the follow-up studies of pregnancies and children born after ICSI are reassuring, still a number of questions remain unanswered, one of them being the concerns in relation to imprinting.125,148–154

7.

8. 9.

Future ICSI performed with ejaculated spermatozoa at first, and later on with epidydimal and testicular spermatozoa, may be considered milestones in infertility treatment for the male patient. Today, very few men cannot be helped to have their own child. And research is ongoing to find other solutions for their fertility problem.155–158 However, some approaches are extremely controversial. ICSI has also triggered basic research in biology and genetics in order to gain more insight into gender development and spermatogenesis. Over the last years many novel genes have been and are being identified. New findings will increase our knowledge and allow more accurate diagnosis and counseling and probably new ways of treatment will become available.

10.

11.

12.

13.

14.

Conclusion In the case of severe male infertility, good clinical practice requires genetic evaluation before, during, and after ART in order to properly treat and counsel the proband, the couple, and eventually the family. The aim is to inform the patients about possible risks, to improve the success rate of the ART treatment, and to avoid the birth of children affected with a severe genetic disease. Moreover, at present, there are still many unknown causes of male infertility. More research, also in the field of genetics, will allow better understanding and definition of the risks of transmitting infertility or possibly other genetic anomalies to the next generations.

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Severe male factor: genetic consequences 121. Bonduelle M, Ponjaert I, Van Steirteghem A, et al. Developmental outcome of children born after ICSI compared to children born after IVF at the age of two years. Hum Reprod 2003; 19: 1–9. 122. Sutcliffe AG, Saunder K, Thornton S, Lieberman BA, Grudzinkas JG. Outcome in the second year of life after in-vitro fertilisation by intracytoplasmic sperm injection: a UK case-control study. Lancet 2001; 357: 2080–4. 123. Belva F, Henriet S, Liebaers I, et al. Medical outcome of 8-year-old singleton ICSI children and a spontaneously conceived comparison group. Hum Reprod 2007; 22: 506–15. 124. Leunens L, Celestin-Westreich S, Bonduelle M, Liebaers I, Ponjaert-Kristoffersen I. Cognitive and motor development of 8-year-old children born after ICSI compared to spontaneously conceived children. Hum Reprod 2006; 21: 2922–9. 125. Sutcliffe AG, Ludwig M. Outcome of assisted reproduction. Lancet 2007; 370: 351–9. 126. Van Steirteghem A, Bonduelle M, Camus M, et al. Outcomes from intracytoplasmic sperm injection. In: Jansen R, Mortimer D, eds. Towards Reproductive Certainty. Fertility & Genetics beyond 1999. Proceedings of the 11th World Congress on in Vitro Fertilization and Human Reproductive Genetics, Sydney, Australia, May 9– 14, 1999. New-York: Parthenon, 1999: 70–6. 127. Martin RH. Genetics of human sperm. J Assist Reprod Genet 1998; 15: 240–5. 128. Aran B, Blanco J, Vidal F, et al. Screening for abnormalities of chromosomes X, Y, and 18 and for diploidy in spermatozoa from infertile men participating in an in vitro fertilization– intracytoplasmic sperm injection program. Fertil Steril 1999; 72: 696–701. 129. Vegetti W, Van Assche E, Frias A, et al. Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men. Hum Reprod 2000; 15: 351–65. 130. Egozcue S, Blanco J, Vendrell JM, et al. Human male infertility: chromosome anomalies, meiotic disorders, abnormal spermatozoa and recurrent abortion. Hum Reprod Update 2000; 6: 93–105. 131. Osmanagaoglu K, Tournaye H, Camus M, et al. Cumulative delivery rates after intracytoplasmic sperm injection: 5 year follow-up of 498 patients. Hum Reprod 1999; 14: 2651–5. 132. Vernaeve V, Verheyen G, Goossens A, et al. How successful is repeat testicular sperm extraction in patients with azoospermia? Hum Reprod 2006; 21: 1551–4. 133. Foresta C, Ferlin A, Gianaroli L, Dallapiccola B. Guidelines for the appropriate use of genetic tests in infertile couples. Eur J Hum Genet 2002; 303–12. 134. Meschede D, Lemcke B, Exeler JR, et al. Chromosome abnormalities in 477 couples undergoing intracytoplasmic sperm injection – prevalence, types, sex distribution and reproductive relevance. Hum Reprod 1998; 13: 576–82. 135. van der Ven K, Peschka B, Montag M, et al. Increased frequency of congenital chromosomal aberrations in female partners of couples undergoing

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intracytoplasmic sperm injection. Hum Reprod 1998; 13: 48–54. Papanikolaou EG, Vernaeve V, Kolibianakis E, et al. Is chromosome analysis mandatory in the initial investigation of normovulatory women seeking infertility treatment? Hum Reprod 2005; 20: 2899–903. Riccaboni A, Lalatta F, Caliari I, et al. Genetic screening in 2,710 infertile candidate couples for assisted reproductive techniques: results of application of Italian guidelines for the appropriate use of genetic tests. Fertil Steril 2008; 89: 800–8. Aytoz A, De Catte L, Camus M, et al. Obstetric outcome after prenatal diagnosis in pregnancies obtained after intracytoplasmic sperm injection. Hum Reprod 1998; 13: 2958–61. Vallance H, Ford J. Carrier testing for autosomalrecessive disorders. Crit Rev Clin Lab Sci 2003; 40: 473–97. Kaback MM. Population-based genetic screening for reproductive counseling: the Tay–Sachs disease model. Eur J Pediatr 2000; 159S3: 192–5. Gason AA, Sheffield E, Bankier A, et al. Evaluation of a Tay–Sachs disease screening program. Clin Genet 2003; 63: 386–92. Liebaers I, Bonduelle M, Van Assche E, et al. How far should we go with genetic screening in assisted reproduction? In: Kempers RD, Cohen J, Haney AF, eds. Fertility and Reproductive Medicine. Proceedings of the XVI World Congress on Fertility and Sterility, San Francisco. Amsterdam: Elsevier Science BV, 1998: 247–54. Verlinsky Y, Cieslak J, Ivakhnenko V, et al. Prevention of age-related aneuploidies by polar body testing of oocytes. J Assist Reprod Genet 1999; 16: 165–9. Munné S, Magli C, Cohen J, et al. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum Reprod 1999; 14: 2191–9. Gianaroli L, Magli MC, Ferraretti AP, et al. Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for what should be proposed. Fertil Steril 1999; 72: 837–44. Staessen C, Platteau P, Van Assche E, et al. Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized controlled trial. Hum Reprod 2004; 19: 2849–58. Mastenbroek S, Twisk M, van Echten-Arends J, et al. In vitro fertilization with preimplantation genetic screening. N Engl J Med 2007; 357: 9–17. Manning M, Lissens W, Bonduelle M, et al. Study of DNA-methylation patterns at chromosome 15q11-q13 in children born after ICSI reveals no imprinting defects. Mol Hum Reprod 2000; 6: 1049–53. Pfeifer K. Mechanisms of genomic imprinting. Am J Hum Genet 2000; 67: 777–87. De Rycke M, Liebaers I, Van Steirteghem A. Epigenetic risks related to assisted reproductive technologies. Risk analysis and epigenetic inheritance. Hum Reprod 2002; 17: 2487–94.

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151. Cox GF, Burger J, Lip V, et al. Intracytoplasmic sperm injection may increase the risk of imprinting defects. Am J Hum Genet 2002; 71: 162–4. 152. Debaun MR, Niemitz EL, Feinberg AP. Association of in vitro fertilization with Beckwith–Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet 2003; 72: 156–60. 153. Maher ER, Brueton LA, Bowdin SC, et al. Beckwith– Wiedemann syndrome and assisted reproduction technology (ART). J Med Genet 2003; 40: 62–4. 154. Moll AC, Imhof SM, Cruysberg JRM, et al. Incidence of retinoblastoma in children born after in-vitro fertilisation. Lancet 2003; 361: 309–10.

155. Ko K, Schöler HR. Embryonic stem cells as a potential source of gametes. Semin Reprod Med 2006; 24: 322–9. 156. Ehmcke J, Wistuba J, Schlatt S. Spermatogonial stem cells: questions, models and perspectives. Hum Reprod Update 2006; 12: 275–82. 157. Kubota H, Brinster RL. Technology insight: in vitro culture of spermatogonial stem cells and their potential therapeutic uses. Nat Clin Pract Endocrinol Metab 2006; 2: 99–108. 158. Nagy ZP, Chang CC. Artificial gametes. Theriogenology 2007; 67: 99–104.

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27 Polar body biopsy Markus Montag, Katrin van der Ven, Hans van der Ven

Introduction Polar body biopsy with subsequent analysis of chromosomal abnormalities was introduced in 1990 by Verlinsky et al.1 and, until today, the group in Chicago has the largest experience in polar body diagnosis2. This technique opened the era of preconception genetic diagnosis as an alternative to preimplantation genetic diagnosis (PGD) of the embryo, which was proposed earlier by Handyside et al.3 It is important to note that polar body diagnosis gives direct information about the first and second polar body and therefore only allows an indirect diagnosis of the maternal genetic or chromosomal constitution of the corresponding oocyte. In contrast, PGD of the embryo gives a direct diagnosis for the embryo and allows the detection of both maternally and paternally derived genetic or chromosomal contributions. Obviously, this is the reason why PGD following removal of 1–2 blastomeres from an embryo is by far the more commonly applied technique worldwide.4 However, in countries with legal restrictions, such as Austria, Germany, Italy, and Switzerland, only polar body diagnosis is possible within the framework of the existing embryo protection laws, provided that the diagnosis is completed prior to fusion of the pronuclei (Austria, Germany, Switzerland)5 or at the oocyte stage (Italy).6 Furthermore, this technique may be more readily accepted by couples with ethical or moral constraints towards the generation and discarding of supernumerary zygotes and embryos, as in the case of PGD.7 This chapter gives an overview of the expectations and limitations of polar body diagnosis, and relevant technical details, with special emphasis on aneuploidy screening.

Clinical application of polar body diagnosis Polar body biopsy has been successfully used for the detection of numerical and structural chromosomal abnormalities in human oocytes2,8 and for the diagnosis of monogenetic diseases.9

Polar body biopsy and detection of numerical chromosomal abnormalities: aneuploidy screening Numerical chromosomal abnormalities are characterized by a false distribution of chromosomes or chromatids in the first or second polar body. These errors are strongly correlated to maternal age. Up to 70% of oocytes from women >40 years old can possess such a disorder.10 This explains why women with advanced maternal age have a lower chance for pregnancy and a higher risk to miscarry once they are pregnant. One possibility to reduce these risks and probably to increase the success rates is a screening for maternally derived chromosomal abnormalities of the oocyte. This can be achieved by polar body diagnosis. During the first meiotic division the diploid chromosome content of an oocyte is reduced to two haploid chromosome sets, one of which is extruded as part of the first polar body. Fluorescent in-situ hybridization (FISH) analysis of the first polar body should reveal two hybridization signals due to the presence of paired chromatids for each of the tested chromosomes. Sperm entry into an oocyte initiates the second meiotic division where the set of paired chromatids is reduced and a single chromatid set becomes part of the second polar body. Therefore, FISH analysis of the second polar body should reveal only one signal for each chromosome due to the presence of single chromatids. After first meiotic division, the number of the chromosomes in the oocyte and the first polar body should be identical and the same holds true for the number of chromatids following the second meiotic division. In general, numerical chromosome aberrations originate more often during the first meiotic division compared to the second meiotic division, although there are chromosome-specific prevalences.11–13 Numerical chromosomal abnormalities can be caused by nondisjunction, meaning that a whole chromosome is not directed to the proper compartment (either oocyte or polar body). Another mechanism is premature chromatid segregation – or predivision, as named by others – of the chromatid dyad structure into two

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single, separated chromatids, which has been suggested to occur frequently prior to the first meiotic division.14 Premature chromatid segregation during meiosis I can either lead to a balanced situation where both chromatids remain in the same compartment, or to an unbalanced situation where the two chromatids are finally allocated to different compartments. Some of the unbalanced segregations which originate in meiosis I in the oocyte can be corrected in meiosis II during formation of the second polar body.15 This explains why the analysis of both polar bodies is advisable in order to achieve a more precise and accurate diagnosis. Polar body biopsy and FISH analysis of the first and second polar body offers the possibility of detecting numerical chromosome aberrations and establishing an indirect diagnosis for the corresponding oocyte. Alterations in the number of signals in the first and second polar body indicate a trisomy, if a single or double-dotted signal is missing in the polar bodies, or a monosomy, if a single or double-dotted signal is found in excess within the polar body. The careful observation and analysis of the number of FISH signals allows classification if the underlying mechanism for the potential trisomy or monosomy is caused by nondisjunction (double dotted-signal) or by unbalanced premature chromatid segregation (single signal16). The introduction of FISH and the availability of commercial multicolor chromosome probes for chromosomes 13, 16, 18, 21, and 22, which show a high prevalence for numerical chromosome aberrations in abortion material, nowadays make possible the simultaneous detection of up to 5 chromosomes in a single hybridization reaction.

Polar body biopsy and detection of structural chromosomal aberrations Structural chromosomal aberrations, e.g. balanced translocations, were found at a higher rate among infertile couples than among the normal population.17–19 Provided that the female is carrier of a balanced translocation, polar body biopsy and subsequent FISH analysis allows selecting against abnormal oocytes using a reliable method for analysis. In the past this has been achieved by case-specific probes which were designed around the chromosomal breakpoints for each individual patient.20 This task was laborious and time-consuming.21 It has also been proposed to inject polar bodies into enucleated oocytes for the production of metaphase chromosomes to enable the direct characterization of the translocation without the need for FISH probes.22 However, the most promising technique, presented by Munné et al, involves the combined use of centromeric and telomeric probes for FISH analysis of the chromosomes involved in the translocation.8 Based on this technique, the authors reported their experience with 35 cases using either polar body biopsy or

embryo biopsy. In the near future, whole-genome amplification and comparative genomic hybridization (CGH) will be the methods of choice if all present problems associated with amplification of DNA from single cells are solved and optimized.23

Polar body biopsy and detection of monogenetic aberrations The detection of monogenetic disorders by polar body or blastomere biopsy requires that the DNA of interest (e.g. the region containing the mutation) from a single cell or the first and second polar body is accurately amplified by polymerase chain reaction (PCR) with locus-specific oligonucleotide primers. Although this approach is feasible, single-cell PCR in general can be subject to a number of problems. In addition to the general danger of contamination of PCR reagents and products with foreign DNA, with a potential resulting misdiagnosis, specific problems need to be addressed in the case of polar body biopsy. A major problem in single-cell PCR is the correct amplification of the region of interest, and it is known from numerous reports that in diploid cells, occasionally one allele will not amplify, also known as allele dropout (ADO).24–28 Although this phenomenon will not lead to a misdiagnosis in homozygous mutant or homozygous wild-type single cells, the situation is different in heterozygous single cells and especially in polar body diagnosis. Recombination and crossing-over of homologous chromatids frequently occurs during meiosis. As a result, the first polar body may consist of one chromatid carrying the mutation of interest and one chromatid carrying the wild-type or normal sequence. In this case, ADO may directly lead to a misdiagnosis, if crossing-over and ADO remain undetected. Only in the case that analysis of the second polar body, which carries either a mutant or a normal chromatid, reveals a discrepant result from the first polar body, the problem would be recognized. Although ADO seems to be a frequent phenomenon in PGD (between 1 and 25% and up to 40%),29 the frequency of ADO in polar body diagnosis is mainly unknown due to the low number of cases. Verlinsky30 reported a frequency of ADO of 6% in 100 polar bodies that were analyzed. Several strategies have been proposed to overcome this diagnostic problem, mainly coamplification of polymorphic markers that are closely linked to the region of interest or the improvement of amplification efficiency through the use of nested primers. The use of PCR conditions that allow for continuous quantification of the PCR product, e.g. with fluorescent primers, will help to determine ADO or cases of preferential amplification of alleles. Although ADO has for long been recognized to be a substantial problem in PGD and especially polar body diagnosis, a systematic evaluation of this phenomenon, which might eventually lead to strategies to decrease ADO rates, has only recently been begun.29

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The use of polar body biopsy for the detection of monogenetic disorders is not the main issue of this chapter. The technique is feasible; however, proper investigation of the first and second polar body with all the necessary controls must be performed. Thus, the time required until reliable results are available is approximately 30–35 hours from the point of follicular puncture. Therefore, this approach is no option for countries with a narrow time frame due to legal constrictions. Furthermore, due to the high risk of an inconclusive diagnosis derived from the analysis of the first and second polar body, a combined approach using PCR on polar bodies for a polar-body-based diagnosis first, followed by PCR on blastomeres to verify the first diagnosis, was proposed.31 However, this approach is questionable due to a higher number of technical manipulations for polar body and blastomere retrieval, and this strategy is once more not possible in certain countries. Because, in polar body diagnosis, only the maternal contribution to a potential genetic disease can be investigated, the isolated application of this technique is limited to selected genetic scenarios, e.g. autosomal dominant diseases with an affected mother or X-linked recessive and dominant disease where the mother is the mutation carrier. In the majority of cases, PGD is the only or the better option to achieve a reliable prognosis about the genetic constitution of the embryo to be transferred.

Polar body biopsy techniques Removal of polar bodies requires access to the perivitelline space through the zona pellucida. An opening in the zona pellucida can be introduced by using acidic Tyrode’s solution as a chemical means.32 The general disadvantage of acidic Tyrode’s solution has been discussed elsewhere, 33 and, in particular, the sensitivity of the oolemma of unfertilized oocytes makes this method unsuitable for polar body biopsy. Another method based on three-dimensional zona dissection was proposed by Cieslak et al.34 Although this method can be performed with simple glass tools, multiple steps including dissection, release, and rotation of the oocyte are needed. This procedure definitely requires skill and time, at least if compared to the recently introduced technique of 1.48-µm diode laser-drilling for zona opening.35–36 We adapted the laser technique for polar body biopsy in 1997.37 Animal experimentation showed the potential of this method for polar body biopsy and for assisted hatching38 and allowed investigation of its proper mode of application.39 We found that the size and position of laser-drilled openings can influence further embryonic development and, in particular, the mode of hatching at the blastocyst stage.40 Due to its ease, laser-assisted biopsy is now widely used for biopsy of blastomeres41,42 and blastocyst

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cells,43 and recently its advantage compared to acidic Tyrode’s solution was reported.44 In our initial presentation in 1998,38 we proposed a very straightforward approach for laser-assisted polar body biopsy; however, in the mean time, we favor an individual procedure that depends on the actual biopsy situation. The first method is preferred if only the first polar body is biopsied (Fig 27.1). The size of the drilled opening is usually in the range of 25–30 µm, but it can be easily adjusted to the diameter of the aspiration capillary. As the capillary can be introduced through the laser-drilled opening, there is no need for a sharp aspiration needle. This allows the use of flame-polished, blunt-ended aspiration needles and greatly reduces the risk of damaging the polar body, the blastomere, or the remaining oocyte or embryo. The procedure becomes safer, more accurate, and more reliable, thus allowing a significant reduction in the number of cells that cannot be reliably diagnosed as a result of technical problems during the biopsy procedure.5 Another benefit is that laser drilling and subsequent biopsy can be performed without changing the culture dish or the capillaries in contrast to zona drilling using acidic Tyrode’s solution. This may help to prevent contamination of samples to be diagnosed by sensitive techniques such as PCR. The simultaneous removal of the first and second polar body is best accomplished if the ooycte is affixed to the holding capillary with the first polar body at the 12 o’clock position and the second polar body to the left of the first polar body but in the same focal plane. A 25–30 µm opening is drilled at 2–3 o’clock and, by pushing the biopsy capillary into the perivitelline space, both polar bodies can be removed simultaneously, provided that the cytoplasmic bridge between the second polar body and the oocyte is not too firm (Fig 27.2). Again we use a blunt-ended capillary, so that even manipulations in direct vicinity to the oolemma do not damage the oocyte. Among the first 200 patients we have treated, we noted in five patients that the oolemma was very sensitive to zona opening and subsequent manipulation. Therefore we developed a third technique to reduce manipulations within the perivitelline space (Fig 27.3). Before opening the zona pellucida, we perform a thinning of the zona over one-fifth of the circumference of the oocyte. We then introduce a small opening (<5 µm) directly opposite to the polar body, which allows entry to the perivitelline space with an aspiration capillary. This method requires the use of a sharp aspiration capillary in order to enable penetration through the narrow channel created by the laser. This method facilitates aspiration of the polar body with minimal intervention; however, both polar bodies can only be removed if they are located close to each other. In all manipulation steps and zona opening techniques we try to stick to two rules:

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Fig 27.1 Polar body biopsy using a laser-drilled opening at the site of the polar body. This method is preferred if only the first polar body is removed. The oocyte is held in a position where the polar body is located at 3 o’clock. The blunt-ended aspiration capillary with an outer diameter of 20 µm is already positioned prior to opening of the zona (a). Two laser shots are usually sufficient to drill an opening that allows for the penetration of the capillary (b), followed by immediate aspiration of the polar body by gentle suction (c).

1.

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We always drill only one opening, as two openings (e.g. to retrieve both polar bodies through separate openings) may cause problems at the time of hatching because the embryo could hatch through both openings simultaneously and therefore may get trapped within the zona.39 We always try to generate a sufficient opening to allow consecutive hatching at the blastocyst stage, because smaller openings (<15 µm) may also cause trapping of the embryo, followed by degeneration.39 This is the reason why we perform zona thinning if only small complete opening is generated in the zona in the third method. The small channel in this approach could trap the embryo during hatching because the entire

thickness of the zona can occasionally be resistant to the embryo. Laser-drilled openings will stay permanently in the zona, and therefore we recommend gentle handling during subsequent transfer of oocytes to other media droplets and even during the embryo transfer. It is also worthwhile mentioning that all manipulation steps require a good operating micromanipulation system and extremely fine adjustable microinjectors, especially for the process of polar body removal. This work will be best supported by using automatic micromanipulators in which certain positions of the capillaries can be stored in a memory in order to speed up the procedure.

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Fig 27.2 Simultaneous biopsy of the first and second polar body. For removal of the first and second polar body the oocyte is held in a position where the polar bodies are located at 12 o’clock (a). An opening is drilled at 1–2 o’clock (b), which allows retrieval of both polar bodies by sliding the capillary over them (c). If the second polar body is still firmly fixed to the oolemma, the capillary with the second polar body already inside is slowly forced towards the left in order to rupture the cytoplasmic bridge. Note the sharp border of the laser-drilled opening (d).

Pretreatment of polar bodies and transfer onto glass slides for aneuploidy screening Once the polar bodies are biopsied, they are placed in a neighboring droplet of medium until all oocytes are biopsied. A special pretreatment of polar bodies, such as hypo-osmotic swelling or proteinase/pronase treatment, is not necessary due to the small cytoplasmic content of polar bodies. For transfer onto the glass

slide, polar bodies are removed individually from their drop and transferred with the aspiration capillary into a tiny drop (0.2 µl) of water placed on a clean glass slide (Fig 27.4). The small volume guarantees that the polar body will attach to the slide within a small area and that the fluid will dry out very fast, which reduces the risk of a dislocation of the polar body on the slide. Nevertheless, the drying process must be observed under a stereomicroscope and the final location of the polar body after air-drying must be marked on top of

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Fig 27.3 Polar body biopsy for oocytes with sensitive oolemma. Oocytes with a sensitive oolemma require as minimal an interaction as possible. For this method we use a beveled aspiration needle, which is positioned at an angle of 30° so that the opening is nearly in parallel to the oolemma and the polar bodies, which are positioned at 12 o’clock (a). The zona is thinned by several laser shots and a small opening is drilled with one single shot at minimal energy (b). Using the sharp tip, the needle can be slit through the small opening and positioned just above the polar bodies (c), which can then be isolated (d).

the slide by encircling with a diamond marker. With some experience, 4–6 polar bodies can be placed within a round area of 10 mm, each encircled with a diamond marker. We recommend performing fixation with 2–3 drops of 10 µl methanol:acetic acid (3:1, ice cold −20oC) and another fixation after air-drying using methanol at room temperature for 5 min. Once the slides are air-dried, 2.5 µl of hybridization solution is placed onto a 12-mm round coverslip, which is then inverted onto the area where the polar bodies are located. The coverslip should be sealed with rubber cement, and additional coverage with a stretch of

parafilm facilitates removal of the coverslip after hybridization. The slide is then placed into a hybridization oven, where co-denaturation of the probe and the DNA of the polar body occurs at a temperature suitable for the probes used (usually around 68–73oC for up to 10 min). Hybridization is usually performed at 37oC. Centromeric probes require only 30–40 min of hybridization, whereas locus-specific probes require longer times. Commercially available multiprobe kits are usually hybridized for 4–8 hours, followed by two rapid washing steps (73oC, 0.7 × SSC and 0.3% NP-40 for 7 min followed by 2 × SSC, 0.1% NP-40

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Fig 27.4 Transfer of isolated polar bodies onto a slide. The transfer of isolated polar bodies from the dish (seen in the background) into the droplet on the slide must be performed on the microscope stage. The set-up shown here allows the dish used for biopsy to be slid backwards. Therefore, the aspiration capillary only needs to be lowered into the droplet for release of the polar body.

Fig 27.5 Identification of the polar body on the slide. This photograph was taken with a 10× phase-contrast objective, and the diamond circle surrounding the polar body can be partially seen. The polar body appears gray-shaded and is marked by an arrow.

for 1 min), which should be carried out exactly as described in the kit’s manual. Following washing, a coverslip and antifade-mounting media must be applied to the slide, which should then be stored immediately in the dark until FISH analysis.

FISH analysis and interpretation of results Prior to analysis of the FISH results, the polar bodies on the glass slide must be located under the microscope.

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Fig 27.6 First polar body with correct signals for chromosomes 13, 16, 18, 21, and 22. This polar body shows two signals for each of the chromosomes under investigation. The picture is a composite overlay, where initially each chromosome probe was assembled as black and white using the appropriate filter set, and prior to overlay, signals were colored using a software program (13, red; 16+18, blue; 21, green; 22, yellow). The signals for 16 and 18 are taken by a dual band pass filter set and therefore cannot be labeled with different colors. This mode of presentation also applies for the following figures in color.

This is rather easy if a circle is made around the area of polar body deposition on top of the slide. The use of a 10× phase-contrast objective usually allows for identification of the diamond circle and even the polar body can be identified in most cases (Fig 27.5). For FISH analysis, a 100× oil immersion objective with good transmission properties for the necessary wavelengths must be used. In the fluorescence viewing mode, the right focal plane can be easily adjusted by focusing the diamond line, followed by searching the polar body within the encircled area. Once the polar body is located, it is recommended to view the different chromosome signals in the order proposed by the manufacturer of the kit, as certain fluorophores will fade more quickly than others. As explained earlier in the section on aneuploidy screening, each chromosome should give two signals in the first polar body and one signal in the second polar body. An example of a first polar body with a correct number of signals for chromosomes 13, 16, 18, 21, and 22 is shown (Fig 27.6), where chromosomes 16 and 18 are detected by centromere-specific probes and chromosomes 13, 21, 22 by locus-specific probes. It can be seen that the signals for chromosomes 16 and 18 can be clearly distinguished, despite the centromeric location of the probe. This is due to an early onset of chromatid separation within the first polar body, which seems to start soon after oocyte retrieval, probably due to in vitro culture.16 In contrast, locus-specific probes will usually give good signals that are easy to evaluate, as the loci are located on the arms of the chromosome.

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Fig 27.7 Second polar body with aneuploidy. This second polar body shows one signal for chromosome X (yellow), one signal for chromosome 21 (green), and two signals for chromosome 13 (red). The close vicinity of the two signals for 13 indicates that the chromatids were not separated during meiosis II and, because both signals are located in the second polar body, the oocyte is missing on 13 and the resulting embryo will have a monosomy 13.

Fig 27.8 Split first polar body. This is an example of the rare event of a meiotic division of the first polar body, where each of the two cells contains one signal for chromosomes 13 (red), 21 (green), and X (yellow). The symmetry of the arrangement is a proof for a division and excludes fragmentation.

Chromosomal abnormalities can also originate in the second polar body, and the example shows one signal for chromosomes 21 (green) and X (yellow) and two signals for chromosome 13 (red) (Fig 27.7). As there is one additional signal 13 in the second polar body, the oocyte is missing one chromatid 13, and

Fig 27.9 Chromosome segregation and trisomy 21. This polar body displays several common features that can be observed during evaluation of fluorescence signals. First, only signals for chromosome 18 are located side by side (blue dotted signals on the left), whereas all other chromosomes underwent predivision of chromatids. Only one signal is present for chromosome 21 (green), which indicates that one additional chromatid 21 is present in the oocyte, and the embryo can develop a trisomy 21 (chromosome 13, red; 16+18, blue; 21, green; 22, yellow – small dots; X, yellow – large dots).

Fig 27.10 Onset of chromatin degeneration. First polar bodies are prone to degeneration, and FISH signals are no longer focused but appear speckled. The example shows three speckled signals for chromosome 13, where due to predivision one additional signal is found in the first polar body, which is missing in the oocyte.

will develop into an embryo with monosomy 13. While performing simultaneous removal of the first and second polar body, special attention must be taken to the recognition of a phenomenon which is

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(c)

Fig 27.11 FISH analysis of a fragmented polar body. This example shows a highly fragmented polar body, where the fragments were located on the slide within a large area and consequently not all fragments could be viewed within one field. Following predivision, both chromatids 18 (blue) are present in separated fragments (a). Two neighboring signals can be found for chromosomes 21 (green), 22 (yellow), and X (bright yellow, very close), but only one signal for 16 (blue ) (b). Chromosome 13 (red) again is located in two different fragments (c).

usually not considered: namely, the division of the first polar body into two cells, where each cell contains one set of chromatids (Fig 27.8). Looking at chromosomes 13 (red), 21 (green), and X (yellow), it is interesting to note that this polar body still displays a symmetric arrangement of chromosomes after fixation and FISH. However, it shows that a division of the first polar body can occur and that one should take care not to consider one of the two resulting cells as second polar body. As mentioned earlier, most signals in the first polar body are split signals due to premature chromatin segregation, and, as demonstrated by several studies, unbalanced predivision of chromatids is the most common origin of aneuploidy.14 The example on display (Fig 27.9) depicts a first polar body where only the two signals for the two chromatids 18 are located side by side, whereas chromosomes 13, 16, 22, and X show a balanced predivision and chromosome 21 an

unbalanced predivision. The corresponding oocyte contains one additional chromatid 21 and therefore can develop a trisomy 21. A frequent problem in analysis of the first polar body is the degeneration of chromatin, which may lead to speckled signals. This is most frequently observed for chromosomes 13 and 22. A diagnosis of aneuploidies or malsegregation is still possible, provided that the speckled regions are well separated from each other due to predivision. An example is given in Fig 27.10, where three individual regions of speckled signals for chromosome 13 can be seen. The corresponding oocyte is missing one chromatid 13 and is at risk for monosomy 13. Another problem is the high degree of fragmentation observed in first human polar bodies (Fig 27.11). Obviously, all fragments can contain chromatin material and therefore it is obligatory to remove all fragments (compare also Fig 27.3d). In such a case, we

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watch the process of air-drying on the slide very carefully and we even make a drawing of the location of the fragments within the encircled area. If only one fragment is overlooked during FISH analysis, one may easily risk a misdiagnosis if one of the chromosomes under investigation is located in the missing fragment. Finally, polar bodies that are very advanced in the process of degeneration possess weak membranes which are likely to rupture during the fixation process. However, the chromatin will usually still affix to the glass slide after drying of the transfer droplet, although the signal will be spread and look like an elongated strand or bundle of DNA (Fig 27.12). Therefore, FISH analysis is still possible, but the signals are scattered all over the encircled area, making interpretation of the results rather difficult.

Our results for aneuploidy screening in women of advanced maternal age Based on our fundamental studies regarding the use of the 1.48-µm diode laser technique for polar body biopsy,37–38 we were the first center in Germany to receive the positive support of an institutional ethics committee to perform polar body biopsy and aneuploidy screening in women of advanced maternal age and in patients with implantation failure.

We use a commercially available kit with probes for chromosomes 13, 16, 18, 21, and 22, which is occasionally modified by addition of a double-color probe for the centromer region of chromosome X, thus enabling the simultaneous analysis of six chromosomes in one single hybridization round.45 Our results are based on 174 treatment cycles, and they clearly show that laser-assisted polar body biopsy is a fast, efficient and atraumatic technique. On average, we need 3–4 min for one individual polar body to accomplish biopsy, pretreatment in a salt solution, and subsequent transfer to a glass slide. The efficacy of the technique is supported by the high rate of successful biopsies. Only 20 polar bodies were lost either during the transfer process or due to rupture after transfer to the slide, and we have obtained FISH results for 95.8% of all oocytes which were biopsied to date (Table 27.1). In our patient cohort, 50% of the successfully diagnosed oocytes (n = 1193) were chromosomally normal (Fig 27.13). For 27% of the oocytes we diagnosed a trisomy, mostly due to a missing chromatid in the polar bodies and trisomy 21 was by far the most common trisomy. We found monosomies in 8% of oocytes and 15% of the oocytes showed complex numerical chromosomal abnormalities involving more than one chromosome. The incidence of aneuploidy was strongly correlated to maternal age and

Table 27.1

Efficacy of laser-assisted polar body biopsy

Treatment cycles with polar body biopsy No of oocytes with biopsy No of oocytes degenerated due to biopsy No of polar bodies lost during biopsy/transfer No of polar bodies without hybridization signals No of oocytes with FISH results

174 1245 5 (0.4%) 20 (1.6%) 27 (2.2%) 1193 (95.8%)

15 50

8

27

Fig 27.12 FISH signals after rupture of the polar body. If a polar body ruptures after transfer onto the slide, one can still find the DNA if the whole area was encircled and if a careful examination in the fluorescence mode is carried out. An example is shown for two areas of DNA which present a positive FISH reaction for probe 21. The two areas are located close to each other and are equal in total intensity, and therefore can be viewed as two signals for 21. However, in such a condition the remaining part of the slide must be examined for additional signals for 21 in order to enable a final diagnosis.

Euploid

Combination

Monosomy

Trisomy

Fig 27.13 Frequency of aneuploidy in 1193 oocytes (mean maternal age: 38.2 years).

ranged from 20 to 30% in women around 30 years old to 70% in women at 45 years old.5 So far, our studies show, that an adequate number of oocytes are required for ICSI and polar body

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biopsy (e.g. 6–8) in order to ensure the transfer of two embryos with a normal chromosome constitution following examination of 5–6 chromosomes. 5 The pregnancy rate also depends on the number of euploid, fertilized oocytes. If it is possible to perform a selection based on pronuclear scoring, 46 in addition to aneuploidy screening, the pregnancy rate can rise up to 50% in selected cases. Unfortunately, this option is not always available, owing to the frequently reduced number of oocytes in patients of advanced maternal age. To date we have performed 174 treatment cycles of polar body biopsy with aneuploidy screening and an embryo transfer was possible in 150 cycles (86.2%). On average, we transferred 1.7 embryos per transfer. This figure seems to be low, but because of preselection by aneuploidy screening, we restricted ourselves to

Table 27.2 Results of polar body (PB) biopsy for aneuploidy screening Mean maternal age Treatment cycles Cycles with embryo transfer No of embryos transferred No of embryos/transfer Biochemical pregnancies

38.2 years 174 150/174 (86.2%) 260 1.73 42/150 (28.0%)

Clinical pregnancies Implantation rate Abortion rate

35/150 (23.3%) 42/260 (16.2%) 6/35 (17.1%)*

*One abortion was induced after prenatal diagnosis of trisomy 16 in a case where only the first polar body was analyzed; five abortions occurred spontaneously: one showed trisomy 4, one was genetically normal, and in another three the material submitted was not sufficient for human genetic investigation

Table 27.3

Reference

transferring a maximum of two embryos in order to avoid a high multiple pregnancy rate. Our clinical pregnancy rate in a patient cohort with a mean maternal age of 38.2 years old is 23.3% at present, and the implantation rate is 16.2% per transferred embryo (Table 27.2). A comparison of our data with those reported in the international literature (Table 27.3) allows the conclusion that laser-assisted polar body biopsy is at least as efficient as the method of zona drilling proposed by Cieslak et al.34 Despite an increase in experience with growing numbers of cycles, the pregnancy rate was slightly decreasing (compare references 5 and 47). We think that this can be explained by the fact that at the beginning of the era of polar body diagnosis in Germany, only our center offered this technique and made the public aware about its possibilities and the potential benefits. Therefore, patients with a longstandinghistory of unsuccessful infertility treatment all over Germany approached our center and asked for polar body diagnosis. However, the underlying infertility problem in most of these patients may not be due to aneuploidy but rather to implantation failure or other problems, because if so, one would have expected a higher rate of previous nidations with subsequent miscarriage due to chromosomal abnormalities. Therefore it is very likely that increasing numbers of ‘bad prognosis’ patients with multiple infertility factors contributed to the decrease of the success rate. It is interesting for us to note that a similar development took place in the very early phase of polar body diagnosis offered by the group of Yuri Verlinsky (see references given in Table 27.3), at a time when Chicago was one of the few centers in the USA offering aneuploidy screening for assisted reproduction.

Success rates of polar body (PB) biopsy and aneuploidy screening in the international literature

Biopsy material

No. of chromosomes

Biopsy using zona drilling by acidic Tyrode’s solution Verlinsky et al54 PB I/I+II 3 Dyban et al55 PB I/I+II 3 Verlinsky et al56 PB I/I+II 3 Verlinsky et al57 PB I/I+II 3 Verlinsky et al58 PB I/I+II 3 Verlinsky et al59 PB I/I+II 3/5 Kuliev et al2 PB I/I+II 3/5 Biopsy using a 1.48-µm diode laser Montag et al5 PB I Montag et al‡ PB I van der Ven et al47 PB I/I+II Abortion rate: *23.7%; †14.3%. ‡ unpublished data.

5 5/6 5/6

367

No. of cycles

No. embryos per transfer

Clinical pregnancy rate (%)

45 161 235 598 659 821 1297

3.1 2.6 2.5 2.6 2.1 2.5 2.35

21.7 14.8 16.0 21.4 22.3 22.2 21.9*

50 110 170

1.9 1.8 1.7

30.9 26.6 23.3†

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Conclusions The pioneering work in polar body diagnosis was performed by Yuri Verlinsky and Santiago Munné and efficient biopsy techniques were elaborated by Cieslak et al34 and our group.37,38 This has led to a variety of (genetic and chromosomal) diagnostic applications following polar body biopsy that have been described in this article. To date, most cases of polar body diagnosis are performed for aneuploidy screening. Although this approach is feasible, one should be reminded that there are only insufficient scientific data available to prove the benefit of polar body diagnosis for aneuploidy screening in terms of higher pregnancy and birth rates. To our knowledge there is no prospective randomized trial to support the sometimes enthusiastic appraisal of this technique. This underlines the fact that such studies are still needed and that the procedure itself must be continuously evaluated. In particular, the phenomenon of the so-called FISH dropout, which was found to be a major problem in FISH analysis of oocytes,48 has never been investigated in polar body FISH. This issue still needs to be addressed, because most results, including ours, have reported a very high rate of missing-chromatid signals during polar body analysis. This leads to the diagnosis of a disomy for the relevant chromosome in the oocyte, and hence trisomy in the embryo. If FISH dropout does occur in polar body diagnosis, then the rate of disomy in the oocyte is overestimated and consequently a large number of oocytes would be discarded due to a misdiagnosis. Another unresolved problem is the freezing of oocytes and embryos after polar body as well as embryo biopsy. As a result of the biopsy procedure, a permanent opening will remain in the zona pellucida until the time of hatching. In many countries, freezing takes place at the pronuclear stage (and is mandatory at that stage for countries with legal restrictions for embryo selection) or at later stages of development. Independent of the stage of freezing, survival rates of frozen–thawed oocytes or embryos after polar body or blastomere biopsy are rather low compared to cells with intact zona.49–51 This is an important issue if the success rate of such methods is expressed by the cumulative pregnancy rate. Patients getting pregnant without preconception or preimplantation genetic diagnosis may have a higher risk of miscarriage or induced abortion due to a chromosomal abnormality. However, it is tempting to speculate that the cumulative pregnancy rate and take-home healthy-baby rate in this group would be at least equal to those of patients with polar body biopsy and aneuploidy screening diagnosis, because these patients will not benefit from the additional chance of cryotransfer cycles. Consequently, the use of polar body diagnosis and aneuploidy screening should be primarily offered to patients at a high risk for chromosomal aberrations and patients who are more likely to benefit from this therapy. 52 The relevant data should be published

continuously in order to enable an evaluation of this technique in the view of evidence-based medicine. Finally, we may conclude, that the use of a noncontact laser for polar body biopsy is a safe and efficient approach.53

References 1. Verlinsky Y, Ginsberg N, Lifchez A, et al. Analysis of the first polar body: preconception genetic diagnosis. Hum Reprod 1990; 5: 826–9. 2. Kuliev A, Cieslak J, Ilkevitch Y, Verlinsky Y. Chromosomal abnormalities in a series of 6733 human oocytes in preimplantation diagnosis for agerelated aneuploidies. Reprod Biomed Online 2002; 6: 54–9. 3. Handyside AH, Kontogianni EH, Hardy K, Winston RML. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990; 244: 768–70. 4. ESHRE PGD Consortium Steering Committee. ESHRE Preimplantation Genetic Diagnosis Consortium: data collection III (May 2001). Hum Reprod 2002; 17: 233–46. 5. Montag M, van der Ven K, van der Ven H. Erste klinische Erfahrungen mit der Polkörperdiagnostik in Deutschland. J Fertil Reprod 2002; 4: 7–12. 6. Benagiano G, Gianaroli L. The new Italian IVF legislation. Reprod Biomed Online 2004; 9: 117–25. 7. Munné S, Sepulveda S, Balmaceda J, et al. Selection of the most common chromosome abnormalities in oocytes prior to ICSI. Prenat Diagn 2000; 20: 582–6. 8. Munné S, Sandalinas M, Escudero T, et al. Outcome of preimplantation genetic diagnosis of translocations. Fertil Steril 2000; 73: 1209–18. 9. Verlinsky Y, Cieslak J, Ivakhenko V, et al. Chromosomal abnormalities in the first and second polar body. Mol Cell Endocrinol 2001; 183S: 47–9. 10. Hassold T, Chiu D. Maternal age-specific rates of numerical chromosome abnormalities with special reference to trisomy. Hum Genet 1985; 70: 11–17. 11. Hassold T, Jacobs PA, Leppert M, Sheldon M. Cytogenetic and molecular studies of trisomy 13. J Med Genet 1987; 24: 725–32. 12. Fisher JM, Harvey JF, Morton NE, Jacobs PA. Trisomy 18: studies of the parent and cell division of origin and effect of aberrant recombination on nondisjunction. Am J Hum Genet 1995; 56: 669–75. 13. Zaragoza MV, Jacobs PA, James RS, et al. Nondisjunction of human acrocentric chromosomes: studies of 432 trisomic fetuses and live-borns. Hum Genet 1994; 94: 411–17. 14. Angell RR. Predivision in human oocytes at meiosis 1: a mechanism for trisomy formation in man. Hum Genet 1991; 86: 383–7. 15. Angell RR. Possible pitfalls in preimplantation diagnosis of chromosomal abnormalities based on polar body biopsy. Hum Reprod 1994; 9: 181–2. 16. Munné S, Dailey T, Sultan KM, et al. The use of first polar bodies for preimplantation diagnosis of aneuploidy. Mol Hum Reprod 1995; 10: 1014–20. 17. Stern C, Pertile M, Norris H, et al. Chromosome translocations in couples with in-vitro fertilization implantation failure. Hum Reprod 1999; 14: 2097–101.

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Polar body biopsy 18. Van der Ven K, Peschka B, Montag M, et al. Increased frequency of constitutional chromosomal aberrations in female partners of couples undergoing intracytoplasmic sperm injection (ICSI). Hum Reprod 1998; 13: 48–54. 19. Peschka B, Leygraaf J, van der Ven K, et al. Type and frequency of chromosome aberrations in 551 couples undergoing intracytoplasmic sperm injection. Hum Reprod 1999; 14: 2257–63. 20. Munné S, Fung J, Cassel MJ, et al. Preimplantation genetic analysis of translocations: case-specific probes for interphase cell analysis. Hum Genet 1998; 102: 663–74. 21. Fung J, Munné S, Duell T, et al. Rapid cloning of translocation breakpoints: from blood to YAC in 50 days. J Biochem Mol Biol Biophys 1998; 1: 181–92. 22. Verlinsky Y, Evsikov S. Karyotyping of human oocytes by chromosomal analysis of the second polar body. Mol Hum Reprod 1999; 5: 89–95. 23. Wells D, Escudero T, Levy B, et al. First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic diagnosis of aneuploidy. Fertil Steril 2002; 78: 543–9. 24. Gitlin SA, Lanzendorf SE, Gibbons WE. Polymerase chain reaction amplification specifity: incidence of allele-dropout using different DNA preparation methods for heterozygous single cells. J Assist Reprod Genet 1996; 13: 107–11. 25. Sermon K, Lissens W, Joris H, et al. Clinical application of preimplantation diagnosis for myotonic dystrophy. Prenat Diagn 1997; 17: 925–32. 26. Ray PF, Ao A, Taylor DM, et al. Assessment of single blastomere analysis for preimplantation diagnosis of the delta F508 deletion causing cystic fibrosis in clinical practice. Prenat Diagn 1994; 18: 1402–12. 27. Rechitsky S, Strom C, Verlinsky O, et al. Allele dropout in polar bodies and blastomeres. J Assist Reprod Genet 1998; 15: 253–7. 28. Hussey ND, Davis T, Hall JR, et al. Preimplantation genetic diagnosis for β-thalassaemia using sequencing of single cell PCR products to detect mutations and polymorphic loci. Mol Hum Reprod 2002; 8: 1136–43. 29. Fiorentino F, Magli MC, Podini D, et al. The minisequencing method: an alternative strategy for preimplantation genetic diagnosis of single gene disorders. Reprod Biomed Online 2003; 9: 399–410. 30. Verlinsky Y. Polar body-based preimplantation diagnosis for X-linked disorders. Reprod Biomed Online 2001; 4: 38–42. 31. Wu R, Cuppens H, Buyse I, et al. Co-amplification of the cystic fibrosis delta F508 mutation with the HLA DQA1 sequence in single cell PCR: implications for improved assessement of polar bodies and blastomeres in preimplantation diagnosis. Prenat Diagn 1993; 13: 1111–22. 32. Gordon JW, Talansky BE. Assisted fertilization by zona drilling: a mouse model for correction of oligospermia. J Exp Zool 1987; 239: 347–81. 33. Montag M, Rink K, Descloux L, et al. The use of a 1.48 µm laser-system in assisted reproduction: laser-drilling of the zona pellucida and laser-assisted immobilization of spermatozoa. Assist Reprod Rev 1999; 9: 205–13. 34. Cieslak J, Ivakhenko V, Wolf G et al. Three-dimensional partial zona dissection for preimplantation

35.

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genetic diagnosis and assisted hatching. Fertil Steril 1999; 71: 308–13. Rink K, Delacrétaz G, Salathé RP, et al. 1.48 µm diode laser microdissection of the zona pellucida of mouse zygotes. Proc SPIE 1994; 213A: 412–22. Rink K, Delacrétaz G, Salathé R, et al. Non-contact micro-drilling of mouse zona pellucida with an objective-delivered 1.48 µm diode laser. Lasers Surg Med 1996; 18: 52–62. Montag M, van der Ven K, Delacrétaz G, et al. Efficient preimplantation genetic diagnosis using laser assisted microdissection of the zona pellucida for polar body biopsy followed by primed in situ labelling (PRINS). J Assist Reprod Genet 1997; 14: 455–6. Montag M, van der Ven K, Delacrétaz G, et al. Laser assisted microdissection of zona pellucida facilitates polar body biopsy. Fertil Steril 1998; 69: 539–42. Montag M, van der Ven H. Laser-assisted hatching in assisted reproduction. Croat Med J 1999; 40: 398–403. Montag M, Koll B, Holmes P, van der Ven H. Significance of the number of embryonic cells and the state of the zona pellucida for hatching of mouse blastocysts in vitro versus in vivo. Biol Reprod 2000; 62: 1738–44. Licciardi F, Gonzalez A, Tang YX, et al. Laser ablation of the mouse zona pellucida for blastomere biopsy. J Assist Reprod Genet 1995; 12: 462–6. Boada M, Carrera M, De La Iglesia C, et al. Successful use of a laser for human embryo biopsy in preimplantation genetic diagnosis: report of two cases. J Assist Reprod Genet 1997; 15: 301–5. Veiga A, Sandalinas M, Benkhalifa M, et al. Laser blastocyst biopsy for preimplantation diagnosis in the human. Zygote 1997; 5: 351–4. Joris H, de Vos A, Janssens R, et al. Comparison of the results of human embryo biopsy and outcome of preimplantation genetic diagnosis (PGD) after zona drilling using acid Tyrode of a laser. Hum Reprod 2000; 15S: 53–4. Montag M, Limbach N, Sabarstinski M, et al. Polar body biopsy and aneuploidy testing by simultaneous detection of six chromosomes. Prenatal Diag 2005; 25: 867–71. Montag M, van der Ven H, on behalf of the German Pronuclear Morphology Study Group. Evaluation of pronuclear morphology as the only selection criterion for further embryo culture and transfer: results of a prospective multicenter study. Hum Reprod 2001; 16: 2384–9. van der Ven H, van der Ven K, Montag M. Clinical experience with laser assisted polar body biopsy. Hum Reprod 2003; 18S: 13–14. Eckel H, Stumm M, Wieacker P, Kleinstein J. Multilocus FISH is a highly reliable method for non-disjunction studies in human oocytes. Hum Reprod 2002; 17S: 190–1. Carson RS, Burgess CM, Glatstein IZ, et al. Preimplantation genetic diagnosis and cryopreservation of embryos. Fertil Steril 1997; 11S: 198. Magli MC, Gianaroli L, Fortini D, et al. Impact of blastomere biopsy and cryopreservation techniques on human embryo viability. Hum Reprod 1999; 4: 770–3.

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51. Lee M, Munné S. Pregnancy after polar body biopsy and freezing and thawing of human embryos. Fertil Steril 2000; 73: 645–7. 52. Munné S, Sandalinas M, Escudero T, et al. Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reprod Biomed Online 2003; 7: 91–7. 53. Montag M, van der Ven K, Dorn C, van der Ven H. Outcome of laser-assisted polar body biopsy. Reprod Biomed Online 2004; 9: 425–9. 54. Verlinsky Y, Cieslak J, Freidin M, et al. Pregnancies following pre-conception diagnosis of common aneuploidies by FISH. Hum Reprod 1995; 10: 1923–7. 55. Dyban A, Freidine M, Severova E, et al. Detection of aneuploidy in human oocytes and corresponding first polar body by fluorescent in situ hybridisation. J Assist Reprod Genet 1996; 13: 73–8.

56. Verlinsky Y, Cieslak J, Ivakhenko V, et al. Birth of healthy children after preimplantation diagnosis of common aneuploidies by polar body fluorescent in situ hybridization analysis. Fertil Steril 1996; 66: 126–9. 57. Verlinsky Y, Cieslak J, Ivakhenko V, et al. Prepregnancy genetic testing for age-related aneuploidies by polar body analysis. Genetic Testing 1997/98; 4: 231–5. 58. Verlinsky Y, Cieslak J, Ivakhenko V, et al. Prevention of age-related aneuploidies by polar body testing. J Assist Reprod Genet 1999; 16: 165–9. 59. Verlinsky Y, Cieslak J, Ivakhenko V, et al. Chromosomal abnormalities in the first and second polar body. Mol Cell Endocrinol 2001; 183S: 47–9.

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28 Clinical application of polar body biopsy Yury Verlinsky, Anver Kuliev

Introduction Introduced 18 years ago, polar body (PB) biopsy has become one of the established approaches for preimplantation genetic diagnosis (PGD).1 The idea of performing PB PGD is based on the fact that polar bodies are the by-products of female meiosis, which allow prediction of the resulting genotype of the maternal contribution to the embryo. Neither the first PB (PB1), which is extruded as a result of the first meiotic division (Fig 28.1a), nor the second PB (PB2), extruded following the second meiotic division (Fig 28.1b), have any known biologic value for pre- and postimplantation development of the embryo. Initially, only the PB1 was tested (Fig 28.1a), based on the fact that in the absence of crossing-over, PB1 will be homozygous for the allele not contained in the oocyte and PB2 (Fig 28.2, left and upper panels). However, the PB1 approach was not applicable for predicting the eventual genotype of the oocyte, if crossing-over occurred, because the primary oocyte in this case will be heterozygous for the abnormal gene (Fig 28.2, middle). As the frequency of crossing-over varies with the distance between the locus and the centromere, reaching as much as almost 50% for telomeric genes, the PB1 approach appeared to be of limited value, unless the oocytes could be tested further on. So, analysis of the PB2 has been introduced (Fig 28.1b) to detect hemizygous normal oocytes resulting after the second meiotic division (Fig 28.2, C1 and C2).2 As described below, the technique currently involves two-step oocyte analysis, which requires sequential testing of PB1 and PB2. It is well known that more than half of patients undergoing in vitro fertilization (IVF) are of advanced reproductive age, i.e. ≥35 years old, being at high risk for producing children with numerical chromosomal abnormalities. Because the majority of numerical chromosomal abnormalities originate in the maternal first meiotic division, study of the outcomes of meiosis I may allow preselection of aneuploidy-free oocytes prior to IVF, avoiding implantation and pregnancy failures caused by chromosomal abnormalities in aging women with infertility problems. Based on these considerations, PB1 testing for chromosomal abnormalities was introduced 12 years ago, demonstrating the reliability of PB1 fluorescence in situ

hybridization (FISH) analysis for prediction of the chromosomal content of oocytes.3,4 The study of metaphase II (MII) oocyte in parallel with their corresponding PB1 showed that each chromosome in the MII oocyte and PB1 was represented by paired FISH signals; as expected, the missing or extra signals in PB1 were opposite to the extra or missing signals in the MII oocyte, respectively. This was also confirmed by other authors,5 and has further been implemented for avoiding the transfer of aneuploid embryos in hundreds of cycles for IVF patients of advanced maternal age.6–8 The data have, however, demonstrated that PB1 analysis fails to detect all numerical chromosome abnormalities in oocytes, because some of them might originate also from the second meiotic division and, therefore, cannot be detected without PB2 testing. Therefore, the current practice includes testing of both PB1 and PB2, to avoid chromosomal abnormalities originating from both the first and the second meiotic divisions (Fig 28.1c). Although more data have to be collected to exclude completely short-term and/or long-term side effects of the procedure, the presently available data provide no evidence for any detrimental effect of the PB biopsy. A special study carried out to evaluate the outcome of the biopsy procedure, based on the analysis of the resulting embryo development, including fertilization rate following PB sampling, cleavage rate, and blastocyst development in hundreds of cases tested, using ICSI (intracytoplasmic sperm injection) embryos as control, showed that the procedures had no detrimental effect on the embryo development or clinical outcome.9 This is also in agreement with the available outcomes of thousands of PGD cycles for single-gene and chromosomal disorders, which showed a comparable prevalence of congenital abnormalities after PGD to that in the general population.

Polar body testing PB1 and PB2 are removed following stimulation and oocyte retrieval using a standard IVF protocol, as described previously.10 Following extrusion of PB1, the zona pellucida is opened mechanically using a microneedle, and PB1 aspirated into a blunt micropipette (Fig 28.1a). The oocyte is then inseminated

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(a)

(b)

(c)

Fig 28.1 Micromanipulations for polar body biopsy. (a) First polar body removal: performed after maturation of oocytes and used in preimplantation genetic diagnosis (PGD) for single-gene disorders. (b) Second polar body removal: performed after intracytoplasmic sperm injection (ICSI) and used in PGD for singlegene disorders. (c) Simultaneous first and second polar body removal: used in PGD for chromosomal disorders.

with motile sperm, or using ICSI, and examined for the presence of pronuclei and the extrusion of PB2, which is removed in the same manner as PB1 (Fig 28.1b). To avoid an additional invasive procedure, both PB1 and PB2 are removed simultaneously for

FISH analysis (Fig 28.1c), and are fixed and analyzed on the same slide. The biopsied oocyte is then returned to culture, checked for cleavage, and transferred, depending on the genotype of the corresponding PB1 and PB2. As mentioned above, for PGD of single-gene disorders, sequential genetic analysis of the PB1 and PB2 is used (Fig 28.1a and b).2,10 Detection of both mutant and normal alleles in the heterozygous PB1, together with the mutant allele in the corresponding PB2, leaves no doubt that the resulting maternal contribution to the embryo is normal, even without testing for the linked markers (Fig 28.2, C2). However, it is ideal to test simultaneously for at least one linked marker to confirm the diagnosis. Alternatively, a mutation-free oocyte may also be predicted when the corresponding PB1 is homozygous mutant, in which scenario the corresponding PB2 should be hemizygous normal, similar to the resulting maternal pronucleus (Fig 28.2, bottom left). However, the genotype of the resulting maternal contribution may be opposite, i.e. mutant, if the corresponding PB1 is in fact heterozygous, but erroneously diagnosed as homozygous normal because of preferential amplification or allele dropout (ADO) of the normal allele. In the above scenario, the extrusion of the normal allele with PB2 would lead to the mutant allele being left in the resulting oocyte (Fig 28.2, C1). Therefore, embryos resulting from oocytes with homozygous mutant PB1 cannot be acceptable for transfer, unless the heterozygous status of PB1 is excluded by the use of closely linked markers. To avoid misdiagnosis completely, sequential PB1 and PB2 analysis may be required combined with multiplex polymerase chain reaction (PCR) to exclude the possibility of an undetected ADO in a heterozygous PB1.10,11 An example of sequential PB1 and PB2 analysis that results in the preselection and transfer of the embryos originating from the mutation-free oocytes is presented in Fig 28.3. For PGD of numerical chromosomal disorders, FISH analysis of PB1 and PB2 is performed, using commercial probes specific for chromosomes 13, 16, 18, 21, and 22 (Abbot, Downers Grove, IL, USA).10 PB1 testing was one of the first approaches used for PGD of translocations, based on the fact that the PB1 never forms an interphase nucleus and consists of metaphase chromosomes.12 It is known that PB1 chromosomes are recognizable when isolated 2–3 hours after in vitro culture, with degeneration beginning 6–7 hours after extrusion.13 Therefore, whole chromosome painting or chromosome segment-specific probes can be applied for testing of maternally derived chromosomal translocations in PB1.12 Although the method resulted in a significant reduction of spontaneous abortions in patients carrying translocations, yielding unaffected pregnancies and births of healthy children, it appeared to be sensitive to malsegregation and/or recombination between chromatids, so requiring a further follow-up analysis of PB2, in order to predict accurately the meiotic outcome following the second

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MI

N

N

T

N N

NN

MII T

T

T

T

T

373

Affected Cross-over MII

T T

MII N

N

NT NT

N T T

N

N

T C1

Affected C2 NT

TT N

N

N

T

Unaffected

Fig 28.2

Unaffected

Possible distribution of thalassemia mutations following I and II meiotic divisions. V95 M

(a) CEN

DXS453

DXS8052 0.31 Mb

DXS8030

CX32 081 Mb

0.17 Mb

DXS441

0.27 Mb

TEL

4.11 Mb

Xq13.1

(b) Markers order DXS453 DXS8052 CX32 DXS8030 DXS559 DXS441 (c)

DXS559

116 127 N 163 137 187

N

N/ V95M

Y

X

113 105 129 127 N V95M 167 163 139 135 194 185

PGD Oocyte #

PREDICTED OOCYTE GEONOTYPE

PB1 105 127 V95M 163 135 185

3

1

PB2 113 129 N 167 139 194

NORMAL*

PB1 105 127 V95M 163 135 185

PB2 113 129 N 167 139 194

NORMAL* ET

6

4

PB1 PB2 113/105 105 129/127 127 N/V95M V95M 167/163 163 139/135 135 194/185 185

NORMAL ET

PB1 113 129 N 167 139 194

PB2 105 127 V95M 163 135 185

AFFECTED*

10

PB1 105 127 V95M 163 135 194/185

12

PB2 113 129 N 167 139 194

NORMAL* RECOMBINANT

PB1 105 127 V95M 163 135 185

PB2 113 129 N 167 139 194

NORMAL*

Fig 28.3 Polar body based PGD for X-linked form of Charcot–Marie–Tooth disease (CMTX1) resulting in transfer of two mutation-free embryos. (a) Schematic representation of CX32 gene and linked markers, showing the complexity of the diagnosis. (b) Family pedigree, showing the carrier mother with V95M mutation and the results of haplotypes analysis established through sperm testing in the father and polar body analysis in the mother. (c) Results of multiplex polymerase chain reaction (PCR) analysis in PB1 and PB2 for the gene and 5 markers, which identified five mutation-free unaffected oocytes, including one recombinant, and one affected. Two of the unaffected oocytes (#3 and #4) were preselected for transfer.

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meiotic division.10,14 However, despite the progress in transforming PB2 into metaphase chromosomes via electrofusion of the PB2 nucleus with a foreign 1-cell human embryo, the proportion of metaphase plates did not exceed 64% even after enucleation of the recipient 1-cell-stage mouse embryo, restricting its usefulness in clinical practice.15 As can be seen from the presented data, the PB approach involving both PB1 and PB2 analysis may currently be applied for PGD of single-gene disorders, aneuploidies, and translocations.

Application of polar body biopsy for preimplantation genetic diagnosis of single-gene disorders The PB approach for PGD may be applied for any maternally derived Mendelian disease. Among the disadvantages of PB-based PGD is that it is not applicable for gender determination and testing for paternally derived mutations, for which purpose blastomere biopsy is used. However, as mentioned above, PB PGD avoids removing cells from embryos, which may affect embryo development, especially when two blastomeres are removed. In addition, PB DNA analysis appears to be more accurate and reliable than genetic analysis in single blastomeres, as it avoids the problem of mosaicism, which is highly prevalent in cleavage-stage embryos, and the high rate of ADO which is at least twice less frequent in PB1 than in blastomeres.16 Also, a considerable proportion of ADO is detectable by sequential analysis of PB1 and PB2, which avoids misdiagnosis due to ADO even in those cases when no informative polymorphic markers are available.10,17 Although the PB approach may be practical for PGD of any maternally derived mutations, it is particularly attractive in the following cases: 1. Specific diagnosis in X-linked disorders 2. PGD for couples with homozygous or doubleheterozygous affected male partners 3. PGD for couples with male and female partners carrying different mutations of the causative gene 4. PGD combined with human leukocyte antigen (HLA)-typing for detecting mutation-free maternalmatch oocytes 5. PGD combined with aneuploidy testing for detecting embryos free of maternal mutation to be tested further by FISH 6. PGD for couples with a religious objection to any micromanipulation and discard after fertilization (see below). For example, initially, PGD for X-linked disorders was performed by gender determination in blastomeres, which was the most straightforward application for PGD from the very beginning, using either PCR or the FISH technique, despite the obvious cost of discarding

50% of healthy male embryos.18,19 On the other hand, testing for X-linked genetic disorders may be entirely limited to oocytes, because the mutations involved are fully maternally derived. This was first applied for ornithine transcarbamylase deficiency (OTC),20 then extended to specific diagnosis of other X-linked disorders,21 and currently comprises experience of specific diagnosis in dozens of cycles performed for OTC, Fragile-X syndrome (XMR1), myotubular myotonic dystrophy, X-linked hydrocephalus, and Xlinked forms of Charcot–Marie–Tooth disease (CMTX1) (see Fig 28.3). A special value of PB biopsy may also be demonstrated by PGD for couples with homozygous or compound-heterozygous affected male partners. This has been performed for affected patients with thalassemia or phenylketonuria (PKU), resulting in unaffected pregnancies and births of healthy children.11,22 Although the risk for producing an affected child in such couples is 50%, the PB strategy allows preselection of mutation-free oocytes, with no further need for embryo testing. Testing is particularly complicated when the parents are carrying different mutations, requiring a complex PGD design to exclude preferential amplification of each of the three alleles tested, as described in PB PGD for PKU.22 As there are still many population groups that object to the presently used methods of embryo biopsy, which makes PGD nonapplicable for them in avoiding the birth of affected children and having unaffected children of their own, the PB approach is the only hope for them to perform genetic diagnosis before embryo formation: i.e. pre-embryonic genetic diagnosis. It was first introduced for sickle cell disease, to predict the potential embryo genotype prior to pronuclei fusion, following ICSI.23 Although pre-embryonic genetic diagnosis can be done by PB1 testing only,24–26 it is not sufficient for the accurate prediction of the embryo genotype without PB2 analysis, which facilitates the identification of the mutation-free zygotes deriving from the heterozygous metaphase II oocytes in PGD for single-gene disorders,11,13,26 and the exclusion from transfer of the embryos originating from the oocytes with meiosis II errors in PGD for chromosomal disorders. Accordingly, only euploid oocytes, or those predicted to have a normal maternal allele, could then be allowed to progress to pronuclei fusion (syngamy), embryo development, and transfer, either in the same, or in a subsequent menstrual cycle, avoiding the formation and possible discard of any affected embryo. This is, principally, a new type of genetic diagnosis, which moves the predictive genotyping to an even earlier stage than a traditional PGD, making the diagnosis more ethically acceptable, as it overcomes the negative reaction to the embryo micromanipulation and discard. It is of note, however, that the above-mentioned case of pre-embryonic genetic diagnosis was not performed on request but necessitated by an incidental hyperstimulation, with the embryo transfer to be postponed to the next cycle anyway.23 So the pronuclear-stage freezing of the preselected mutation-free oocytes was

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the only realistic option to resume their culture and transfer in a subsequent cycle, which resulted in an unaffected pregnancy and birth of a healthy child. Eventually, the actual pre-embryonic diagnosis was done for Sandhoff disease in a couple with a religious objection to discarding the embryo.27 In this case, 18 oocytes were obtained in a standard IVF protocol, from which PB1s were removed following maturation 4–5 hours after aspiration. PB2s were then removed 6 hours after ICSI, and both PB1 and PB2 were tested for maternal mutation, simultaneously with linked polymorphic markers. Based on the results of PB1 and PB2 analysis, the oocytes predicted to contain the maternal mutation were frozen at the pronuclear stage (prior to embryos formation), while the oocytes, predicted to be mutation free, were cultured and confirmed to be unaffected using embryo biopsy. Two of these embryos reaching blastocyst stage were transferred back to the patient, resulting in a singleton pregnancy and birth of a healthy mutation-free child, which opens the prospect of PGD in the communities where no embryo testing and disposal is presently acceptable (Fig 28.4).

Application of polar body biopsy for preimplantation genetic diagnosis of chromosomal disorders Our data show that approximately half of oocytes from patients of advanced reproductive age undergoing IVF are aneuploid, suggesting a strong indication for PGD in this group of patients.28,29 However, the prevalence of aneuploidies varies from 40% in patients <35 years old to >70% in those of ≥40 years old (Fig 28.5).28,29 With the current practice of transferring as few embryos per cycle as possible to avoid multiple pregnancies, the incidental transfer of aneuploid embryos could lead to IVF failure, because aneuploidies may be responsible for failure of implantation or spontaneous termination of established pregnancies. The standard embryo selection practice based on morphologic criteria, which correlate poorly with genotype, cannot exclude that at least one of two or three transferred embryos may actually be aneuploid with compromised developmental potential. As can be seen from Fig 28.6, more than one-third of oocytes have chromosomal errors after meiosis I, suggesting a potential for improving the chances of aging patients to become pregnant by PB1 testing alone prior to IVF. However, chromosomal errors originate from meiosis II as frequently as in meiosis I, making it mandatory that both PB1 and PB2 are tested (Fig 28.7).28,29 Contrary to the expected age-related increase of chromosomal nondisjunctions, our data revealed mainly chromatid errors (Fig 28.6). Only 2.5% of oocytes were with missing chromosomes, and only 0.2% with an extra chromosome. So, chromatid errors are in fact the major origin of aneuploidies, at least in IVF patients of advanced maternal age.3–5,28–30 Follow-up FISH analysis of the embryos originating from such oocytes confirmed

375

that the observed errors in premature division of chromatids of the same homolog at meiosis I do actually result in embryo aneuploidies.7 Whatever the actual mechanism of chromatid errors, they seem not to be attributed to an artifact of oocyte aging, and their transfer should be avoided. Polar body diagnosis for chromosomal disorders has to date been applied in more than 3000 clinical cycles for PGD of chromosomal disorders (Table 28.1). This involved FISH analysis for aneuploidy in 17 329 oocytes, from which 5039 were preselected and transferred in 2399 cycles, resulting in 674 clinical pregnancies and the birth of more than 600 healthy children. An average pregnancy rate of approximately 28% in the whole series seems quite acceptable, taking into consideration that the majority of patients were ≥35 years old, representing the major indication for PGD at the present time. In the group of patients with the above indication, the average age was approximately 39 years old, suggesting that aneuploidy testing may not only allow avoiding the birth of children with age-related aneuploidies but also improve the pregnancy rate in IVF patients of advanced reproductive age. Although intrinsic genetic parameters cannot explain completely the reason why as many as 90% of oocytes fail to produce a viable embryo, at least a proportion of these oocytes could be detected by PB analysis and avoided in fertilization and transfer. Together with other predictive factors, such as different clinical and epigenetic characteristics, preselection of euploid oocytes by PB1 and PB2 sampling may allow distinguishing in the future of those few oocytes with maximum potential to result in a clinical pregnancy and the birth of a healthy child. Although there was no control group of patients to evaluate the clinical significance of the above PB testing, the observed pregnancy rate is quite acceptable for aging patients undergoing routine IVF. This is in accordance with the reported data on positive pregnancy outcomes following aneuploidy testing at the cleavage stage in groups of aging or poor-prognosis IVF patients.31–33 As mentioned above, PB testing is also one of the methods of choice for PGD of translocations, which has actually been introduced through PB1 testing.12 A considerable impact of PGD on the reproductive outcome of carriers of balanced translocations was observed from the very beginning of the application of PGD for maternally derived translocations, which was obvious from the reduction of spontaneous abortion rates.12,25 As the carriers of balanced translocations have an extremely poor chance of having an unaffected pregnancy, PGD provides them with the realistic option of having an unaffected pregnancy from the onset. We have to date performed 58 PGD cycles for couples with maternally derived translocations, using PB1 and PB2 FISH analysis. Of 667 oocytes tested in these cycles, FISH results were available in 529 (79%), allowing preselection of only normal or balanced embryos for transfer in 34 (71.4%) cycles. This resulted in 11 clinical pregnancies, resulting in the

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Markers order: D5S1982 D5S1988 HEXB 16Kb Del HEXB 1200V D5S2003 D5S349 D5S1404

(a)

117 206 1207V 130 102 165

115 194 N 116 104 193

1.1

2.1 117 115 206 203 1207V1b Kb Del 130 132 102 98 165 185

115 203 16Kb Del 2.1 32 y.o. 132 98 185 2.2 PGD

117 201 N 128 98 185

(b) Oocyte # 1

117 201 N 128 98 185

N

(c)

3

4

5

6

7

8

9

10

11

13

14

15

16

17

115 203 Del 132 98 185

117 201 N 128 98 185

117 201 N 128 98 185

117 201 N 128 98 185

115 203 Del 132 98 185

117 201 N 128 98 185

115 203 Del 132 98 185

117 201 N 128 98 185

115 203 Del 132 98 185

115 203 Del 132 98 185

115 203 Del 132 98 185

115 203 Del 132 98 185

117 201 N 128 98 185

115 203 Del 132 98 185

117 201 N 128 98 185

N

N

M

N

M

N

M

M

M

N

M

N

M

N

Fr

Fr

Fr

Fr

Fr

M Fr

Fr

18

Fr

Embryo #

115 194 N 116 104 193

117 201 N 128 98 185

115 194 N 116 104 193

117 201 N 128 98 185

115 194 N 116 104 193

117 201 N 128 98 185

115 194 N 116 104 193

117 201 N 128 98 185

115 194 N 116 104 193

117 201 N 128 98 185

115 194 N 116 104 193

117 201 N 128 98 185

117 206 1207V 130 102 165

117 201 N 128 98 185

117 206 1207V 130 102 165

117 201 N 128 98 185

Normal

Normal

Normal

Normal

Normal

Normal

Carrier

Carrier

ET

Fr

Fr

Fr

Fr

ET

Fr

Fr

Fig 28.4 Pre-embryonic diagnosis for Sandhoff disease. (a) Family pedigree with mutation and haplotype analysis based on parental (1.1 and 1.2) and affected child’s (2.1) genomic DNA testing. The markers’ order is presented on the upper left for father and upper right for mother. Maternal and paternal mutations and the linked markers are shown in italic type, while normal alleles and their linked markers are shown in bold type. (b) Results of sequential first and second polar body analysis of 16 oocytes, showing 8 normal (bold) and 8 mutant oocytes (italic) which were frozen prior to syngamy. (c) Blastomere analysis of embryos, resulting from the mutation-free oocytes, which confirms the polar body diagnosis. Two of these embryos (# 1 and # 10), free of both maternal and paternal mutations, were transferred, resulting in birth of an unaffected child. The remaining 6 embryos, two of which were heterozygous, were frozen for a future use by the couple.

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377

Percent abnormal oocytes

80

70

119 733

327

196

60 928 50 890

681

821

783

873

693

40 294 30 34

35

36

37

38

39

40

41

42

43

44

Age (years) n = the number of oocytes analyzed

45

+

Fig 28.5 The linear relationship between increasing maternal age and the percentage of aneuploid oocytes. Aneuploid rates range from 39% at age 34 years old to as high as 72% for women ≥45 years old based on studies of up to 5 chromosomes (i.e. chromosomes 13, 16, 18, 21, and 22). A total of 1551 cycles were analyzed.

58.3%

20.1%

0.2%

100%

6.4%

2.5% Complex 12.5%

Fig 28.6

Meiosis I errors observed in first polar body (PB1) fluorescence in situ hybridization (FISH) analysis.

14.9%

36.8%

27.6% 37.8% 34.6%

48.3%

First polar body Disomy

Nullisomy

Second polar body Complex aneuploidy

Fig 28.7 Types of aneuploidies in first and second polar bodies. Disomy in PB1 or PB2 infer nullisomy in resulting oocytes, while nullisomy in PB1 or PB2 infer disomy in resulting oocytes.

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Table 28.1

Preimplantation diagnosis for chromosomal disorders by polar body biopsy Cycles (n)

Oocytes studied (n)

Resulting normal embryos transferred (n)

Transfers (n)

Pregnancies (n)

Babies (n)

Aneuploidies Translocations

3084 58

17329 667

5039 62

2399 36

674 11

608 10

Total

3142

17996

5101

2435

685

618

delivery of 10 unaffected children and three of which were spontaneously aborted, which is in agreement with the overall experience of PGD for translocations, suggesting a more than three-fold reduction of the spontaneous abortion rate after PGD, compared with the spontaneous abortion rate before PGD.34,35

3.

4.

Conclusion Polar body analysis has become one of the established methods of PGD for genetic and chromosomal disorders. Despite the limitation of this method to the testing of maternally derived abnormalities, it is currently applied in thousands of PGD cases. Because PB testing requires additional experience in the micromanipulation of oocytes, involving PB1 and PB2 removal, which is not part of a routine IVF procedure, application of the method has been restricted to a few centers. However, for some countries this is the only option for PGD, as no embryo biopsy is allowed owing to various social or ethical reasons. In addition, because the PB method avoids the removal of any material from the embryo, it has a clear advantage over PGD at the cleavage stage. PB testing is in fact complementary to blastomere biopsy in cases of PGD for single-gene disorders combined with aneuploidy, or preimplantation HLA-typing, especially when the parents carry different causative genes. PB testing is of special value in the case of aneuploidy, because testing for aneuploidies by blastomere biopsy has an important limitation due to an extremely high mosaicism rate at the cleavage stage. To differentiate incidental mitotic errors at the cleavage stage from the constitutive chromosomal abnormalities deriving from female meiosis, combined PB and blastomere testing for aneuploidies may in future be required, taking into consideration the 50% aneuploidy rate in oocytes and embryos from women of advanced reproductive age. Further understanding of the nature, mechanisms, and biologic significance of aneuploidies in preimplantation development will help in evaluation of the usefulness of different PGD methods.

References 1. Verlinsky Y, Ginsberg N, Lifchez A, et al. Analysis of the first polar body: preconception genetic diagnosis. Hum Reprod 1990; 5: 826–9. 2. Verlinsky Y, Rechitsky S, Cieslak J, et al. Preimplantation diagnosis of single gene disorders by two-step oocyte

5.

6.

7.

8.

9.

10. 11. 12.

13.

14.

15.

16.

17.

genetic analysis using first and second polar body. Biochem Mol Med 1997; 62: 182–7. Verlinsky Y, Cieslak J, Ivakhnenko V, et al. Pregnancies following pre-conception diagnosis of common aneuploidies by FISH. Hum Reprod 1995; 10: 1923–7. Dyban A, Fredine M, Severova, E, et al. Detection of aneuploidy in human oocytes and corresponding first polar bodies by FISH. J Assist Reprod Genet 1996; 13: 73–8. Munné S, Daily T, Sultan KM, Grifo J, Cohen J. The use of first polar bodies for preimplantation diagnosis of aneuploidy. Hum Reprod 1995; 10: 1014–120. Verlinsky Y, Cieslak J, Ivakhnenko V, et al. Birth of healthy children after preimplantation diagnosis of common aneuploidies by polar body FISH analysis. Fertil Steril 1996; 66: 126–9. Verlinsky Y, Cieslak J, Ivakhnenko V, et al. Preimplantation diagnosis of common aneuploidies by the first and second polar body FISH analysis. J Assist Reprod Genet 1998; 15: 285–9. Verlinsky Y, Cieslak J, Ivakhnenko V, et al, Prevention of age-related aneuploidies by polar body testing of oocytes. J Assist Reprod Genet 1999; 16: 165–9. Cieslak J, Tur-Kaspa I, Ilkevitch Y, et al. Multiple micromanipulations for preimplantation genetic diagnosis do not affect embryo development to the blastocyst stage. Fertil Steril 2006; 85:1826–9. Verlinsky Y, Kuliev A. Atlas of Preimplantation Genetic Diagnosis. New York: Parthenon, 2000. Verlinsky Y, Kuliev A. Practical Preimplantation Genetic Diagnosis. Berlin & New York: Springer, 2006. Munné S, Morrison L, Fung J, et al, Spontaneous abortions are significantly reduced after preconception genetic diagnosis of translocations. J Assist Reprod Genet 1998; 15: 290–6. Verlinsly Y, Kuliev A. Preimplantation Diagnosis of Genetic Diseases: a New Technique for Assisted Reproduction. New York: Wiley-Liss, 1993. Munné S, Bahce M, Schimmel T, et al. Case report: chromatid exchange and predivision of chromatids as other sources of abnormal oocytes detected by preimplantation genetic diagnosis of translocations. Prenat Diagn 1998; 18: 1450–8. Verlinsky Y, Evsikov S. Karyotyping of human oocytes by chromosomal analysis of the second polar body. Mol Hum Reprod 1999; 5: 89–95. Rechitsky S, Strom C, Verlinsky O, et al. Allele drop out polar bodies and blastomeres, J Assist Reprod Genet 1998; 15: 253–7. Rechitsky S, Verlinsky O, Amet T, et al. Reliability of preimplantation diagnosis for single gene disorders. Mol Cell Endocrinol 2001; 183 (suppl 1): S65–8.

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Clinical application of polar body biopsy 18. International Working Group on Preimplantation Genetics (IWGPG). Preimplantation Genetic Diagnosis – Experience of Three Thousand Clinical Cycles. Report of the 11th Annual Meeting International Working Group on Preimplantation Genetics, in conjunction with 10th International Congress of Human Genetics, Vienna, May 15, 2001. Reprod Biomed Online 2001; 3: 49–53. 19. European Society of Human Reproduction and Embryology PGD Consortium Steering Committee. ESHRE Preimplantation Genetic Diagnosis Consortium: data collection III (May 2001). Hum Reprod 2002; 17: 233–46. 20. Verlinsky Y, Rechitsky S, Verlinsky O, Strom C, Kuliev A. Preimplantation diagnosis for ornithine transcarbamylase deficiency. Reprod Biomed Online 2000; 1: 45–7. 21. Verlinsky Y, Rechitsky S, Verlinsky O, et al. Polar body based preimplantation diagnosis for X-linked genetic disorders. Reprod Biomed Online 2002; 4: 38–42. 22. Verlinsky Y, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis for PKU. Fertil Steril 2001; 76: 346–9. 23. Kuliev A, Rechitsky S, Verlinsky O, Strom S, Verlinsky Y. Preembryonic diagnosis for sickle cell disease. Mol Cell Endocrinol 2001; 183: S19–22. 24 Verlinsky Y, Cieslak J, Freidin M, et al. Pregnancies following pre-conception diagnosis of common aneuploidies by fluorescent in-situ hybridization. Hum Reprod 1995; 10: 1923–7. 25. Munné S. Preimplantation genetic diagnosis of numerical and structural chromosome abnormalities. Reprod Biomed Online 2002; 4: 183–96.

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26. Montag M, van der Ven K, Dorn C, van der Ven H. Outcome of laser-assisted polar body biopsy and aneuploidy testing. Reprod Biomed Online 2004; 9: 425–9. 27. Kuliev A, Rechitsky S, Laziuk K, et al. Pre-embryonic diagnosis for Sandhoff disease. Reprod Biomed Online 2006; 12: 328–33. 28. Kuliev A, Cieslak J, Ilkevitch Y, Verlinsky Y. Nuclear abnormalities in series of 6733 human oocytes. Reprod Biomed Online 2003; 12: 328–33. 29. Kuliev A, Cieslak J, Verlinsky Y. Frequency and distribution of chromosomal abnormalities in human oocytes. Cytogenet Genome Res 2005; 111: 193–8. 30. Angel R, Xian J, Ledger W, Baird T. First meiotic division abnormalities in human oocytes: mechanism of trisomy formation. Cytogenet Cell Genet 1994; 65: 194–202. 31. Munné S, Fisher J, Warner A, et al, and referring centers PGD group. Preimplantation genetic diagnosis significantly reduces pregnancy loss in infertile couples: A Multi-Center Study. Fertil Steril 2006; 85: 326–32. 32. Gianaroli L, Magli MC, Ferraretti A, et al. The beneficial effects of PGD for aneuploidy support extensive clinical application. Reprod Biomed Online 2005; 10: 633–40. 33. Verlinsky Y, Tur-Kaspa I, Cieslak J, et al. Preimplantation testing for chromosomal disorders improves reproductive outcome of poor-prognosis IVF patients. Reprod Biomed Online 2005; 11: 219–25. 34. Munné S, Sandalinas M, Escudero T, et al. Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reprod Biomed Online 2003; 7: 91–7. 35. Verlinsky Y, Cieslak J, Evsikov S, Galat V, Kuliev A. Nuclear transfer for full karyotyping and preimplantation diagnosis for translocations. Reprod Biomed Online 2002; 4: 300–5.

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29 Preimplantation genetic diagnosis for infertility (PGS) Santiago Munné

Introduction The incidence of chromosomal abnormalities in cleavage-stage embryos produced in vitro, 50–70% depending on maternal age, is considerably higher than that of spontaneous abortions, indicating that a sizable percentage of chromosomally abnormal embryos are eliminated before any prenatal diagnosis. Also, data from oocyte donation shows that in women of advanced maternal age (AMA) the decline in pregnancy is largely caused by failing oocyte quality.1 Thus by selecting chromosomally normal embryos for replacement, preimplantation genetic diagnosis (PGD) for infertility (or PGS) should, using appropriate methods, (i) increase implantation rates, (ii) reduce spontaneous abortion rates, (iii) reduce aneuploid conceptions, and (iv) improve delivery rates in ART cycles.2 Despite large studies indicating the advantages of this approach, the notion that PGS is beneficial is not yet uniformly shared, and results vary between PGS centers. In this chapter, the different steps that compose PGS will be evaluated to determine which are appropriate for ART improvement. There is new evidence that biopsy of two cells per embryo, while not reducing error rates, can reduce the implantation potential of an embryo. How the cells are biopsied can also negatively affect embryo development. Biopsied cells must be fixed for fluorescence insitu hybridization (FISH) analysis and some fixation methods produce fewer false positive errors than others. Once fixed, the FISH procedure needs to analyze a minimum number of chromosomes, apparently at least eight chromosome pairs, to result in improvement of implantation rates. But not all analyzers do the same good job. There are reports indicating FISH error rates between 5 and 50%, this sometimes being confused with true mosaicism.3,4 Obviously high error rates will preclude any benefit of a screening technique. Finally, once chromosomally normal embryos have been selected for replacement, special care

should be taken in doing the transfer. Reports following appropriate methods have shown that PGS does indeed increase implantation rates, and reduces spontaneous abortion and trisomic conception rates,3,5–8 but large studies are still needed to show a significant improvement in delivery rates. However, in a single or double embryo transfer (SET or DET) system, this should be achieved in centers with no limit on the number of embryos that can be replaced. In addition to infertile couples, PGS has proved to significantly reduce the risk of miscarriage in women with idiopathic repeated miscarriage (RM) or for patients with RM due to chromosome translocations.9,10

Chromosome abnormalities and embryo selection In most IVF laboratories one of the most powerful tools to improve results is embryo selection, based on morphological and developmental characteristics. However, the implantation potential of human embryos produced in vitro remains low even when most laboratories use morphological selection, generally about 29% for patients <35 years old, 21.8% for those 35–37, 14% for 38–40 and 7.7% for 41–42 years olds. One reason for the limited potential of morphological selection is that the majority of human embryos produced in vitro are chromosomally abnormal.11–19 In a recent study involving over 6000 embryos,12 it was shown that chromosome abnormalities were widespread regardless of maternal age and morphology. For example, only 44% of the best embryos, according to morphology, in young patients (<35 years old) were chromosomally normal, and decreased subsequently with age and decreasing embryo developmental characteristics (Fig 29.1).12 Thus, in that study, although morphology is correlated with euploidy and implantation potential, its use as a selection tool for replaced embryos can only improve the implantation potential by a few percentage points (i.e. for 35–37 years of age,

This chapter has drawn upon material from Santiago Munné, ‘Preimplantation genetic diagnosis for infertility’, chapter 13 in Joyce Harper (ed.), Preimplantation Genetic Diagnosis, 2nd edition, © Cambridge University Press (in press), reproduced with permission.

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% abnormal embryos 90 80 70

56%

60 50 40 30 20 10 0 35–37

38–40

>=41

Maternal age arrested

slow

dysmorphic

from an average of 37% normal embryos, to 44% in those with the best morphology). Extending culture to day 5 can improve implantation rates for patients with multiple day-3 embryos and good morphology. However, embryos of lower quality can implant and develop successfully if replaced on day 3, and delay to day 5 might be deleterious in these embryos.20 Studies on chromosome abnormalities in blastocysts derived from PGS cases indicate that some chromosome abnormalities do not reach blastocyst, such as pure monosomies and haploidies, but these studies have not been fully confirmed.21–23 There are emerging technologies attempting to assess embryo quality noninvasively.24 As with extended culture, it is unlikely that all chromosome abnormalities will be detected. To start with, there are two types of chromosome abnormalities of very different origins; one is aneuploidy, generated during gametogenesis and accounting for 20–50% of chromosome abnormalities depending on maternal age.12 The other is postzygotic abnormality, accounting for 30% of chromosome abnormalities and not linked to maternal age but to dysmorphism,12 and is probably caused by spindle abnormality that also causes dysmorphism.25 The latter are more to be correlated with abnormal development, and can be screened against by noninvasive methods, morphological selection, and extended culture, but not most trisomies, which can and do implant. Thus PGS alone or in combination with other selection techniques has the potential to select the most euploid embryos and improve ART outcome. A growing body of evidence shows that PGS increases the implantation rate while reducing trisomic conceptions and miscarriage. However, despite large studies indicating the advantages of aneuploidy screening, the notion that PGS is beneficial is not yet uniformly shared, caused by the differing results of studies that

Good

Fig 29.1 Chromosome abnormalities by age and morphology. Adapted from Munné et al.12

used different procedures and methods; some being more effective than others.

Differences in methods between optimal and poor PGS results PGS results do not depend only on the PGS laboratory, because many factors, including hormone stimulation, biopsy techniques, fixation methods, and embryo replacement skills, can substantially affect the outcome of a cycle. In this section several key aspects needed to ensure ART improvement through PGS will be discussed.

Which cells and how many to biopsy? First (or first and second) polar body,26–28 and day-3 single-cell embryo biopsy are the most common cells used for assessment of euploidy in human embryos.2,29 More recently, some programs have applied polar body biopsy combined with day-3 single-cell analysis either with acid tyrode30 or mechanical,31 day-3 two-cell biopsy,32 or blastocyst biopsy.33 Each method has advantages and disadvantages regarding damage caused, quality of information obtained, and quantity of cells analyzed. While first and second polar body analysis are the less invasive techniques, especially if the biopsy is performed mechanically, they do not provide information on postzygotic abnormalities, which affect 30% of embryos.11 Blastocyst biopsy is theoretically more benign than cleavage stage biopsy, since the inner cell mass is not biopsied, but not all embryos that are euploid reach the blastocyst stage (while if transferred before, they could implant),20 and the techniques for trophectoderm biopsy are unknown to the vast majority of embryologists. Furthermore, if the PGS were performed by a reference laboratory (e.g. transporting

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Table 29.1 Cells survived after thaw (%)

383

Effect of cell loss on implantation* Implantation rate (%)

Procedures with unique transfers (n)

≤50 51–75 76–99 100

9 32 32 103

Sac

7.7 15.3 27.6 33.6

Fetal heart beat

Pregnancy rate with fetal heart beat (%)

7.7 13.7 27.6 30.0

11.11 18.75 37.50 49.51

*Only thaws and transfers with uniform embryo cohorts are included. From: Cohen et al.35

the fixed cells for analysis), the technique would require day-6 replacement. There are no reports on the effect of embryo biopsy done without PGD and PGS selection on implantation. Some studies followed embryos only to blastocyst formation.34 The best indirect assessment of the damage caused by embryo biopsy is the study by Cohen et al35 comparing the effect of cell loss after freezing and thawing. In that study, cycles in which ‘pure’ replacements (all embryos match the criteria) of either embryos with all cells intact, embryos with one cell lost, or with two cells lost, and three cells lost were compared. As seen in Table 29.1, cycles that had embryos replaced with no cells lost, had a 30% implantation rate (+ fetal heart beat), compared with 28% if all embryos replaced have lost one cell, but only 14% if all embryos replaced had lost two cells. In some studies, single-cell embryo biopsy appears to minimize reduction in implantation potential that is more than compensated by the selection of euploid embryos.5–7 However, two-cell biopsy seems to reduce the implantation potential by more than half,35 and embryo biopsy by inexperienced hands can be very detrimental, even when withdrawing a single cell.36–39 Blastocyst biopsy is starting to be used clinically,33 producing several cells for analysis; while this certainly will reduce the risk of misdiagnosis, it is unlikely to show good implantation. However, no controlled studies are available yet about its effect on outcome.

How to open the zona Mechanical biopsy40 seems intuitively to be the less detrimental means of embryo biopsy, but is the hardest to learn. Chemical zona opening using Tyrode’s solution41 is widely used, although both methods are being progressively substituted by laser biopsy,42 which is the simplest to use. Mouse models are not appropriate for evaluation of human embryo biopsy because of the difference in zona pellucida characteristics and the inability of mouse blastomeres to recover following acidosis.43 There are very few publications comparing the three methods in relation to ART outcome.44,45 Chatzimeletiou et al44 suggested that a safe working distance for the laser is crucial in order to prevent

immediate or long-term adverse effects on the development of lased and biopsied human embryos. Another study compared acidified Tyrode’s medium and laser biopsy; and found that making the hole between two cells obtained identical pregnancy rates, but with a slight increase in broken cells after acid (5%) than after laser (2%) (p<0.05).45 These observations certainly suggest that specialized training is necessary for the correct performance of zona opening, regardless of the method used, and lack of such training is surely one of the primary reasons for unsatisfactory implantation obtained in some centers after PGS.

How to remove the cell Once the zona pellucida has been breached, blastomeres can be biopsied either by suction, liquid displacement46 or by exerting pressure against the zona pellucida with the micropipette.47 The first method seems the less detrimental since it hardly disturbs the other cells, unless the embryo is compacted; while the last is certainly the most disruptive. No studies have compared the three methods. The majority of normally developing human embryos have been found to undergo compaction on day 4 of development48 but the first immature tight junctions appear at the six-cell stage.49 Compaction complicates cell biopsy and results in high rates of cell lysis. This has been circumvented by using Ca2+/Mg2+-free medium, which breaks the tight junctions. However, extended time in such media was found to impair embryo development.50 Thus the embryos must be less than 5 minutes in Ca2+/Mg2+free medium before the biopsy, and after the biopsy the embryos should immediately be washed to remove this media.51–53 The composition of the Ca2+/Mg2+-free medium is also important. Hill and Li54 found that a Ca/Mg-free biopsy medium with sodium lactate, sodium pyruvate, and alanyl-glutamine, produced significantly higher pregnancy rates than simpler media. Polar body (PB) biopsy is usually done by suction or liquid displacement.55 A specific problem of first PB biopsy is to coordinate it with ICSI. Inexperienced personnel should be reluctant to perform PB biopsy before

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ICSI, since the position of the first polar body is indicative of the spindle position and results may be jeopardized if the sperm injection disturbs the spindle.56 In certain cases, results must be obtained before ICSI,57 but a bridge of microtubules between the egg chromosome spindle and the polar body may exist, and if the polar body is pulled the egg may be damaged. If ICSI is performed too late, in vitro oocyte aging may occur, producing nondisjunction. Experienced personnel usually perform first and second polar body biopsy together at the zygote stage.55 Again it is clear that inexperienced hands doing biopsy can easily cause very low implantation.

Cell fixation Biopsied polar bodies are extremely small and are therefore usually fixed by letting the cell dry on the slide; then applying Carnoy’s fixative (3:1 methanol:acetic acid). Since the chromatin structure of the first polar body degenerates within 24 hours after egg retrieval (and sometimes is already degenerated) blastomere fixation methods are not effective. However, if the first PBs are fixed a few hours after retrieval and have not degenerated, metaphase chromosomes can be obtained using improved techniques58 and FISH signals can be better observed. This method is also suitable for chromosome painting for PGS of maternal translocations when the translocation breakpoints are not terminal. For blastomere fixation the original method (method1) using Carnoy’s fixative59 although improved with several modifications is still considered difficult to use successfully when individuals do it sporadically; so two alternative methods have been developed.60–62 Method-2 uses Tween-20, a detergent, and HCl, and method-3 uses a combination of Tween-20 and HCl, and Carnoy fixative. The mixture in method-1 of fixative with the drop of hypotonic containing the blastomere produces turbulences in which the cell may be lost. This risk is about 3% in expert hands63 but it is certainly higher unless continuously practiced.62,64 In contrast, methods2 and -3 overcome the turbulence step and are easily learned. However, the purpose is to provide a fixed nucleus with the highest chance of producing reliable FISH results. Comparing the three techniques, Velilla et al63 found that the average diameter of the fixed nuclei was 59, 31, and 46 microns respectively, resulting in 14%, 58%, and 39% overlaps between chromosomes, and 10%, 30%, and 17% errors, respectively. So it appears that method-1 is more effective in reducing errors and minimizing no results but involves a higher learning curve. It is also possible that changes in FISH protocols may allow better results using the other two methods (Gary Harton, personal communication).

Number of chromosomes analyzed Fluorescence in situ hybridization (FISH) is currently the best method to analyze PBs and blastomeres, since

the former have poor quality metaphases and, in the latter, metaphases are produced in low rates even after culture.51 The human eye can only detect five fluorescent dyes in the visible spectrum, the number of chromosome DNA probes used simultaneously is thereby limited to five. This requires two or more rounds of hybridization to analyze extra chromosomes. However, more hybridization rounds on the same cell mean more errors, and therefore PGS laboratories usually analyze a maximum of 12 chromosomes. The number and types of chromosomes analyzed is of course critical for ART improvement. The chromosomes most involved in aneuploidy at the cleavage-stage are in order 22, 16, 21, and 15.65 When combined with those whose abnormalities can reach term (X, Y, 13, 18, 21) they make eight the minimum number of probes that should be used to detect aneuploidy with PGS. Although a reduction in spontaneous abortions has been reported using only five probes (X, Y, 13, 18, 21),6 any studies obtaining improved implantation have used probes for at least the eight critical chromosomes (X, Y, 13, 15, 16, 18, 21, 22).5,7,8,10 This is because the five-chromosome test detects only 28–31% of chromosome abnormalities detected in fetuses, 70–72% with nine probes (the previous eight plus 17) and 79–80% with the 12-probe test (the eight plus 17, 14, 8, and 20).66,67 However, spontaneous abortion data does not coincide entirely with that obtained by comparative genome hybridization (CGH) in oocytes, where the nine-test detects only 57% abnormal eggs and the 12-test only 67%68–70 (Table 29.2). This is because some trisomies and monosomies survive better than others to the first trimester.21,65 PGS analysis of all chromosomes has been attempted using CGH. At first, because of the time taken, embryo biopsy was followed by freezing;71–73 but thawing attrition was found to eliminate the advantages of the PGS selection. Next, attempts were made to analyze first polar bodies and replace selected embryos on day 5;74 but the technique was found to be too labor intensive to be practical and was also abandoned. With the advent of better freezing methods, CGH is being revisited.75,76 Once more, there is no standard number of chromosomes analyzed between IVF programs, which will mean yet another variation in PGS results between centers and CGH is not yet a practical alternative.

No results, and result rescue The rate of undiagnosed cells mostly depends on proper biopsy and fixation techniques. The rate of no diagnosis ranges from a very low 1% of embryos undiagnosed77 to a very large 20% of embryos undiagnosed.36 Even with the best of fixations, FISH signals may overlap or split. Size analysis and distance between signals has been used in the past as criteria for scoring dubious results.78 A much better alternative is to use ‘no result rescue’ (NRR), which consists of rehybridizing a nucleus for

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Table 29.2

385

Abnormalities detected by FISH and CGH Eggs + PB analyzed

Egg/PB that will be classified abnormal by FISH using:

Ref

Total

Abortion

4 chrom. XY, 13, 15, 18

68 69 70

25 21 100

11 21 22

4 3 13

4 9 13

6 11 14

6 12 18

Total

146

54

20(37%)

26(48%)

31(57%)

36(67%)

Table 29.3

Error rates reported by different centers, and differences between studies

Ref

No. of probes

Embryos reanalyzed

4 168 81 169 19 3

10 5 8 6 to 9 8 9+NRR

29 55 228 885 853 212

Type of embryos

6 chrom. 5 chr+16, 22

8 chrom. 6 chr+15, 17

11 chrom. 8 chr+14, 20, 8

Abnormals missed 5 9 4

Day reanalyzed

discarded PGS* discarded discarded, PGS PGS PGS

5 5 3 4 4 4

Fixation type Twin-20/HCl Twin-20/HCl Twin-20/HCl Carnoy Carnoy Carnoy

18

Embryos without results

Discordance**

24% n/a 28% n/a n/a 3%

50.0% 40.0% 24.5% 7.2% 8.6% 5.2%

*PGS: embryos classified by PGS as chromosomally abnormal. **Discordance: normal for abnormal or abnormal for normal, NRR: no result rescue.3

which there is an uncertain signal for a specific chromosome with a probe for the same chromosome which binds to a different locus.3,79,80 A recent study by Colls et al3 involving 34 225 embryos shows that with this method, nuclei with inconclusive results are reduced from 7.5% to 3.1%, and FISH errors from 13.6% to 4.7%. As yet, this is by no means a standard procedure, and shows another area of potential error in the application of PGS.

Number of analyzers Counting FISH dots is not as simple as it seems. There is a blind spot where the optical nerve is collected and passed through the human retina; in certain situations a FISH signal becomes invisible. Also, signals on the periphery of the nucleus, usually in micronuclei, may be missed by an analyzer. Thus it is paramount that two analyzers score all samples independently. In one study, doing NRR with two analyzers who scored the signals independently, the individual error rate was 10.3% compared to the combined analysis of 4.7% (Zheng et al, personal communication). This is quite a surprising outcome, and this important factor is not generally recognized.

Error rate criteria and differences between centers The error rate of fixation and FISH technique should be continuously evaluated by the PGS laboratory and can include:

(a) (b) (c) (d) (e)

reanalysis of all embryos not replaced after PGS, reanalysis on day 3 or day 4, using standard FISH and fixation methods, clinical confirmation of normality or abnormality, previous published criteria of defining a mosaic embryo as abnormal (i.e. 81), although this point may be debatable and instead (f) the previously used <3/8 abnormal cells to define normality, this could be raised to 50% since hardly anyone would accept replacement of such an embryo. Once similar criteria are used, errors will then be caused either by the same technique, or mosaicism. Each PGS laboratory has a different error rate, and values range from 4 to 7%3,19,57 to 50%4,23,81 (Table 29.3). An error rate close to 50% is in fact equivalent to no diagnosis at all. Thus it is paramount for a patient to be aware of the error rate of the PGS center that will perform the procedure. Some centers have been known to quote error rates from the literature instead of their own error rate, which can certainly be misleading. Potential differences between PGS centers can occur according to how the reanalysis is performed. Overall these differences can be because of: (1) use of unsuitable fixation techniques that are more prone to errors; (2) use of unsuitable probe combinations, including mixtures of colors, or polymorphic probes which are more prone to errors; (3) failing to reanalyze all embryos not replaced after PGS, thus overestimating chromosome

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Table 29.4

Estimated risk of PGS misdiagnosis due to mosaicism

Risk of classifying an abnormal embryo as normal

2N/POL (detrimental) Chaotic (detrimental) Split (detrimental) Mitotic aneupl (all) Meiotic & mitotic aneupl (all) TOTAL

Overall frequency (A)

% Normal cells (B)

Risk of misdiagnosis (A×B)

3.7% (70/1903) 12.7% (242/1903) 0.3% (5/1903) 6.6% (126/1903) 0.6% (12/1903) 23.9% (455/1903)

34.8% 9.8% 29.8% 24.2% 12.2% 18.0%

1.3% 1.2% 0.1% 1.6% 0.1% 4.3%

% Abnormal cells (B)

Risk of misdiagnosis (A×B)

23.1% 24.9% 26.7% 23.6%

0.9% 0.3% 0.1% 1.3%

Risk of classifying a mostly normal embryo as abnormal Overall frequency (A) 2N/POL (benign) Chaotic (benign) Split (benign) TOTAL

3.9% (74/1903) 1.3% (24/1903) 0.2% (3/1903) 5.3% (101/1903)

Total misdiagnosis rate due to mosaicism

5.6%

These were embryos fully trisomic or monosomic for a specific chromosome and in addition mosaic for the same chromosome. Data from: Munné et al.169 The risk of misdiganosis was calculated by multiplying column A by column B.

abnormalities by analyzing embryos with a higher likelihood of being abnormal; (4) reanalyzing on day 5 instead of day 3 or 4 some abnormal embryos will not survive to day 5,21 and an embryo misdiagnosed as abnormal (but being normal) will be more likely to reach blastocyst stage, but the ones arresting will not be counted in the denominator, thus overestimating the frequency of errors); (5) using ‘cytogenetics confirmation’ criteria (an abnormal to be confirmed should have the same identical abnormality detected by PGS) instead of a ‘clinical confirmation’ criteria (example monosomy 18 by PGS and chaotic 100% abnormal by reanalysis, abnormality is confirmed). So there are plenty of reasons why patients should investigate the details of actual procedures and results at the clinic they attend, and take the time to understand the potential differences.

Mosaicism and error rates Mosaicism rates vary widely in the literature. Some of the differences between centers are due to population; others to hormonal stimulation and the general quality of embryos produced in those centers. For example, it is well known that changing intrafollicular and laboratory conditions may alter the rate of mosaicism.82 Also, as mentioned previously, mosaicism frequencies often change according to the developmental stage of the embryo. Most large studies on cleavage stage embryos indicate mosaicism rates around 25–30%.11–14,19,57,83 Mosaicism

can produce false positive or negative results. It has been estimated that the false positive error rate is 4.3% and the false negative error rate 1.3%57 (Table 29.4). The low error rate caused by mosaicism compared to its 30% frequency can be explained by the fact that the majority of mosaic embryos have only abnormal cells (Table 29.5).3 Even considering 50% abnormal cells as the cut off for defining normalcy, the total error rate can be as low as 7%3 (Table 29.5), of which 5% is due to mosaicism and the rest to other technical problems. Even lower error rates have been recently reported by Magli et al.19 Thus, the effect of mosaicism on PGS errors have been overestimated; providing appropriate methods are used, it is included in the overall <10% error rate.

Results of PGS for aneuploidy: trisomic offspring, spontaneous abortions, and implantation Reduction in trisomic offspring The first proposed use of PGS for chromosome abnormalities was as an alternative to prenatal diagnosis. In order to determine if PGS significantly reduces the risk of aneuploid conceptions, a suitable control group is needed. However, there is scant data on chromosomally abnormal conceptions, stratified by age, from IVF databases such as SART. Since IVF pregnancies are usually closely monitored, a second-best control group would be pregnancies diagnosed through early prenatal diagnosis. Using such a

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Table 29.5

Error rate and % of abnormal cells in mosaics

592 abnormal embryos from PGS were reanalyzed and found to be: normal mosaic <38% abnormal mosaic 38–49% abnormal mosaic 50–99% abnormal mosaic 100% abnormal homogeneously abnormal False positive error with 38% threshold as abnormal False positive error with 50% threshold as abnormal

13 15 12 124 297 131 4.7% 6.8%

In Colls et al.3

control population, Munné et al8 found that the expected risk of aneuploid conceptions for chromosomes X, Y, 13, 18 and 21 in a group of 2279 PGS cycles was 4.7%, based on the data of Eiben et al84; but after PGS the observed rate of trisomic conceptions was significantly lower, 1.2% (p<0.001). A continuation study on 2300 fetuses resulting from PGS cycles, including the previously published data of Munné et al8 indicated a reduction in aneuploid conceptions from an expected rate of 2.6% to an observed rate of 0.5% (p<0.001) (Table 29.6). Data from other centers include a 1.2% error rate in 170 pregnancies from women with an average age of 36 years77 while data from polar bodies indicated no errors from 376 babies born.85 Although the prevention of trisomic conceptions is usually lumped together with the concept of improving ART outcome, it is an indication in itself. For example, a recent study86 reported that in a survey from The Netherlands, where PGS is covered by health insurance, 87% of sub-fertile patients would undergo PGS if pregnancy rates were unchanged and 100% of Down’s syndrome were detected, and 36% if pregnancy was reduced from 20% to 14%. If 80% of Down’s were accurately screened, and there was no change in pregnancy rate, 75% of subfertile women would undergo PGS. That is akin to the 81% screening potential reported by Munné et al.8

Decrease in spontaneous abortions Most studies about pregnancy loss agree that a prior miscarriage with advanced maternal age, are the major risk indicators for spontaneous abortion.87–91 SART92 data indicates that 13.3% of ART pregnancies in patients younger than 35 years of age result in miscarriage, 17.7% in patients 35–37, 26.2% in 38–40, 39.4% in 41–42, and 53.3% in patients 43–44 years old. The German registry91 reports even higher rates of miscarriage after IVF, with 23.9% in patients 35–36 years old, 26.3% in 37–38, 36.6% in 39–40, 43.1% in 41–42, and 56.1% in women over 42 years of age.

387

In sporadic miscarriages among the general population, chromosome abnormalities range from 39% to 76%, depending on the study.93–98 Some of these studies, particularly earlier ones, appear to underestimate chromosome abnormalities. This is because conventional karyotyping requires tissue culture, which is prone to maternal contamination and fails up to 25% of the time, failing more often if the embryo is chromosomally abnormal.97,99 Chromosome studies in spontaneous abortions of ART patients also indicate a high rate of chromosome abnormalities,67,100 with 65–71% of spontaneous abortions being chromosomally abnormal, and increasing with maternal age, from 65% in women 39 years old and younger to 82% in women 40 years old and older.100 Because of the high rate of chromosome abnormalities in spontaneous abortions, PGS should substantially reduce the rate of miscarriage in infertile patients undergoing ART. Indeed, FISH with probes for chromosomes 13, 15, 16, 18, 21, 22, X, and Y can detect 72–83% of the chromosomally abnormal fetuses routinely detected by karyotyping in women of advanced maternal age.66,67 Since this combination of probes is our current standard, PGS should also eliminate close to 80% of all chromosomally abnormal embryos at risk of causing a miscarriage. In a multicenter study, controls were compared with a test group undergoing embryo biopsy and PGS for aneuploidy of chromosomes X, Y, 13, 18, and 21,6 and the results revealed a significant reduction in spontaneous abortions, from 23% in the controls to 9% in the PGS group (p<0.05) and a significant increase in ongoing pregnancies (10.5% vs 16.1%, p<0.05). In another study, an abortion rate of only 9% was reported after PGS of aneuploidy for 343 cases in women >36 years old101,102 instead of an expected 16%. While these may not be large differences in real numbers, for individual patient couples the improvements are extremely important. Even so, in two studies, pregnancy outcome in 191 couples after PGS was compared with previous pregnancy history. In one of them, Gianaroli et al77 reported a 11.4% pregnancy loss compared to 88.5% before PGS (p<0.001). Verlinsky et al85 reported a pregnancy loss of 28% after PGS of aneuploidy in a population with an average maternal age of 37, and compared that to the previous reproductive history of that same group (68%, p<0.001). These two studies have been criticized on grounds of comparing a self-selected population with prior negative reproductive history with their next cycle, overestimating the true baseline of spontaneous abortions in that group. Once more, the individual success is of paramount importance. A more recent study8 comparing SART data92 on spontaneous abortions with PGS data on 522 pregnancies from 100 IVF centers, revealed a significant decrease in spontaneous abortions after PGS, from 19% to 14.1% (n=382) (p<0.05) in women 35–40 years old, and from 40.6% to 22.2% (n=140) (p<0.001) in women

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Table 29.6

Expected and observed aneuploid conceptions after PGS

Age <35 35–39 >39 Total

Conceptions after PGS

Expected aneuploidies*

891 906 503

× 1.2% =10.7 × 1.3% =11.8 × 7.3% =36.7

59.2/2300 (2.6%)

11/2300 (0.5%)***

Observed aneuploidies**

p<0.001

*Eiben et al.84 **Munné et al.8 and unpublished data. ***Observed aneuploidies: 4 XO, 1 XXY, 1 T13, 1 T18, 4 T21.

Table 29.7 IVF clinic PGD cycles

Comparison of non-PGS and PGS cycles from five IVF centers

Age group

Loss rate (%)

Non-PGD cycles live birth (%)

Loss rate (%)

Live birth (%)

1 2 3 4 5

38–42 38–42 38–42 38–42 38–42

505 210 1204 509 191

27% 36% 34% 29% 25%

35% 14% 12% 15% 17%

70 72 120 236 208

22% 27% 15% 26% 16%

40% 15% 23% 22% 25%

Total

38–42

2619 30%

18%

706 21%

24%

Total PGS results were siginificantly better for pregnancy rate (p<0.01), miscarriage rate (p<0.01), and pregnancy to term (p<0.001).

>40 years old. This study had the limitation that not all centers in the SART database contributed to the PGS group. To try to solve that problem a similar study103 compared SART data from 2003–2005 from five IVF centers with extensive experience in biopsy (at least 50 cases in that period and at least 10% of their IVF cycles being PGS), with the PGS data from those same centers and same years, for patients 38–42 years old. There was a significant reduction in spontaneous abortions from 30% in non-PGS cycles to 21% in PGS cycles (p<0.01) (Table 29.7). In each of the studies showing an improvement after PGS, the protocols used were of the highest standard, and the personnel well-practised in PGS procedures.

Increase in implantation, pregnancy, and ‘take home’ baby rates Reviewing the literature there have been two starkly different conclusions: a first group of investigators represents and supports the hypothesis that PGS for aneuploidy improves implantation and reduces miscarriage rates,3,5–8,10,77,85,103 and a second group were not able to demonstrate any significant differences between control and PGS patients32,104 or showed a negative effect.36 There are critical differences between these groups of studies, which will be reviewed in this section.

There have been basically three types of comparative trial investigations of PGS for aneuploidy. (1) Trials that used retrospective analyses or compared patients only to their prior reproductive history;8,10,77,85,103 (2) those that used prospective datasets comparing patients that accepted PGS with patients that declined it;3,5–7 and (3) prospective data in a randomized fashion.32,36,104 Some retrospective studies are problematic. For instance, studies comparing previous pregnancy history before and after PGS have the obvious bias that most patients choosing PGS had prior poor results without PGS.77,85 Other retrospective studies may contain inclusion biases. For instance, Munné et al8 compared cycles from 100 IVF clinics performing PGS to the whole SART database for the year 2000 (>300 clinics), and detected a significant decrease in spontaneous abortions after PGS. It can be argued that IVF centers performing PGS were not representative of the whole IVF population represented by SART. Possibly that problem was solved in a later study, which compared PGS cycles from five IVF centers, to cycles from those same centers that did not have PGS, showing again a decrease in spontaneous abortions and an increase in take-home baby rates.103 Although that paper can also be criticized on the grounds that PGS cycles usually should have larger cohorts of embryos than those without PGS, the literature indicates higher

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Table 29.8

389

Summary of prospective studies comparing PGS and control ART outcome Study 16

25

37

4110

532

63

736

one 4–8** no**

one 8 yes

one 8 yes

one 8 yes

two 6 no

one 8 yes

one 8 no

yes n/a n/a

yes 3.1% 6.7

yes 4.4% 8.9

yes/no† n/a n/a

no 4.7% 5.9

yes 4.7% 7.2

no 20.1% 5.4

comp

comp

comp

rand

rand

comp

rand

117 117 3.6

127 135 3.0a

138 138 3.7a

28 29 n/a

141 148 2.8a

100 100 2.4a

402 unclear*** 1.9

3.1

1.8a

2.0a

n/a

2.0a

1.5a

1.8

13.7% 17.6% 29.9% 35.9% 33.8%c 15.0%c 10.6%c

12.4%a 24.2%a 25.1% 29.1% 20.6% 5.4% 10.2%a

10.6%c 17.6%c n/a n/a n/a n/a n/a

n/a n/a 20.7% 43.0% n/a n/a n/a

11.5% 17.1% 27.7% 19.6% 25.6% 25.0% 10.4%b

20%d 31%d 32% 35% 28%d 6%d 14.1%d

14.7%e 16.8% to 6%e ¥ 84 unclear*** 21.4% unclear*** n/a

15.9%c

22.5%a

n/a

n/a

16.5%b

28.9%d

n/a

22.2%

20.0%

n/a

n/a

20.6%

26%

16.4%

32.5%

27.6%

n/a

n/a

14.9%

31%

unclear***

Characteristics Cells biopsied Chromosomes analyzed Analysis of 15, 22 chromosomes Fixation type appropriate No result rate Average number embryos PGS group Type of study Results Cycles retrieved control Cycles retrieved PGS Average number embryos replaced control Average number embryos replaced PGS Implantation rate control Implantation rate PGS Pregnancy rate control Pregnancy rate PGS Pregnancy loss rate control Pregnancy loss rate PGS Ongoing implantation rate* control Ongoing implantation rate* PGS Ongoing pregnancy rate control Ongoing pregnancy rate PGS

comp: prospective non-randomized comparative study; rand: prospective randomized: study. *Fetus ongoing ≥12 weeks/embryos replaced. p<0.001,b p=0.06,c p<0.05,d p<0.025, e = 59% implantation reduction caused by the biopsy alone, when no PGS analysis was performed. **Only 31/117 cycles with 8 probes. ***Pregnant cycles. † Different centers in this multicenter study used different fixation types. ¥ There were three subgroups of cycles in the PGS group, the first with two normal embryos replaced (16.8% implantation rate), the second with two undetermined embryos replaced (6% implantation rate), and the rest. ***Because PGS cycles were reported together with a subgroup of cycles with no PGS analysis (see ¥), PGS pregnancy, miscarriage and ongoing pregnancy rates cannot be assessed properly.

a

rates of chromosome abnormalities in larger cohorts of embryos than in small ones,105,106 which should in fact affect PGS cycles more negatively. A larger cohort of embryos per procedure is of course one of the prerequisites for optimal PGS results. Regarding prospective nonrandomized and prospective randomized studies, there are many differences between them, some extremely important: (I) the number of cells being biopsied, (II) the number and type of chromosome probes applied, (III) the form of randomization, (IV) the type of cell fixation used,

(V) the error rate and undiagnosed rate of embryos, (VI) the number of embryos available for biopsy (Table 29.8). As shown in Table 29.8 studies in group II used inappropriate methodology resulting in a lack of positive results. These methodology problems included for example biopsying two cells per embryo,32 which according to Cohen et al35 most probably immediately eliminates any potential beneficial effect of the PGS selection. Indeed, the same group32 have just published a study on a clinically randomized trial (CRT) comparing PGS results applying one-cell and two-cell biopsy.107 This new

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study107 found a significant negative influence on embryo development from day 3 to day 5 after two-cell biopsy compared to one-cell biopsy (p<0.007), particularly in embryos with poorer morphology (p<0.0001). The implantation rate between one-cell biopsy (23.5%) and two-cell biopsy (17.3%) was not significant, and pregnancy rate was just below significance (p=0.068). However, comparing Goossens et al107 study to their previous one,32 both reported exactly the same implantation rate after biopsy of two-cells (17%), while in the Staessen et al32 study they reported 11% for the control group. If the implantation rate (11%) of that control group is compared to the one-cell group implantation rate in Goossens et al107 (23.5%) the difference is statistically significant, demonstrating that in their hands twocell biopsy is detrimental, and that implantation rate significantly increases after PGS with single-cell biopsy (Fig 29.2). Interestingly, error rates for both FISH results were similar between one-cell and two-cell biopsies,107 thus making two-cell biopsy unnecessary. Several type-II studies have a low average number of embryos produced in the PGS cases,36 resulting in an extremely limited potential selection. Prior studies have indicated that in order to improve pregnancy outcomes, a minimum number of 2-PN embryos (n = 8) is necessary7 (Table 29.9). Similarly, Tur-kaspa108 indicated that best results are obtained with a minimum of 13 oocytes. This is because, when the total number of embryos is low (four or less), most control group embryos are replaced and the total number of normal embryos replaced is similar in control and PGS groups. PGS is a selection tool to improve ART outcome, if there are not enough embryos to do a selection, pregnancy rates cannot improve. Other methodology problems in type-II studies were that chromosomes 15 and 22 were not analyzed.32,36 These chromosomes account for 24% of chromosome abnormalities detected in IVF spontaneous abortions67 and at least 10% of day-3 abnormal embryos65 (Table 29.10). Also, their fixation methods were not the ones with the lowest error rate in Staessen et al32 and Mastenbroek et al.36 Finally, probably the most damaging shortcoming of the Mastenbroek et al36 study is its four-fold higher rate of undiagnosed embryos (20%) compared to other studies in Table 29.8 (3–5%) and the 1.8–2.5% of Goossens et al.107 In addition, by replacing these undiagnosed embryos, they created a third arm in their study, that is embryos biopsied but undiagnosed. No other study to date had this arm, and it would be unethical to design a priori such a study knowing that biopsy may be detrimental, but not offering PGS to balance or compensate in excess for that damage. Interestingly, this third arm (biopsy, no PGS) showed in their hands that the biopsy method used by Mastenbroek et al.36 produced a 59% reduction in implantation potential (from 14.7% in controls to 6% in this group), something that has not occurred in other studies. After PGS, and even not analyzing 15 and 22, and offering it to patients with very few embryos, PGS selection was actually able to compensate

25.0% 23.5% 20.0%

10.0%

17.3%

17.1%

15.0% 11.5%

5.0% 0.0%

Control

2-Cells biopsied

2-Cells biopsied

1-Cell biopsied

Staessen et al (2004) Goossens et al(2007)

Fig 29.2 Comparison of no PGS, one cell biopsy + PGS, and two cell biopsy + PGS.

for the biopsy damage (assuming it was similar in the ‘biopsy and no PGS’ and ‘biopsy and PGS’ groups) producing 16.8% implantation rates, but not enough to be better than controls. None of the studies mentioned above is perfect, but comparison studies using appropriate methodology do clearly indicate that PGS is beneficial for some patients. Randomized studies with appropriate technology and skills are needed.

Suggestions for appropriate RCT Studies reporting PGS results should include the following: (1) Patients’ maternal ages should be at least 35 and older, although a perfect study might limit age to 38–42, where chromosomes abnormalities are more frequent. (2) A minimum of eight zygotes or six embryos with six or more cells each on day 3 should be available for each procedure. (3) The PGS center(s) should be skilled and have a proven low rate of undiagnosed embryos (<5%), including two persons scoring results. (4) The biopsy method(s) used should minimize embryo damage and be of one cell only. (5) The fixation method(s) used must be appropriate. (6) The PGS laboratory(ies) reporting should have error rates of <10%, confirmed by reanalyzing all abnormal embryos detected. (7) Reporting IVF centers should have sufficient experience with PGS, either (a) showing positive results with comparison studies or (b) showing similar pregnancy rates between PGS of single gene defects and non-biopsied cycles. (8) Chromosomes analyzed should at least include X, Y, 13, 15, 16, 18, 21, 22, using a maximum of two hybridization steps; and also using ‘no result rescue’ to reduce error rates in a third hybridization if needed.3

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Table 29.9 zygotes

Pregnancy and implantation rates in PGS patients and their respective controls, depending on the number of 2PN

Pregnancy a Groups Total*** <8 zygotes ≥8 zygotes

391

Implantation b

Control

PGS

Control

PGS

29% (30/103) 25% (14/55) 33% (16/48)

31% (32/103) 14% (6/43) 43% (26/60)

10% (38/367)** 9% (17/194) 12% (21/176)

20% (39/201)** 19% (10/54) 20% (29/147)

a

Cycles pregnant with FHB, b FHBs per embryos replaced. **p< 0.005. *** Patients 35 and older with less than two previously failed IVF cycles. Average maternal age in both groups was 40 years. From Munné et al.7

Table 29.10 Chromosome specific aneuploidy rates in human cleavage-stage embryos

Chromosome

Number of analyzed embryos

XY 14 6 18 1 4 7 13 17 15 21 16 22

1308 279 194 1607 550 236 235 1350 218 638 1548 1209 818

Total

1607

Aneuploid (%)* 0.8% 1.1% 1.5% 1.5% 2.0% 2.5% 2.6% 2.8% 2.8% 3.6% 3.7% 4.4% 6.2%

*

Double aneuploidies counted twice, once for each chromosome. From Munné et al.65

Other indications for using PGS with infertility As well as advanced maternal age and prevention of trisomic conceptions, there are two other indications for PGS: recurrent pregnancy loss (RPL) and repeated implantation failure (RIF). A third indication, male factor, has had such a limited number of cases studied, that the ART outcome cannot be reasonably assessed.

Recurrent pregnancy loss (RPL) Patients with normal karyotypes that are unfortunate enough to have three or more consecutive spontaneous abortions of less than 20–28 weeks’ gestation are considered to have RPL.116,117 There is little evidence of endometrial rejection or a defective endometrium and 50% of cases remain classified as having unknown etiology.118–120 The studies suggest these couples may be more prone to having chromosomally abnormal conceptions. Chromosome abnormalities are the major cause of miscarriage, with 99% of chromosomally abnormal pregnancies miscarrying94 compared with 7% chromosomally normal.121 Also, about 85% of miscarriages are embryonic losses with <9 weeks of gestation, before fetal heart beat is detected,122 and of those, karyotype studies report 87% chromosome abnormalities.123 CGH studies, which preclude potential maternal contamination, report even higher rates.98,124,125 It cannot be assumed that only chromosome abnormalities are involved. The frequency of abnormal embryonic karyotypes has been found to be higher in sporadic abortions (63–76%) than in RPL (40–60%).94–96,98 Furthermore, women aborting 2–4 consecutive pregnancies had 60% chromosomally abnormal fetuses but women with >4 miscarriages had only 29% abnormal.96 It is important to note that the Ogasawara et al96 study involved all kinds of RPL patients, and not only idiopathic ones. All previously mentioned studies were performed on products of conception from clinically recognized pregnancies. Studies on RPL cleavage-stage embryos have consistently indicated either more chromosome abnormalities than in control groups126–129 or similar rates in fertile RPL patients to those infertile ones.8,10

Repeated implantation failure (RIF) Studies on RIF,5,7,53,75,109,110 male factor,111,112 previous trisomic conception,113 and even egg donation114 have been mostly limited to analyzing chromosome abnormalities in these embryos, so little evidence has been obtained regarding its effect on ART outcome, which in many cases may have other causes.115 Thus, in this section only RPL will be discussed.

PGS can reduce spontaneous abortions in patients with idiopathic RPL The main goal of most patients undergoing PGS for RPL is to prevent another miscarriage; so a few studies have evaluated spontaneous abortion rates after PGS.10,130 Other studies were uncontrolled104 and/or did not assess miscarriages,110,129 and so cannot provide evidence here.

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N=122 100% With ≥3 90% previous 80% losses 70%

94%

89%

85%

60% 50% 40%

44%

39%

33%

30% 16%

20% 10% 0%

before PGD*

12%

8% <36

<36

total

p<0.05

p<0.001

p<0.001

observed after PGD**

expected after PGD*

Munné et al10 offered PGS to idiopathic RPL patients and compared pregnancy loss with that expected according to their previous number of miscarriages and maternal age, and according to prediction from the study by Brigham et al.131 These RPL patients had experienced an average of 3.9 previous pregnancies before the PGS cycle, of which 87% were lost. Based on the Brigham et al.131 formula, the RPL group expected losses of 36.5%; after PGS the observed loss rate was only 16.7% (p=0.028). In the ≥35 subgroup, the expected loss in the next pregnancy was 44.5% compared to an observed 12% (2/17) (p=0.007) after PGS. When the data of Munné et al10 was combined with newer unpublished in-house data, the results were even better, showing that PGS for idiopathic RPL can reduce miscarriage rates in all age groups (Fig 29.3). Recently, Garrisi et al130 have studied the effect of PGS reduction of spontaneous abortions in relation to previous miscarriages. Their study found that PGS significantly reduces miscarriage rates in patients with 3–5 previous miscarriages, but not significantly in patients with 2 or more than 5 (Table 29.11). This agrees with Ogasawara et al96 who showed that women with 5 or more miscarriages had more chromosomally normal miscarriages than those with 3–4. In addition, Garrisi et al130 analyzed the effect of PGS reduction in miscarriages in relation to the fertility status of the patient. They found that patients with RPL that became pregnant after IVF treatment had a significant reduction in miscarriages after PGS, from 41% to 17% (p<0.005) (Table 29.12). Overall, it is reasonable to conclude that RPL with idiopathic etiology in women of advanced maternal age is mostly a problem of recurrent chromosomally abnormal embryos. This is in agreement with previous observations in translocation carriers in which PGD of translocations significantly reduced spontaneous abortions.9,132 Guidelines that only take into consideration randomized clinical trials do not show belief that

Fig 29.3 Reduction in spontaneous abortions after PGS. ∗Munné et al,10 ∗∗Brigham et al.131

PGS can reduce spontaneous abortions.133 But the evidence at the time supported the concept that PGS can reduce spontaneous abortions and no other studies denied this, so PGD/S guidelines currently indicate that PGS should be offered for idiopathic RPL.134

PGS can reduce spontaneous abortions in patients with RPL caused by translocations The unbalanced products of a translocation are usually lethal and present a real risk of RPL to the patient. Among 1284 couples with recurrent miscarriage, 58 (4.5%) were carriers of translocations. In the next pregnancy the couples who were carriers of reciprocal translocations miscarried significantly more often (68%) than couples without structural abnormalities (28%) (n=1184) (p<0.001).135 It has been shown that PGD for patients with translocations substantially increase their chances of sustaining a pregnancy to full term.9,132 Data from another group showed that in 45 pregnancies, 18% spontaneously aborted, this was much lower than the 88% of pregnancy loss in these patients prior to undertaking PGD procedure (p<0.001).85,136 For most translocation patients, the risk of consecutive pregnancy loss is their major incentive for enrolling in a PGD program. Indeed, about 2.7% to 4.7% of RPL patients carry structural chromosome abnormalities.135,137–140 There is ample evidence that PGD of translocations substantially increases a couple’s chances of sustaining a pregnancy to full term, with couples with translocations showing on average 85% pregnancies lost before PGD, and 0–25% after PGD.9,10,85,132,136,141–144 In the last review of 471 PGD translocation patients, only 7% (9/129) pregnancy loss was observed after PGD141 (Table 29.13). A study specific of patients with RPL due to translocations and with no history of live births (0/117) showed that after PGD only 5.3% were lost.144 In addition, the cumulative pregnancy rate was 57.6% and a

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Table 29.11

393

PGS results according to previous number of miscarriages

Number of prev spontaneous abortions

Cycles

% Lost expected

% Lost after PGS

P

95 169 15

32% 41% 44%

26% 23% 47%

NS p<0.025 NS

2 3–5 >5 From.130

Table 29.12

PGS results according to fertility

Method of conception

Cycles

IVF Natural

56 113

% Lost expected

% Lost after PGS

p

% Deliveries

40% 41%

14% 17%

NS p<0.005

34% 38%

From.130

Table 29.13

Type Robertsonian Reciprocal

Outcome of PGS for translocations

Average age

Cycles

Number transferred

34.0 36.1

133 338

16 106

Not pregnant 67 153

Pregnant

Miscarried

Ongoing pregnancy

% Loss

% Ongoing

50 79

2 7

48 72

2% 2%

36.1% 21.3%

In Munné.141

cumulative ongoing pregnancy rate was 54.5% in the short period of time of an average of 1.24 IVF cycles. PGD results can be compared to an expected 26–68% pregnancy loss (p<0.001) for patients when they are not treated by PGD,135,137,140 with the advantage that time to conception is much faster with IVF–PGD than without it (Table 29.14). While patients can have a successful outcome without PGD, the psychological pain inflicted by repeated spontaneous abortions is considerable, so the shorter this period, the better.

Towards a full chromosome count Current FISH tests can effectively analyze up to 12 probes, which means that only about 67% (67) of known chromosome abnormalities in spontaneous abortions are detected. Tests that can score all 24 chromosome types in a time frame compatible with day-3–5 transfer are highly desirable. Several strategies have been suggested,145 including: (i) quantitative fluorescence multiplex PCR (QF-PCR),146,147 (ii) cell conversion,148,149 (iii) comparative genome hybridization (CGH),150–152 (iv) FISH or SKY with 24 probes,153–156 and (v) DNA microarrays.157

Of these techniques, only (iii) CGH has been applied clinically72–74,76 although (v) DNA microarrays still hold real potential for clinical application. Of the others, (i) QF-PCR has not been able to give results on single cell; (ii) cell conversion, which involves the fusion of cells to oocytes or polar bodies to convert them to metaphase stage, is not conducive for application in reference laboratories and requires highly skilled personnel; in (iv) the rate of conversion to obtain karyotypable or SKY-quality metaphases is too low; and the same problem applies to polar bodies fixed in different conditions.153,157

Comparative genomic hybridization (CGH) CGH is promising,150 as a DNA-based method capable of accurately determining total or partial aneuploidy by detecting losses or gains in all 46 chromosomes. The combination of a specific type of PCR which amplifies the entire genome (whole genome amplification) and FISH technology, enabled the application of CGH for the detailed investigation of oocytes, PBs and embryos.68–71,158,159 The technique has been successfully tested in human blastomeres and babies have been born after PGS using CGH.71–73,152,159

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Table 29.14

Ref.

Pregnancy outcome in translocation carriers after PGD or no-PGD treatment

Patients (cycles)

Patients conceiving a child (per cycle; cumulative)a

Risk of miscarriage

Time frame

14 (22% 33%) 71 (21% 30%) 17 (47% 59%)

17% (3/18)** 9% (7/79) 0% (0/17)

1.4 cycles 1.4 cycles 1.2 cycles

15 (32%, n/a) 32 (n/a, 68%) 25 (n/a, 74%) 18 (n/a, 64%)

68% (15/47) 65% (62/95) 26% (11/43) 38% (13/34)

11.5 months d 23.3 months c 6 years 4.2 yearsg

48 (36%,55%)

4% (2/50)

1.4 cycles

7 (n/a, 64%) 8 (n/a, 67%)

36% (4/11) 31% (4/13)

23.3 monthsh 4.2 yearsg

Reciprocal translocation with PGD 143 141 144

43 (64) 239 (338) 29 (36)

without PGD 135 170b 140e 137f

47 47 41 28

Robertsonian translocations with PGD 141

88 (133)

without PGD 170b 137f

11 12

a

First pregnancy after ascertainment of carrier status. Cumulative success rate. c As reported by Sugiura-Ogasawara et al.135 d Although Sugiura-Ogasawara et al135 do not mention it, if to achieve 95 pregnancies took 23.3 months, to achieve 47 is estimated to take about 11.5 months. e Data includes: 28 reciprocal translocations, 5 pericentric inversions, 4 paracentric inversions, 3 Robertsonian translocations, and 1 marker chromosome. Inversions and Robertsonian translocations produce less chromosomally abnormal pregnancies and therefore are more likely to succeed. f While Sugiura-Ogasawara et al.135 patients were not treated for RM, Stephenson and Sierra137 were treated for different conditions other than translocations, thus the lower miscarriage rate compared to Sugiura-Ogasawara et al.135 g Average age at start 29.8 years and 34 years at end, without differentiating reciprocal and Robertsonian. h As reported by Sugiura-Ogasawara et al135 for reciprocal translocations, and thus it could be different. ** Lim et al143 did not use appropriate methods of PGS for translocation, which, for interphase FISH analyses require the use of two proximal and one distal probe to the breakpoint or one proximal and two distal, in order to detect all possible unbalanced embryos; hence the high rate of spontaneous abortions in this study. b

But CGH is labor intensive and the technique requires five days to yield results; so, after biopsy, the embryos must be frozen and then thawed for replacement, bringing increased embryo loss which does not compensate for improved embryo selection potential.22,160,161 It has therefore mostly been abandoned. To avoid cryopreservation, CGH analysis of first polar bodies (PBs) was proposed and applied,74 but meiosis-II errors cannot be detected; some studies indicate that these account for half of the chromosome abnormalities;161 and the time required made its widespread application unrealistic. Recently, Sher et al76 used an improved method of oocyte and embryo freezing, termed vitrification,162,163 which produced >85% survival rates, and combined it with CGH for the analysis of first PBs. Even though the pregnancy rates reported in this study were acceptable, the methodology was not validated because the freeze-thaw

losses obviated the CGH benefits, as has been found in other investigations involving CGH examination of oocytes and PBs.68–70,164 Additionally, examination of first PBs means that, just as with the study by Wells et al,74 only errors affecting chromosomes during meiosis-I could be detected. However, an ongoing clinical trial involving the use of CGH for the analysis of both first and second PBs, in combination with vitrification of zygotes generated from poor prognosis IVF patients, has demonstrated that meiosis-II errors are actually more frequent than meiosis-I errors (55.3% vs 39.2%) (Fragouli and Wells, unpublished). This suggests that in order for CGH to significantly improve clinical outcome, the examination of both PBs is necessary. It therefore remains to be seen if CGH of PBs is capable of improving ART outcome more than the FISH technology currently used.

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Table 29.15

395

Embryos classified on day 3 by PGS that reach blastocyst stage

Ref

Average maternal age

Technique (chromosomes analyzed)

% Euploid reaching blastocyst

% Abnormal reaching blastocyst

Euploid

Abnormal

23 22 21 76

38.8 34.8 38.5 27.5

FISH (5) FISH (6) FISH (9) CGH (24)

63% (54/86) 29% (24/70) 68% (40/59) 93% (25/27)

20% (55/281) 19% (14/73) 16% (30/185) 21% (11/52)

n/a* 63% (24/38) 57% (40/70) 69% (25/36)

n/a* 37% (14/38) 43% (30/70) 31% (11/36)

59% (143/242)

19% (110/591)

62% (89/144)

38% (55/144)

Total

% Blastocyst that are

*Not all euploid replaced on day 5, thus % blastocyst cannot be calculated.

Interestingly, it seems that zygotes diagnosed as normal by CGH of both PBs reach blastocyst stage in much higher numbers than those selected for FISH (Table 29.15), thus indicating a better selection, since all chromosomes are analyzed with CGH. However, differences in maternal age in the studies (compared in Table 29.15) may also play a part.

DNA microarrays The use of DNA microarrays in PGS to determine chromosomal copy number has been proposed.145,156 However, this technology has only recently been successfully applied to single cells,165–167 although not yet clinically. Two types of DNA microarray platforms have been used for this purpose: array comparative genomic hybridization (array CGH) and single nucleotide polymorphism (SNP) arrays. Both approaches require a whole genome amplification (WGA) step prior to use with single cells. Copy number aberrations are detected in much the same way using array CGH as in conventional CGH. Namely, test and reference DNA are differentially labeled, co-precipitated, and co-hybridized onto a target representative of the genome. However, the targets used in a CGH are bacterial artificial chromosome (BAC) clones spotted onto the arrays, permitting a much higher degree of resolution than is possible by cytogenetic means. Using optical methods, the relative abundance of each probe is measured, intensity ratios are calculated, and chromosomal status is determined. SNP arrays are spotted with oligonucleotide probes targeting regions of copy number variation. Prior to hybridization onto the array, the test sample’s DNA is digested with a restriction enzyme and the resulting adaptor-ligated fragments are amplified. Thereafter, the amplified DNA is fragmented and labeled. Copynumber assessment of scanned images is performed using genotyping software. There are pros and cons to the use of each platform. For example, SNP chips are particularly susceptible to noise and bias due to their short target size. This drawback would be particularly pronounced in amplified single cell preparations. BAC CGH arrays are very robust, producing a high signalto-noise ratio, better reproducibility and lower standard

deviations. The use of FISH mapped BACs further improves the reliability and specificity of CGH arrays, which are preferable to the use of genomic databases and representations, as is the case with SNP arrays.

PGS versus non-invasive embryo selection techniques A wide variety of techniques are used to select human embryos for the highest implantation potential. The oldest methods are based on morphology assessment20 and have been studied extensively. But chromosome abnormalities bear little relation to morphology,11,14,19 viz. the best morphology embryos in women aged 35–37 and older are 56% chromosomally abnormal compared to 75% for those that arrest.103 In these studies poor morphology is often linked to mosaicism and other postmeiotic abnormalities, but not to aneuploidy. Another selection technique that can screen for some chromosome abnormalities is blastocyst morphology; however, Table 29.15 shows that 38% of all blastocysts are still aneuploid in older patients. A recent book edited by Elder and Cohen24 reviews other selection techniques such as metabolomics; however, as with blastocyst selection, embryos implanted can still miscarry in high numbers or carry chromosome abnormalities (Table 29.15).

Conclusion A sizable fraction of embryos produced during ART procedures are chromosomally abnormal. Current techniques based on morphology and developmental assessment do not screen for the majority of these chromosome abnormalities; thus PGS for infertility selects chromosomally normal embryos for replacement, expecting an improvement of implantation, a reduction in spontaneous abortions and trisomic offspring, and an improvement in so-called take-home babies. However, differences in techniques have produced conflicting results. Some techniques seem to be better than others and can be summarized as: (1) appropriate maternal age, (2) minimum number of zygotes and embryos to be biopsied, (3) a low rate of undiagnosed

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embryos, (4) a single cell biopsied, (5) appropriate fixation method, (6) the error rate should be <10% with all embryos reanalyzed except those replaced, (7) extensive and positive experience with PGS, (8) the analysis of at least chromosomes X, Y, 13, 15, 16, 18, 21, and 22 with NRR where appropriate. Under these conditions PGS seems to produce positive ART outcomes. However, no fully randomized study has been performed to date under these conditions. For a fraction of patients choosing PGS, high takehome baby rates is not the major goal; we see that 83% of patients wish to prevent trisomic conceptions as long as pregnancy rates do not decrease; while 36% would choose it even if there were a reduction in pregnancy rates.86 Also, patients with idiopathic RPL or previous miscarriages are reluctant to experience the trauma again and choose PGS to avoid pregnancy loss if at all possible, irrespective of potentially lower pregnancy rates. Techniques analyzing all chromosomes have the promise of furthering the selection potential of the technique while minimizing error rates.

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139. Carp HJA, Dirnfeld M, Dor J, Grudzinskas JG. ART in recurrent miscarriage: preimplantation genetic diagnosis/screening or surrogacy? Hum Reprod 2004; 19: 1502–5. 140. Goddijn M, Joosten JHK, Knegt AC, et al. Clinical relevance of diagnosing structural chromosome abnormalities in couples with repeated miscarriage. Hum Reprod 2004; 19: 1013–17. 141. Munné S. Preimplantation genetic diagnosis for translocations. Hum Reprod 2006; 21: 839–40. 142. Gianaroli L, Magli C, Fiorentino F, et al. Clinical value of preimplantiation genetic diagnosis. Placenta 2003; 24: S77–83. 143. Lim CK, Jum JH, Min DM, et al. Efficacy and clinical outcome of preimplantation genetic diagnosis using FISH for couples of reciprocal and Robertsonian translocations: the Korean experience. Prenat Diagn 2004; 24: 556–61. 144. Otani T, Roche M, Mizuikea M, et al. Preimplantation genetic diagnosis significantly improves the pregnancy outcome of translocation carriers with a history of recurrent miscarriage and failing to produce a live birth. Reprod Biomed Online 2006; 13: 879–94. 145. Harper JC, Wells D. Recent advances and future developments in PGS. Prenat Diagn 1999; 19: 1193–9. 146. Mansfield ES. Diagnosis of Down syndrome and other aneuploidies using quantitative polymerase chain reaction and small tandem repeat polymorphisms. Hum Mol Genet 1993; 2: 43–50. 147. Sherlock J, Cirigliano V, Petrou M, et al. Assessment of diagnostic quantitative fluorescent multiplex polymerase chain reaction assays performed on single cells. Ann Hum Genet 1998; 62: 9–23. 148. Verlinsky Y, Evsikov S. Karyotyping of human oocytes by chromosomal analysis of the second polar body. Molec Hum Reprod 1999; 5: 89–95. 149. Willadsen S, Levron J, Munné S, et al. Rapid visu-

alization of metaphase chromosomes in single human blastomeres after fusion with in-vitro matured bovine eggs. Hum Reprod 1999; 2: 470–5. 150. Kallioniemi A, Kallioniemi OP, Sudar D, et al. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 1992; 258: 818–21. 151. Wells D, Sherlock JK, Handyside AH, Delhanty DA. Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genome hybridization. Nucleic Acid Res 1999; 27: 1214–18. 152. Voullaire L, Wilton L, Slater H, Williamson R. Detection of aneuploidy in single cells using comparative genome hybridization. Prenat Diagn 1999; 19: 846–51. 153. Márquez C, Cohen J, Munné S. Chromosome identification on human oocytes and polar bodies by spectral karyotyping. Cytogenet Cell Genet 1998; 81: 254–8. 154. Sandalinas M, Márquez M, Munné S. Spectral karyotyping of unfertilized and non-inseminated oocytes. Molec Hum Reprod 2002; 8: 580–5. 155. Fung J, Weier HUG, Goldberg JD, Pedersen RA. Multilocus genetic analysis of single interphase cells by spectral imaging. Hum Genet 2000; 107: 615–22. 156. Weier HUG, Munné S, Lersch RA, et al. Towards a full karyotype screening of interphase cells: ‘FISH

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157.

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and chip’ technology. Molec Cell Endocrinol 2001; 183: S41–5 Pinkel D, Landegent J, Collins C, et al. Fluorescence in-situ hybridization with human chromosomespecific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc Natl Acad Sci USA 1988; 85: 9138–42. Fragouli E, Wells D, Whalley KM, et al. Increased susceptibility to maternal aneuploidy demonstrated by comparative genomic hybridization analysis of human M-II oocytes and first polar bodies. Cytogenet Genome Res 2006; 114: 30–8. Wells D, Delhanty JDA. Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Mol Hum Reprod 2000; 6: 1055–62. Munné S, Wells D. Questions concerning the suitability of comparative genome hybridization for preimplantation genetic diagnosis. Fertil Steril 2003; 80: 871–2. Verlinsky Y, Kuliev A. Preimplantation diagnosis for aneuploidies using fluorescence in-situ hybridization or comparative genomic hybridization. Fertil Steril 2003; 80: 869–70. Yoon TK, Chung HM, Lim JM, et al. Pregnancy and delivery of healthy infants developed from vitrified oocytes in a stimulated in-vitro fertilization embryo transfer program. Fertil Steril 2000; 74: 180–1. Yoon TK, Lee DR, Cha KY, et al. Survival rate of human oocytes and pregnancy outcome after vitrification using slush nitrogen in assisted reproductive technologies. Fertil Steril 2007; 88: 952–6.

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164. Fragouli E, Delhanty JD, Wells D. Single cell diagnosis using comparative genomic hybridization after preliminary DNA amplification still needs more tweaking: too many miscalls. Fertil Steril 2007; 88: 247–8. 165. Kearns WG, Pen R, Benner A, et al. Comprehensive genetic analyses using a modified whole genome amplification protocol and microarrays to identify genetic disorders and determine embryo implantation from single cells. Fertil Steril 2007; 88(Supp. l): S236–7. 166. Treff NR, Su J, Mavrianos J, et al. Accurate 23 chromosome aneuploidy screening in human blastomeres using single nucleotide polymorphism (SNP) microarrays. Fertil Steril 2007; 88(Suppl. 1): S1. 167. Munné S, Steuerwald NM, Wells D, et al. Comprehensive aneuploidy screening in single cells using microarray comparative genomic hybridization methods implications for preimplantation genetic diagnosis Fertil Steril 2007; 88(Suppl. 1): S86. 168. Li M, Marin DeUgarte C, Surrey M, et al. Fluorescence in-situ hybridization reanalysis of day-6 human blastocysts diagnosed with aneuploidy on day 3. Fertil Steril 2005; 84: 1395–400. 169. Munné S, Sandalinas M, Escudero T, et al. Chromosome mosaicism in cleavage stage human embryos: evidence of a maternal age effect. Reprod Biomed Online 2002; 4: 223–32. 170. Sugiura-Ogasawara M, Suzumori K. Can preimplantation genetic diagnosis improve success rates in recurrent aborters with translocations? Human Reprod 2004; 19: 2171–2.

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30 Genetic analysis of the embryo Yuval Yaron, Veronica Gold, Ronni Gamzu, Mira Malcov

Introduction For couples at risk of transmitting a genetic disease, preimplantation genetic diagnosis (PGD) and transfer of disease-free embryos offers an alternative to prenatal diagnosis by chorionic villous sampling (CVS) or amniocentesis, followed by therapeutic abortion of affected fetuses. Molecular PGD was initially employed for embryo sexing in couples at risk for X-linked diseases. The technique used polymerase chain reaction (PCR) to amplify Y chromosome-specific sequences, and only embryos determined to be females were transferred.1 During the last two decades, the range of genetic abnormalities that can be detected by PGD has dramatically increased. Virtually all genetic disorders for which the mutation has been detected are amenable to PGD. Moreover, it is now possible to perform combined PGD and human leukocyte antigen (HLA) typing. This may prove beneficial in cases where a child is affected with a genetic disease amenable to bone marrow transplantation. In this approach, future siblings may be not only free of the disease but may also be suitable donors for the affected sibling. This approach has been successfully employed for Fanconi’s anemia.2 However, the use of PGD for HLA typing, particularly in the absence of a genetic disease, and its use in screening embryos for susceptibility to cancer and late-onset diseases as well as for gender selection raise important ethical concerns. Despite its promise, PGD is still limited by technical difficulties due to the minute amount of genetic material, and the inherent pitfalls of the PCR, such as amplification failure, allele dropout (ADO), and foreign DNA contamination. There is also a rather narrow window of opportunity to perform diagnosis within hours to enable embryo transfer without jeopardizing pregnancy rates. This chapter reviews the various aspects of the genetic analysis of preimplantation embryos. Chromosomal analysis of the embryo is discussed in a separate chapter.

Basic principles of preimplantation genetic diagnosis Polymerase chain reaction Single-cell molecular analysis for PGD was made possible by the polymerase chain reaction, first introduced in

the mid-1980s. The technique enriches a DNA sample for one specific oligonucleotide fragment called the PCR product or amplicon. The technique uses a pair of short oligonucleotide fragments, called primers, that are homologous to stretches of genomic DNA at a locus of interest. The PCR thermocycler is programmed to perform successive cycles consisting of denaturation, at temperatures > 90°C, during which the double-stranded template DNA melts into two separate single strands: annealing, in which the primers attach to their region of homology; and extension, during which new nucleotides (dNTPs) are added in succession to recreate a doublestranded DNA molecule by the enzymatic action of the thermostable Taq polymerase. The resulting new strands serve as templates for the subsequent cycles. After 30–40 such cycles, the initial minute quantity of DNA is amplified to the extent that it can actually be visualized by methods such as radioactive labeling, ethidium bromide, or silver staining. The PCR products may further be subjected to a variety of analytic techniques that determine the presence of point mutations, small deletions, or insertions, or for analysis of linked polymorphic genetic markers. Finally, the precise composition of the amplified fragment may be studied by direct sequencing. The number of cycles that may be performed in standard PCR is limited by a gradual decline in amplification efficiency with each subsequent cycle. This is partly due to the decrease in the activity of the Taq polymerase over time. Another reason is the ‘fraying’ of the amplicon edges by the exonuclease activity of Taq polymerase. This causes the amplicons to become unsuitable templates for further amplification because their primer annealing sites become eroded. As a result of these limitations, when the number of initial DNA template molecules is limited, as in a singlecell PGD analysis, the quantity of amplified DNA may be insufficient for a complete molecular analysis. The twostep, nested-primer PCR approach offers a solution to this problem, by allowing sufficient amplification of even a single DNA copy. The method employs a first pair of outer primers, designed to amplify the region of interest in the primary PCR reaction. The PCR product of the primary PCR reaction is then further amplified using a second set of inner or nested primers. The use of nested primers that are proximal to the annealing site of the outer primers increases amplification efficiency

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since the nested primers anneal to sites that have not been eroded. This technique also decreases the rate of nonspecific amplification.

Pitfalls of PCR in PGD The precise diagnosis by PCR relies on several key elements: adequately functioning reagents such as primers, dNTPs, and Taq polymerase; the presence of an adequate tested DNA template; and the lack of any DNA contamination. Perturbations in any of these elements may lead to misdiagnosis. In particular, PCR for PGD has three potential pitfalls: amplification failure, allele dropout (ADO), and contamination.

Amplification failure Amplification by PCR is unsuccessful in approximately 10% of isolated blastomeres, regardless of their genotype. The main reasons for amplification failure include biopsy technique, premature cell lysis, lysis protocol used, and PCR conditions.3,4 There appears to be an association between embryo or blastomere morphology and the success rate of PCR amplification. Cells that appear to be anucleate and those derived from arrested or fragmented embryos have a low amplification efficiency.5,6 In such cells, the DNA may be degraded or entirely absent. Adequate positive and negative controls must be used, to establish and fine-tune the PCR protocol and to ensure the integrity of the results. This is of particular importance in cases where the diagnosis is based on detection of deletions, such as in Duchenne muscular dystrophy (DMD). When in such cases an allele is not amplified, one must be certain that this is indeed due to a deletion and not secondary to amplification failure.

Allele dropout Allele dropout occurs when only one of the two alleles present in a cell is amplified to a detectable level. ADO is equally likely to affect either of the alleles in a heterozygous cell and thus it is not possible to predict which allele will be ‘dropped-out’ in a given reaction. The most significant implication of ADO is misdiagnosis of heterozygous embryos, particularly in PGD of dominant disorders. In such cases, the absence of the mutated allele due to ADO may result in misdiagnosis of an affected fetus as a normal one. Likewise, ADO may be responsible for misdiagnosis of recessive disorders in affected compound heterozygotes, where if only one of the mutations is detected, the embryo may be mistaken for a heterozygote.7 The reported frequency of ADO varies widely. In most experiments the rate of ADO is reported to be 5–20%, although, in some cases ADO has been shown to affect over 30% of single-cell amplifications6,8–12 or none of the cells.13 The causes of ADO are still not fully understood. Current hypotheses include inaccessibility of the DNA template due to imperfect denaturing temperature or

incomplete cell lysis and DNA degradation prior to PCR. Ray and Handyside9 demonstrated that an increase in denaturing temperature from 90oC to 96oC during PCR may be associated with a four-fold reduction in ADO at the cystic fibrosis locus and an 11-fold reduction at the β-globin locus. The use of alkaline lysis buffer or lysis buffer containing proteinase K and detergent has also been suggested to reduce ADO.9,14 Degenerated and apoptotic cells show increased ADO probably due to partial degradation of the DNA strands. It has been suggested that ADO is higher in blastomeres than in other cell types.11 This may be explained, at least partly, by the higher rate of haploidity of blastomeres.15 In cases of diagnosis of dominant disorders or recessive diseases when the parents do not carry the same mutation, measures should be taken to avoid or reduce the risk of ADO. A number of PGD protocols have been suggested that achieve this goal, most based on advanced techniques such as multiplex PCR, quantitative fluorescent (QF) PCR, reverse transcription (RT) PCR and others, as will be described in the following sections. Other less sensitive detection methods may ‘overlook’ the minimally amplified allele, resulting in ADO.8,12,16 The significant frequency of ADO resulting in misdiagnosis has led many PGD centers to use two cells from each embryo for genetic analysis.

Contamination Contamination is one of the greatest obstacles to the analysis of specific genes in single cells.17 In the setting of PGD, there may be three main sources for possible contamination. First, paternal genome contamination may arise from the fact that many spermatozoa are still embedded in the zona pellucida after in vitro fertilization (IVF), and may thus be mistakenly sampled with the blastomere, second polar body, or trophoectoderm cells during embryo biopsy. Intracytoplasmic sperm injection (ICSI) using a single sperm that is injected into the oocyte completely abolishes this possibility. Accordingly, most PGD units are now routinely using ICSI for all PGD cases in which diagnosis relies on PCR. The second source of possible contamination may arise from maternal cumulus cells adherent to the oocytes. Stripping of the cumulus cells from the zona pellucida is performed mechanically and/or by enzymes to reduce this risk. Finally, external contamination either from laboratory technicians or from PCR products generated during previous experiments is yet another source of contamination. The risk of external contamination is influenced by the number of PCR cycles required for sufficient amplification of the DNA. Thus, with a starting template of only one genome, the risk of contamination with exogenous DNA sequences is a particularly concerning problem that must be avoided by the use of adequate safety measures, as will be described below.

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Advanced molecular methods for preimplantation genetic diagnosis Multiplex PCR Multiplex PCR refers to the simultaneous amplification of more than one fragment in the same PCR reaction using more than one pair of unrelated primers.8,11,12,18 One or more primer pairs amplify the DNA fragment containing the locus to be tested, while the other(s) serve as a positive control within the same reaction. Amplification of multiple loci within the same multiplex PCR reaction is possible in single blastomeres. This requires careful primer design and reaction optimization to ensure that all primers sets amplify efficiently under the same conditions, including annealing temperatures and concentrations of the different reagents in the PCR buffer, such as MgCl2. Careful design of primers is mandated in order to avoid primer-dimer formation, interaction between different PCR products, and interaction of primers with products. The primers should be designed such that the product of each PCR primer pair is of a different size so that it may be distinguished by gel electrophoresis. Alternatively, different fluorescent tags can be used for each primer pair. Successful multiplex PCR reactions enable simultaneous assessment of numerous loci.18 Multiplex-PCR reaction may include assays for specific gene defects, unique sequences of specific chromosomes, and linked-informative polymorphic markers. This allows both the analysis of the disease mutation, assessment of aneuploidy, as well as reduction in the risk of contamination and ADO.10,11,19–22 This strategy is particularly useful for the PGD of dominant disorders, in which one primer set amplifies the region of mutation, while the other amplifies a polymorphic marker that is linked with the tested gene.20,23 The probability of ADO affecting both mutation site and the linked polymorphic site is very low and thus the mutant allele is more likely to be detected.

Fluorescent PCR The PCR products are commonly separated by gel electrophoresis, and their migration depends chiefly on their size. The standard visualization techniques include radioactive labeling, ethidium bromide, or silver staining. These techniques are rather insensitive, requiring a relatively large amount of DNA. Moreover, they cannot distinguish between products of a relatively similar size or provide an adequate estimate of quantity. Fluorescent PCR employs primers tagged with a fluorescent dye, which label the resulting amplicons, enabling detection by fluorescence-based DNA sequencers using a module such as GeneScan. A laser beam scans the acrylamide gel as the fluorescent products pass across the laser path by means of electrophoresis. The different fluorescent dyes absorb the light at a particular wavelength and emit fluorescence at a different wavelength. The emitted light passes through a filter, is digitally amplified, and

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analyzed by a computer. With this technique, it is possible to separate, detect, and analyze the fluorescentlabeled PCR products with sensitivity 1000 times greater than that achieved using conventional methods.24 This method also has a higher fragment-size resolution and is able to distinguish between products having a size difference of even 1–2 bp. Thus, several primer sets can be multiplexed even if their product sizes only vary slightly. This approach significantly reduces the likelihood of ADO resulting from preferential amplification, since even minimally amplified alleles are detected.8,12,16 In addition, since the detection efficiency is several magnitudes higher, fewer PCR cycles are required, thereby reducing the risk of contamination. Moreover, since fewer cycles are needed, less time is required for the complete analysis. Using this approach, Sermon et al16 have successfully reduced the rate of ADO by a factor of four in the diagnosis of myotonic dystrophy, and Findlay et al8 reported an accurate diagnosis in as much as 97% of the cases.

Quantitative fluorescent PCR Quantitative fluorescent (QF) PCR provides information on the ploidity of the cell.25 It amplifies specific DNA sequences unique for each chromosome, such as short tandem repeat (STR) markers which are composed of a varying number of nucleotide repeats (2–5 bp) and are highly polymorphic. Normal individuals are usually heterozygous for such polymorphic markers, i.e. have a different number of repeats, and therefore have differentsized alleles. During the initial exponential phase of PCR amplification, the amount of DNA product is proportional to the original number of repeats.26 Disomic individuals thus produce different-sized alleles with a ratio of 1:1, whereas trisomic DNA samples produce either three alleles of different lengths at a ratio of 1:1:1 (trisomic triallelic), or two alleles of the same size at a ratio of 2:1 (trisomic diallelic).25 This method has been successfully used in prenatal diagnosis of aneuploidy.27 In PGD however, QF-PCR is only reliable in identifying triallelic trisomies, since the interpretation of diallelic trisomies is problematic due to the possibility of preferential amplification.12

Whole genome amplification The most significant limitation of single-cell analysis is the small amount of DNA. As mentioned previously, multiplex PCR is one way to overcome this problem. In addition, methods designed to achieve nonspecific amplification of the entire genome, i.e. whole genome amplification (WGA), have been developed.28,29 These techniques amplify a large proportion of the entire genome, thereby allowing further analyses by specific PCR reactions, allowing confirmation of diagnosis by alternative methods or the analysis of other genes. There are several WGA techniques.

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Primer extension preamplification PCR Primer extension preamplification (PEP) is a WGA method designed mainly for single cells. Using randomsequence primers of 15 bp it has been claimed to amplify at least 70% of the genome in more than 30 copies.28 This however, is likely to be a rather conservative estimate since Paunio et al30 reported that PEP yields at least 1000 copies of the genome, and Wells et al29 have suggested that more than 90% of genomic sequences are represented in PEP amplification products. One of the drawbacks of PEP is the time required, which is usually more than 12 hours. Sermon et al31 have successfully adopted a modified protocol that requires less than 6 hours, and Tsai32 has improved the efficiency by further technical modifications. Several autosomal recessive, dominant, and X-linked disorders have been successfully detected in single cells using PEP, including Tay–Sachs disease, cystic fibrosis, hemophilia A, Duchenne muscular dystrophy (DMD), and familial adenomatous polyposis coli (FAP).10,33,34

Degenerate oligonucleotide primed PCR A second form of WGA, called degenerate oligonucleotide primed PCR (DOP-PCR), has been recently applied to PGD.29,35 DOP-PCR utilizes a universal primer with a 6 bp degenerated region of all possible nucleotide combinations, flanked with a GC-rich sequence to improve the hybridization. It amplifies a similar proportion of the genome as does PEP, but to a greater extent, providing sufficient DNA for over 100 subsequent PCR amplifications,29 or for other analytical procedures such as comparative genomic hybridization (CGH). It has been shown that using a combination of DOP-PCR, CGH, and QF-PCR, it is possible to determine the copy number of each chromosome and conduct various molecular studies on single cells and blastomeres.29,36

The Φ29 enzyme has been widely preferred over Bst DNA polymerase due to its superior sequence fidelity38,43 and its higher processivity (number of nucleotides incorporated per single DNA polymerase/DNA-binding event), the highest one described for a DNA polymerase.37,44,45 This attribute explains its low amplification bias (less than three-fold) compared with DOP and PEP-PCR methods (102- and 106-fold).37 Hellani et al46 and Handyside et al47 published the first successful reports of single-cell MDA from lymphocytes and blastomeres with further analysis of array CGH and nested PCR for 20 different loci, respectively. Despite these advantages, ADO is not completely eliminated, with ADO rates of 10–31%, not different from those of other single-cell PCR techniques.43 In the setting of PGD, MDA has several significant advantages. It obviates the need to set up unique singlecell protocols, such as that following MDA, and secondround PCR may employ standard PCR protocols commonly used in molecular laboratories. In addition, the large quantity of DNA uniformly representing the entire genome allows subsequent analyses of a variety of other loci,43 both for diagnosis as well as research. In 2006 Renwick et al coined the term preimplantation genetic haplotyping (PGH), which utilizes MDA with subsequent multiplex PCR of a fixed set of numerous diseaseassociated polymorphic markers. This facilitates determination of the high-risk haplotype by linkage analysis using a single protocol for each disease, without the need to establish a specific protocol for each different mutation.48 Although the same test can be applied to several couples without considering or even identifying the mutation they carry, it has several limitations. It requires additional informative family members to determine phase (i.e. to determine which is the mutation-associated parental haplotype). It requires the use of several informative disease-linked markers. Additionally, the occurrence of recombination events could lead to misdiagnosis.43

Multiple displacement amplification

Polymorphic markers

Multiple displacement amplification (MDA) is a recently developed nonPCR-based method that has been utilized for clinical samples with limited DNA content, providing high yield of relatively long fragments (>10 kb) with uniform and reliable representation across the genome.37 In MDA (Fig 30.1), annealing of exonuclease-resistant random hexamers to DNA template is followed by stranddisplacement DNA synthesis at a constant temperature of 30°C, without the need of prior DNA denaturation.37,38 The strand-displacing mechanism is accomplished by the Φ29 DNA polymerase37 or the Bacillus stearothermophilus (Bst) DNA polymerase large fragment.39 This mechanism allows increasingrandom priming events that form a network of hyperbranched DNA structures which generate thousands of copies of the original DNA in only a few hours.39,40 It appears that MDA is more advantageous, owing to decreased rates of unspecific amplification artifacts,41 incomplete coverage of loci,30 strong amplification bias,37 and short length of the DNA products.42

Multiplex PCR and WGA enable both the analysis of the tested gene for mutation as well as the analysis of polymorphic genetic markers such as STRs, also known as microsatellites, in a process referred to as ‘DNA fingerprinting.’ This technique is useful for ruling out contamination from various sources described earlier, and thus improves reliability of the diagnosis. The amplification of one or more highly polymorphic STRs allows the determination of the source of DNA amplified.49 As mentioned previously, polymorphic STRs consist of a varying number of repeats of a 2–5 bp motif, present in introns throughout the genome. At each informative STR locus, each parent has two alleles of varying repeat number, resulting in two amplicons of different lengths in each individual. The resulting embryo will have inherited only one allele from each parent. Any deviation from the expected inheritance of one allele from each parent is indicative of contamination – maternal, paternal, or external.8,12,49

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Fig 30.1 MDA: the genomic target DNA is reacted with random hexamer primers, Φ29 DNA polymerase and dNTPs at a constant temperature of 30°C (1). The priming occurs at multiple sites on the DNA (2) – only one strand is shown. The Φ29 polymerase extends the annealed primers simultaneously (3). When the enzyme meets newly synthesized double-stranded DNA it displaces the strand and continues replicating (4), while additional hexamer primers bind to displaced strands, and a hyperbranched structure is formed (5). This cascade of exponential amplification generates large quantities of ~10kb DNA products from the genomic input DNA.

Polymorphic STRs can also be used in the actual diagnosis when the exact mutation causing the disease is unknown. In such cases, polymorphic markers in close proximity or within the disease locus are used to evaluate whether the embryo has inherited the affected allele. Intragenic markers and tightly linked ones are preferred, as they are unlikely to be separated from the mutation by recombination during meiosis. In order to perform such linkage analysis, the parents and both healthy and affected sibs are analyzed to determine which polymorphic marker is inherited along with the disease. Such a strategy has been used for the diagnosis of Marfan syndrome, the first autosomal dominant disorder to be tested by PGD50 and DMD. In the latter, only 60% of DMD patients exhibit detectable large-scale deletions in the

dystrophin gene. Since it is the largest known human gene, spanning more than 2 million base pairs, it is often impossible to detect small deletions or point mutations.51 Linkage analysis has also been suggested for the diagnosis of disease with large trinucleotide repeat expansions, such as fragile X and myotonic dystrophy.16,52 Single-cell analysis of the expanded portion of the disease gene itself often leads to misdiagnosis due to problems in amplifying the extremely large repeats.53

Mutation analysis All the above-mentioned PCR techniques amplify the DNA of a single cell to a detectable level. In disorders caused by large-scale deletions, such as DMD or

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spinal muscular atrophy (SMA), the actual PCR amplification reaction is sufficient for making a diagnosis since it is based on the lack of amplification of the corresponding deleted portion of the gene. In other disorders caused by trinucleotide expansion, such as fragile X or myotonic dystrophy, the disease allele is significantly larger than the normal one, and amplicon size may also be diagnostic. More commonly, however, the amplified fragment harboring the mutation is indistinguishable from the normal one using the standard visualization methods such as gel electrophoresis. In such cases, further analysis of the amplified fragment is required for mutation detection. Whenever the targeted mutation is precisely known, specific methods can be devised for the detection of the particular mutation. This is preferred to scanning methods that are used to search for mutations that have not been characterized. Scanning methods include heteroduplex analysis, single-strand conformational polymorphism (SSCP), denaturant gradient gel electrophoresis (DGGE), and others. These methods are based on the fact that the normal DNA strands, mutant DNA strands, and various combinations thereof often have varying electrophoretic migratory properties under different conditions, allowing distinction between them. These techniques often assist in scanning for a mutation in diseases that are caused by numerous different mutations. Whereas PGD using these techniques has been reported in conditions such as β-thalassemia,54–56 it is preferred to limit their use to initial mutation screen in the affected family members. Once the specific fragment of the gene harboring the mutation has been detected by these methods, further analysis is mandated using direct sequencing. The latter provides bona fide evidence of the mutation, and also facilitates the development of direct diagnostic techniques such as restriction endonuclease (RE) digestion of DNA or the amplification refractory mutation system (ARMS).

Restriction endonuclease digestion Alteration in the DNA sequence caused by mutations may often lead to creation or abolition of specific restriction endonucleases (RE) recognition sites. These bacterially derived enzymes recognize specific DNA sequences and cleave the DNA strand at or near to the recognition site. When the precise mutation is known, a restriction enzyme may be selected which differentially cleaves the normal DNA strand but not the mutant one, or vice versa. Following electrophoresis, it is possible to distinguish the digested from the non-digested products and thereby detect the presence or absence of the mutation. Many mutations alter the recognition site of at least one of the many possible, commercially available, restriction enzymes. As an example, the ZFX and ZFY genes located on the X and Y chromosome, respectively, can be distinguished according to difference in the size of the fragments

produced by the restriction enzyme HaeIII. This allows sex determination to be performed more accurately than based on the presence or lack of amplification of the Y chromosome-specific SRY gene.

Amplification refractory mutation system The amplification refractory mutation system (ARMS) employs three primers in the PCR reaction: a common primer, which anneals upstream of the mutation site; and two other primers, which differ slightly, each specific for either the normal or mutant alleles. The sitepecific primers may be designed to vary in length, to contain a restriction site, or to be tagged by different fluorescent markers.12 Any of these methods would facilitate the distinction of amplicons produced by either the normal or mutant allele. Since this test results in selective amplification of both the mutant and normal alleles, it is considered to be a safer method than the detection of the mutant allele alone. Using this technique in the multiplex PCR approach, it is possible to identify several different mutations, such as for cystic fibrosis, in a single-cell PCR reaction.57

Mini-sequencing Mini-sequencing (SnaPshot) permits analysis of very small DNA fragments amplified by PCR, based on primer extension. It has been suggested that smaller amplicons have lower ADO rates. This would potentially improve the reliability of PGD without the need for extensive optimization for individual mutations. Bermudez et al report single-cell protocols for the diagnoses of cystic fibrosis, sickle cell anemia, and βthalassemia using this technique.58

DNA microarray technology DNA microarrays or ‘chips’ allow the simultaneous detection of up to thousands of different polymorphisms or mutations in defined genes. Numerous oligonucleotide probes (usually 20–25 bp) are arrayed in microscopic predefined regions on a solid surface such as a thumbnail-sized glass slide. The probes are complimentary to known mutations in defined genes or single nucleotide polymorphisms (SNPs) throughout the genome. The microarray is hybridized with a fluorescentlabeled tested DNA and the fluorescent signal is detected and digitally analyzed. Hybridization is indicative of a match between the tested DNA and the specific oligonucleotide probe. For each possible mutation, several slightly varied probes may be used to increase sensitivity.

Array CGH Aneuploidy, chromosome number imbalance, represents a major cause of spontaneous abortions.59–61 Fluorescence in situ hybridization (FISH) for detection of aneuploidy is discussed elsewhere in this textbook.

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1

2

3

Log2 fluorescence ratio

4

5

Chromosomes

Fig 30.2 Array CGH: test and reference genomes (1) are fluorescently labeled with Cy5 (red) and Cy3 (green), respectively (2). The labeled DNAs are simultaneously hybridized to mapped DNA fragments with whole genome representation (BAC clones, cDNA library or oligonucleotides) that were previously immobilized on a microarray chip (3). The resulting fluorescence intensities are measured by a computerized microarray scanner (4). In cases of copy number losses only hybridization by the reference DNA will be achieved, resulting in the detection of the green signal. In cases of copy number gains, the amplified (test) DNA region will strongly compete for the target DNA specific region, resulting in a predominantly red signal. The scanned images are subsequently analyzed by specialized software (5), which accurately calculate the ratio of test to reference copy numbers for each chromosome (aCGH profile). The images obtained are converted into graphics where deviation from the low and high predefined thresholds represents the deletions or duplications, respectively, for each chromosome.

The major drawback of FISH, however, lies in the fact that only a limited set of chromosomes can be analyzed in a single cell, usually 5–10. Comparative genomic hybridization (CGH) following whole genome amplification (WGA) is an alternative to interphase FISH62 that may be used to screen for all aneuploidies in single blastomeres63–65 and polar bodies.66,67 In CGH, test and reference DNA samples are labeled with two different fluorochromes and co-hybridized to normal human metaphase spread on a microscope slide.68,69 A computerized imaging system calculates the fluorescence ratio for each fluorochrome at each chromosomal locus. Deviation from a 1:1 ratio indicates a change in DNA copy number (i.e. deletion, duplication, trisomy, etc.).69 Array CGH (Fig 30.2) is a simpler more uniform technique that employs selected genomic regions

printed onto a solid surface as hybridization probes. This eliminates the use of metaphase spreads that are nonuniform, and enables higher resolution, depending on the number, density, and size of the genomic probes printed on the array.68–70 Le Caignec et al68 detected chromosomal imbalances from single lymphoblasts, fibroblasts, and blastomeres by array CGH following WGA by MDA. This approach may be preferable to array CGH following DOP-PCR that was reported by Hu et al.71 Array CGH has the potential to become an important method for aneuploidy diagnosis and screening in the setting of PGD, to a greater extent than standard FISH, allowing a larger number of abnormalities to be detected.68 It has been suggested that full genome aneuploidy screening for embryo selection would enhance implantation rates.68

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Laboratory techniques in preimplantation genetic diagnosis Preimplantation genetic diagnosis at the single-cell level is a multistep complex procedure. The various pitfalls outlined previously necessitate adequate calibration of the techniques employed to avoid misdiagnosis. Owing to ethical limitations, single human blastomeres are difficult to obtain; therefore, different PGD centers have developed different protocols, and there is as yet no uniform method. Because of the numerous genetic disorders amenable to PGD, it is impossible to provide suitable protocols for all. Instead, some of the commonly used laboratory methods are described in the following section.

General safety measures It is highly recommended that a physically separated site be used for template preparation, PCR assembly, and product analysis. Equipment and reagents used for single-cell PCR should be solely reserved for this purpose and should never be allowed to come into contact with previously amplified DNA samples. To avoid contamination, laboratory technicians should wear disposable outer clothing, caps, masks, shoe covers, and powder free gloves, which are kept in the room. In order to avoid external contamination from previously amplified DNA, some centers use a room kept under constant positive pressure. All equipment and required disposable supplies such as tubes, racks, and pipettes are to be kept in the room. Glassware should be sterilized and aerosol resistant pipette tips should be used. All reagents and solutions should be DNA-free, sterilized by autoclaving and filtered through a 0.22 µm filter or by ultraviolet (UV) irradiation. All reagents should be prepared in a fume hood equipped with UV light. These safety measures, however, should not be considered a substitute for efforts to avoid the possibility of external contamination occurring in the first place. The PCR reagents should be rigorously tested prior to any clinical case to ensure that they have not become contaminated. It is recommended that all PCR reagents (minus Taq polymerase) be prepared in excess and aliquoted to reduce the number of pipettings and samplings from the stock preparation. Sample aliquots may then be tested while the remainder is frozen until use. To detect contamination in the analyzed sample, a negative control should be used consisting of all PCR reagents, substituting the template DNA or blastomere with an aliquot of the final blastomere wash buffer. To eliminate contamination by sperm, ICSI is employed.

Choice of positive controls A variety of cells harboring the mutation of interest may be used as positive controls, such as buccal cells,

cumulus cells, lymphocytes, and lymphoblasts. To reduce the chance of misdiagnosis due to ADO, it is possible to biopsy and analyze two blastomeres from the same embryo.8,10,72 The isolated single cells may also be used for calibration of the PGD techniques and for testing the precision, sensitivity, and reliability of the single-cell PCR strategy. Buccal cells may be obtained from patients by mouth-washing with double-distilled water or by scraping the inside of the cheek with a sterile cotton swab and suspending the smear in phosphate-buffered saline (PBS). The suspension is centrifuged at 7.5g for 5 minutes. The cell pellet is washed three times in PBS, and cells are resuspended and isolated using a pulled-glass micropipette under an inverted microscope. Single cells are then washed several times in PBS microdrops to ensure that, indeed, only a single cell is aspirated and transferred to sterile PCR tubes for further use.73–75 Cumulus cells may be obtained by incubating the retrieved oocyte in IVF culture medium supplemented with 80 IU hyaluronidase. Separated cumulus cells are then rinsed with IVF culture medium, washed in PBS, and transferred to sterile PCR tubes using a pulled-glass micropipette under a stereomicroscope.30 Lymphocytes may be isolated from peripheral blood by the Ficoll–Paque method, washed three times in PBS, resuspended, and diluted in culture medium on a glass slide. Individual cells are then selected using a pulled-glass micropipette under an inverted microscope, washed three times in PCR buffer (50 mmol/l KCl, 10 mmol/l Tris-HCl, pH 8.3) supplemented with 0.01% polyvinylpyrrolidone (PVP), and transferred to sterile PCR tubes for further use. Lymphocytes may be used fresh or frozen–thawed. For freezing, lymphocytes are washed three times in PBS, resuspended in autologous plasma, and 20 µl of concentrated lymphocytes are added to 40 µl of fetal calf serum, 120 µl of RPMI medium and 20 µl of dimethylsulfoxide (DMSO), and kept in liquid nitrogen until required. Cells can be stored for up to 1 year. Thawing is performed by several washes with culture medium.76 A lymphoblast cell-line carrying the known mutation is probably the best choice, since its establishment provides a perpetual source of cells with a known genetic composition. The cell-line is achieved by transformation of peripheral blood lymphocytes with the Epstein–Barr virus (EBV).77 Once the cell-line is established, single cells may be aspirated and transferred to 1.5-ml Eppendorf tubes, washed three times with PBS, resuspended in 50 µl PBS, and kept at 4°C until use.78

Embryonic cell isolation Embryo biopsy is described in detail in Chapter 14. For the purpose of genetic analysis of the embryo, the single biopsied nucleated cells are washed several times in droplets of PCR buffer (50 mmol/l KCl, 10 mmol/l Tris-HCl, pH 8.3) supplemented with 0.01% PVP or 4

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mg/ml bovine serum albumin (BSA) in a Petri dish using a pulled micropipette. PVP or BSA are used in order to prevent adherence of the cells to the pipette. The isolated cell is transferred in a minimal volume of washing buffer to a PCR tube containing lysis buffer or water, and can be frozen immediately at −80°C until use. Alternatively, the cells can be lysed immediately and then frozen.8,9,52,76,79–80

Cell lysis Lysis of the single embryonic cells and exposure of their genetic material to the PCR reagents is one of the most critical steps, and greatly affects ADO rates, and efficiency and reliability of PGD.14 Among the several options, the three most commonly used lysis solutions are water, alkaline lysis buffer, and proteinase K/SDS buffer. There is as yet no consensus as to which is superior. Water Single blastomeres are washed three times in PBS transferred under visual control by pulled micropipettes to PCR tubes containing 60 µl of biotechnology-grade water. An aliquot from the last washing droplet is added to a PCR tube containing 60 µl water, to serve as a negative control. Lysis is accomplished by two cycles of freezing in liquid nitrogen and thawing, and then boiling for 10 minutes. Lysates can be stored until use at −20°C.8 Alkaline lysis buffer Single cells are transferred as above to PCR tubes containing 5 µl of alkaline lysis buffer (ALB – 200 mmol/l KOH, 50 mmol/l dithiothreitol [DTT]). For immediate use, samples are placed at −80°C for at least 30 minutes and undergo immediate lysis by incubation at 65°C for 10 minutes. Alternatively, samples may be immediately lysed, frozen, and stored (not longer than 1 week) at −80°C until further processing.76,81 After lysis, 5 µl of neutralization buffer (300 mmol/l KCl, 900 mmol/l Tris-HCl, pH 8.3, 200 mmol/l HCl) is added. Lysates are centrifuged briefly and placed on ice for immediate use or stored at −20°C until use.82 Proteinase K/SDS buffer Single blastomeres are washed three times in PBS or PCR buffers supplemented with 0.01% PVP or BSA and transferred individually to PCR tubes containing 5 µl of proteinase K/SDS buffer (17 mmol/l sodium dodecylsulfate [SDS] and 400 ng/ml proteinase K).73,83 Samples are incubated at 50°C for 1 hour followed by denaturation at 99°C for 15 minutes to inactivate the enzyme. Lysates can be stored at −80°C until used.14,75

Primary and nested PCR conditions For the primary PCR reaction, the following are mixed with the biopsied cell lysate to a final volume of 50 µl: PCR buffer (10 mmol/l Tris-HCl, 50 mmol/l KCl, and 2.5 mmol/l MgCl2, pH 8.3), 0.3 mmol/l dNTP, 1–2 U Taq polymerase, and 0.5 mmol/l outer primers. It is recommended to perform optimization of the reaction by using

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different MgCl2 concentrations and different pH conditions. Amplification efficiency can be improved by addition of one or more of the following ingredients: glycerol, gelatin, betain, DMSO, (NH4)2SO4, or detergent. The PCR-thermocycler program begins with a prolonged stage of initial denaturation at 95°C for 6 minutes. This has been shown to correlate with reduction in ADO rates.81 This is followed by 30 cycles of denaturation at 94°C for 1 minute, annealing at 52–65°C (according to the primer melting temperature) for 1 minute, and extension at 72°C for 1 minute. Final extension at 72°C for 10 minutes is usually performed. Specificity of the reaction can be improved by using ‘hot start.’ For the secondary or nested PCR, 2–5 µl of the primary PCR product serves as template to be used with the nested primers. In the nested-PCR reaction, the duration and temperature of the initial denaturation step may be reduced and MgCl2 concentration can be lowered. DMSO is not required for this step. Other reagents and PCR conditions may be similar to those used in the primary PCR reaction.5,13

Multiplex PCR According to the standard protocol, each 50 µl reaction includes 1–1.5 U of Taq polymerase, 0.3 mmol/l for each dNTP, 0.5–2.5 mmol/l, MgCl2, and 0.1–0.5 mmol/l of each primer. The reaction 10 × PCR buffer is usually composed of 500 mmol/l KCl, 100 mmol/l Tris-HCl, pH 8.3, but at least one of the following ingredient 5 is usually added: glycerol, gelatin, betain, DMSO (NH4)2SO4, and detergent. The PCR-thermocycler program begins with initial denaturation at 96°C for 5 minutes (ensuring appropriate accessibility to the DNA strands). This is followed by 30 cycles at 94°C for 45 seconds, 52–56°C for 60 seconds, and 72°C for 60 seconds. Final extension of 5–15 minutes at 72°C is usually performed. If ethidium bromide gel electrophoresis analysis is performed, a nested PCR is usually required. After primary PCR is performed, a 2–5 µl aliquot of the product serves as a DNA template for a nested-PCR reaction.

Primer extension preamplification PCR This method is based on multiple rounds of extensions using a random mixture of 15-base oligonucleotides as primers. Theoretically, the mixture contains up to 1 × 109 different primers. The PEP-PCR reaction in a final volume of 60 µl includes 33 mmol/l random primers, 10 × PCR buffer (100 mmol/l Tris-HCl, pH 8.3, 25 mmol/l MgCl2, 1 mg/ml gelatin, and 500 mmol/l KCl), 0.1 mmol/l dNTPs, and 5 U of Taq polymerase. The PCR buffer should be K+-free if the cell was lysed by an alkaline lysis buffer. The reaction is carried out in 50 cycles of the following: denaturation at 92°C for 1 minute, annealing at 37°C for 2 minutes, a programmed ramping step of 10 s/°C until 55°C, and extension at 55°C for 4 minutes.28,30,34 Improvement of amplification can be achieved by raising the denaturation temperature, elongating the

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denaturation period, raising the pH buffer from 8.3 to 8.8, modifying the MgCl2 and gelatin concentrations, reducing the KCl concentration and using a more thermostable DNA polymerase, and one that has minimal exonuclease activity. Addition of glycerol, betain, BSA, detergents, spermidine, and (NH4)2SO4 may also improve the product yield. Primers should be dissolved in Tris-HCl 5–10 mmol/l, pH 8.3, and not in TE buffer to prevent the chelation of Mg2++ ions by ethylene diaminetetracetic acid (EDTA). The PEP-PCR product should produce an even smear on ethidium bromide gel electrophoresis. A 2–10 µl aliquot of the PEP product serves as a template for subsequent PCR reactions amplifying the mutation-containing fragment, linked polymorphic markers, and for sex determination.

Degenerated oligonucleotide primed PCR DOP-PCR is based on multiple rounds of extensions using a universal primer containing a 6 bp degenerate region representing all possible nucleotide combinations, flanked with a GC-rich short sequence to improve hybridization to genomic DNA. The DOP-PCR reaction mixture in a final volume of 100 µl contains 2.0 mmol/l degenerated primers, 10 × PCR buffer (100 mmol/l TrisHCl, pH 8.3, 25 mmol/l MgCl2, and 500 mmol/l KCl, but the buffer should be K+-free if the cell was lysed by alkaline lysis buffer), 0.2 mmol/l dNTPs, and 2.5 U of Taq polymerase.29 Thermal cycling conditions are as follows: prolonged initial denaturation step at 94°C for 9 minutes, then 8 cycles of denaturation at 94°C for 1 minute, annealing at 30°C for 1.5 minutes and extension at 72°C for 3 minutes, followed by 50 cycles of denaturation at 94°C for 1 minute, annealing at 62°C for 1 minute and extension at 72°C for 1.5 minutes. Final extension at 72°C for 8 minutes.17 As for PEP, amplification efficiency may be improved by adding and changing the reaction ingredients and by gradually increasing the extension time after the first 10 cycles.

Multiple displacement amplification MDA is based on DNA amplification using a bacteriophage DNA polymerase and exonuclease-resistant phosphorothioate-modified random hexamer oligonucleotide primers in an isothermal strand displacement reaction. It is achieved using bacteriophage Φ29 DNA polymerase, hexamer primers, and reaction buffer, according to the manufacturer’s instructions (GenomiPhi v2 DNA Amplification Kit, GE Healthcare or Repli-G kit, Qiagen, Crawley, UK). The samples are incubated at 30°C for 2–6 hours, followed by 3–10 minutes incubation at 65°C to inactivate the enzyme. Purified products can undergo subsequent diverse analyses as CGH.

Fluorescent PCR Fluorescent PCR is performed in a final volume of 25 µl of 10 × PCR buffer containing 15 mmol/l MgCl2 and 0.2

mmol/l of each dNTP, and fluorescent-tagged primers at a final concentration of 0.05 mmol/l. After a ‘hot-start,’ 0.6–1.5 U of Taq polymerase is added to the reaction mix. Initial denaturation is first performed at 95°C for 5 minutes, followed by 36 cycles of denaturation at 94°C for 60 seconds, annealing at 60°C for 60 seconds, and extension at 72°C for 60 seconds. The reaction is completed with a final extension at 70°C for 10 minutes. Owing to its high sensitivity, nested PCR is usually not necessary.12,72,84

Restriction enzyme digestion For each different restriction enzyme, different conditions, such as buffer, temperature, and concentration, are specified in the commercially available kits. Some PCR reagents may interfere with the digestion reaction. To avoid this, PCR products can be purified by absorption of the DNA fragments onto glass fibers in the presence of chaotropic salts, then washed and eluted with a low-salt buffer or water. The isolated fragment may then be subjected to the restriction enzyme and buffer, incubated for 1–2 hours at 37°C, and resolved by electrophoresis on agarose or acrylamide gels.

Product detection Ethidium bromide gel electrophoresis An aliquot of the PCR products is applied to an agarose or acrylamide gel containing 0.05% ethidium bromide, and visualized under UV light. One lane is provided for a ‘DNA ladder’ containing a mixture of DNA fragments of known sizes. This allows the determination of the size, presence, and a measure of quantity of the resulting fragments. This technique, however, is neither sensitive nor accurate because it does not detect PCR products if the amplification yield is low, nor does it allow distinction between alleles differing in length by a few base pairs. GeneScan Following fluorescent PCR, size-separation is performed on an acrylamide gel or using a capillary method available in some sequencers. Fragment sizes are automatically determined for each PCR product. Each primer set is labeled with a different fluorescent marker; therefore, the products may be distinguished according to their specific emission wavelengths. The relative quantity of each PCR product may also be determined by the relative intensities of their fluorescence. Using a weight marker standard within each lane makes it possible to distinguish between products with a size difference of as little as 1–2 bp. The results are demonstrated as a diagram with colored peaks.8,12

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Genetic analysis of the embryo 2. Verlinsky Y, Rechitsky S, Schoolcraft W, Strom C, Kuliev A. Preimplantation diagnosis for Fanconi anemia combined with HLA matching. JAMA 2001; 285: 3130–3. 3. Sermon K, Lissens W, Nagy ZP, Van Steirteghem A, Liebaers I. Simultaneous amplification of the two most frequent mutations of infantile Tay–Sachs disease in single blastomeres. Hum Reprod 1995; 10: 2214–17. 4. Kontogianni EH, Griffin DK, Handyside AH. Identifying the sex of human preimplantation embryos in X-linked disease: amplification efficiency of a Y-specific alphoid repeat from single blastomeres with two lysis protocols. J Assist Reprod Genet 1996; 13: 125–32. 5. Cui KH, Matthews CD. Nuclear structural conditions and PCR amplification in human preimplantation diagnosis. Mol Hum Reprod 1996; 2: 63–71. 6. Ray PF, Ao A, Taylor DM, Winston RM, Handyside AH. Assessment of the reliability of single blastomere analysis for preimplantation diagnosis of the ∆F508 deletion causing cystic fibrosis in clinical practice. Prenat Diagn 1998; 18: 1402–12. 7. Grifo JA, Tang YX, Munné S, et al. Healthy deliveries from biopsied human embryos. Hum Reprod 1994; 9: 912–16. 8. Findlay I, Ray P, Quirke P, Rutherford A, Lilford R. Allelic drop-out and preferential amplification in single cells and human blastomeres: implications for preimplantation diagnosis of sex and cystic fibrosis. Hum Reprod 1995; 10: 1609–18. 9. Ray PF, Handyside AH. PCR from single cells for preimplantation diagnosis. In: Elles R ed. Methods in Molecular Biology: Molecular Diagnosis of Genetic Disease. New Jersey: Humana Press, 1996. 10. Ao A, Wells D, Handyside AH, Winston RM, Delhanty JD. Preimplantation genetic diagnosis of inherited cancer: familial adenomatous polyposis coli. J Assist Reprod Genet 1998; 15: 140–4. 11. Rechitsky S, Strom C, Verlinsky O, et al. Allele dropout in polar bodies and blastomeres. J Assist Reprod Genet 1998; 15: 253–7. 12. Sherlock J, Cirigliano V, Petrou M, Tutschek B, Adinolfi M. Assessment of diagnostic quantitative fluorescent multiplex polymerase chain reaction assays performed on single cells. Ann Hum Genet 1998; 62: 9–23. 13. Dreesen JC, Bras M, de Die-Smulders C, et al. Preimplantation genetic diagnosis of spinal muscular atrophy. Mol Hum Reprod 1998; 4: 881–5. 14. El-Hashemite N, Delhanty JD. A technique for eliminating allele specific amplification failure during DNA amplification of heterozygous cells for preimplantation diagnosis. Mol Hum Reprod 1997; 3: 975–8. 15. Harper JC, Coonen E, Handyside AH, et al. Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos. Prenat Diagn 1995; 15: 41–9. 16. Sermon K, De Vos A, Van de Velde H, et al. Fluorescent PCR and automated fragment analysis for the clinical application of preimplantation genetic diagnosis of myotonic dystrophy (Steinert’s disease). Mol Hum Reprod 1998; 4: 791–6.

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Textbook of Assisted Reproductive Technologies gene deletions using whole genome amplification. Nat Genet 1994; 6: 19–23. Telenius H, Pelmear AH, Tunnacliffe A, et al. Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes Chromosomes Cancer 1992; 4: 257–63. Voullaire L, Wilton L, Slater H, Williamson R. Detection of aneuploidy in single cells using comparative genomic hybridization. Prenat Diagn 1999; 19: 846–51. Dean FB, Hosono S, Fang L, et al. Comprehensive human genome amplification using multiple displacement amplification. Proc Natl Acad Sci USA 2002; 99(8): 5261–6. Spits C, Le Caignec C, De Rycke M, et al. Wholegenome multiple displacement amplification from single cells. Nat Protoc 2006; 1: 1965–70. Lage JM, Leamon JH, Pejovic T, et al. Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH. Genome Res 2003; 13: 294–307. Hughes S, Lim G, Beheshti B, et al. Use of whole genome amplification and comparative genomic hybridisation to detect chromosomal copy number alterations in cell line material and tumour tissue. Cytogenet Genome Res 2004; 105: 18–24. Cheung VG, Nelson SF. Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA. Proc Natl Acad Sci USA 1996; 93(25): 14676–9. Telenius H, Carter NP, Bebb CE, et al. Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics 1992; 13: 718–25. Coskun S, Alsmadi O. Whole genome amplification from a single cell: a new era for preimplantation genetic diagnosis. Prenat Diagn 2007; 27(4): 297–302. Blanco L, Bernad A, Lazaro JM, et al. Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. J Biol Chem 1989; 264: 8935–40. Rodríguez I, Lázaro JM, Blanco L, et al. A specific subdomain in phi29 DNA polymerase confers both processivity and strand-displacement capacity. Proc Natl Acad Sci USA 2005; 102: 6407–12. Hellani A, Coskun S, Benkhalifa M, et al. Multiple displacement amplification on single cell and possible PGD applications. Mol Hum Reprod 2004; 10: 847–52. Handyside AH, Robinson MD, Simpson RJ, et al. Isothermal whole genome amplification from single and small numbers of cells: a new era for preimplantation genetic diagnosis of inherited disease. Mol Hum Reprod 2004; 10: 767–72. Renwick PJ, Trussler J, Ostad-Saffari E, et al. Proof of principle and first cases using preimplantation genetic haplotyping – a paradigm shift for embryo diagnosis. Reprod Biomed Online 2006; 13: 110–19. Pickering SJ, McConnell JM, Johnson MH, Braude PR. Use of a polymorphic dinucleotide repeat sequence to detect non-blastomeric contamination of the polymerase chain reaction in biopsy samples for preimplantation diagnosis. Hum Reprod 1994; 9: 1539–45.

50. Harton GL, Tsipouras P, Sisson ME, et al. Preimplantation genetic testing for Marfan syndrome. Mol Hum Reprod 1996; 2: 713–15. 51. Lee SH, Kwak IP, Cha KE, et al. Preimplantation diagnosis of non-deletion Duchenne muscular dystrophy (DMD) by linkage polymerase chain reaction analysis. Mol Hum Reprod 1999; 4: 345–9. 52. Sermon K, Lissens W, Joris H, et al. Clinical application of preimplantation diagnosis for myotonic dystrophy. Prenat Diagn 1997; 17: 925–32. 53. Daniels R, Holding C, Kontogianni E, Monk M. Single-cell analysis of unstable genes. J Assist Reprod Genet 1996; 13: 163–9. 54. El-Hashemite N, Wells D, Delhanty JD. Single cell detection of β-thalassaemia mutations using silver stained SSCP analysis: an application for preimplantation diagnosis. Mol Hum Reprod 1997; 3: 693–8. 55. Vrettou C, Palmer G, Kanavakis E, et al. A widely applicable strategy for single cell genotyping of β-thalassaemia mutations using DGGE analysis: application to preimplantation genetic diagnosis. Prenat Diagn 1999; 19: 1209–16. 56. Kanavakis E, Vrettou C, Palmer G, et al. Preimplantation genetic diagnosis in 10 couples at risk for transmitting β-thalassaemia major: clinical experience including the initiation of six singleton pregnancies. Prenat Diagn 1999; 19: 1217–22. 57. Scobie G, Woodroffe B, Fishel S, Kalsheker N. Identification of the five most common cystic fibrosis mutations in single cells using a rapid and specific differential amplification system. Mol Hum Reprod 1996; 2: 203–7. 58. Bermudez MG, Piyamongkol W, Tomaz S, et al. Single-cell sequencing and mini-sequencing for preimplantation genetic diagnosis. Prenat Diagn 2003; 23: 669–77. 59. Hassold T, Chen N, Funkhouser J, et al. A cytogenetic study of 1000 spontaneous abortions. Ann Hum Genet 1980; 44: 151–78. 60. Chandley AC. Infertility and chromosome abnormality. Oxf Rev Reprod Biol 1984; 6: 1–46. 61. Jacobs PA. The chromosome complement of human gametes. Oxf Rev Reprod Biol 1992; 14: 47–72. 62. Kallioniemi A, Kallioniemi OP, Sudar D, et al. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 1992; 258: 818–21. 63. Wells D, Delhanty JD. Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Mol Hum Reprod 2000; 6: 1055–62. 64. Voullaire L, Slater H, Williamson R, Wilton L. Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization. Hum Genet 2000; 106: 210–17. 65. Wilton L, Williamson R, McBain J, Edgar D, Voullaire L. Birth of a healthy infant after preimplantation confirmation of euploidy by comparative genomic hybridization. N Engl J Med 2001; 345: 1537–41. 66. Wells D, Escudero T, Levy B, et al. First clinical application of comparative genomic hybridization and polar body testing for preimplantation genetic

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diagnosis of aneuploidy. Fertil Steril 2002; 78: 543–9. Fragouli E, Wells D, Thornhill A, et al. Comparative genomic hybridization analysis of human oocytes and polar bodies. Hum Reprod 2006; 21: 2319–28. Le Caignec C, Spits C, Sermon K, et al. Single-cell chromosomal imbalances detection by array CGH. Nucleic Acids Res 2006; 34: e68. Wilton L. Preimplantation genetic diagnosis and chromosome analysis of blastomeres using comparative genomic hybridization. Hum Reprod Update 2005; 11: 33–41. Bejjani BA, Shaffer LG. Application of array-based comparative genomic hybridization to clinical diagnostics. J Mol Diagn 2006; 8(5): 528–33. Hu DG, Webb G, Hussey N. Aneuploidy detection in single cells using DNA array-based comparative genomic hybridization. Mol Hum Reprod 2004; 10: 283–9. Findlay I, Quirke P. Fluorescent polymerase chain reaction: Part I. A new method allowing genetic diagnosis and DNA fingerprinting of single cells. Hum Reprod Update 1996; 2: 137–52. Holding C, Bentley D, Roberts R, et al. Development and validation of laboratory procedures for preimplantation diagnosis of Duchenne muscular dystrophy. J Med Genet 1993; 30: 903–9. Findlay I, Lilford R. Sources and detection of contamination in preimplantation diagnosis. Proc XII Annual Scientific Meeting of the Fertility Society of Australia 1994: 101. Ioulianos A, Wells D, Harper JC, Delhanty JD. A successful strategy for preimplantation diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. Prenat Diagn 2000; 20: 593–8.

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76. Hussey ND, Donggui H, Frieland DA, et al. Analysis of five Duchenne muscular dystrophy exons and gender determination using conventional duplex polymerase chain reaction on single cells. Mol Hum Reprod 1999; 5: 1089–94. 77. Ventura M, Gibaud A, Le Pendu J, et al. Use of a simple method for the Epstein–Barr virus transformation of lymphocytes from members of large families of Reunion Island. Hum Hered 1988; 38: 36–43. 78. Van de Velde H, Sermon K, De Vos A, et al. Fluorescent PCR and automated fragment analysis in preimplantation genetic diagnosis for 21-hydroxylase deficiency in congenital adrenal hyperplasia. Mol Hum Reprod 1999; 5: 691–6. 79. Salido EC, Yen PH, Koprivinkar K, Yu LC, Shapiro LJ. The human enamel protein gene amelogenin is expressed from both X and Y chromosomes. Am J Hum Genet 1992; 50: 303–16. 80. Ao A, Handyside AH. Cleavage stage human embryo biopsy. Hum Reprod Update 1995; 1: 3. 81. Ao A, Ray P, Harper J, et al. Clinical experience with preimplantation genetic diagnosis of cystic fibrosis (delta F508). Prenat Diagn 1996; 16: 137–42. 82. Cui XF, Li HH, Goradia TM, et al. Single-sperm typing: determination of genetic distance between the G gamma-globin and parathyroid hormone loci by using the polymerase chain reaction and allelespecific oligomers. Proc Natl Acad Sci USA 1989; 86: 9389–93. 83. Han S, Zhong XY, Troeger C, et al. Current application of single-cell PCR. Cell Mol Life Sci 2000; 57: 96–105. 84. Findlay I, Quirke P, Hall J, Rutherford A. Fluorescent PCR: a new technique for PGD of sex and single-gene defects. J Assist Reprod Genet 1996; 13: 96–103.

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31 Proteomic analysis of the embryo Mandy Katz-Jaffe

Introduction Unlike the human genome, which is relatively fixed and steady throughout the human body, the human proteome (protein complement to the genome) is, by several orders of magnitude, more complex, diverse, and dynamic. Any single gene can produce a heterogeneous population of proteins that can be further modified by post-translational modifications such as phosphorylation. The result is a human proteome estimated at considerably over a million proteins to only ∼25 000 human genes.1 Several studies have indicated that the genome’s transcriptome (mRNA expression levels) does not necessarily predict the abundance or functional activity of proteins;2,3 rather, it is the human proteome that significantly contributes to physiological homeostasis in any cell or tissue.4 Various biological conditions, including age, gender, diet, lifestyle, medication, and disease, directly impact the composition of the human proteome in any particular cell or tissue, generating a unique proteomic signature.5 The characterization of protein signatures during embryonic development has the potential to address a variety of unresolved topics, with the ultimate goal of expanding our knowledge of embryonic cellular processes and the evolution of viability assays. Relatively little is known regarding the proteome of the human preimplantation embryo – in particular, the protein production of the blastocyst just prior to implantation. The task begins with identifying the proteins expressed, including those proteins changing in response to internal and external stimuli. These individual proteins can then be quantified and characterized, at the same time examining their interactions during embryonic development. In order to elucidate embryonic cellular architecture and function, a detailed understanding of the complexity at the protein level is essential. Of particular interest is the cell surface proteome, as it may pinpoint key molecules associated with implantation, including cell surface receptors, as well as the protein–protein interactions occurring between the developing embryo and the surrounding maternal environment. Zeptoproteomics is the term that has been coined to define proteomic technology optimized to analyze

protein expression in a limited number of cells.6 The preimplantation embryonic stage represents the most difficult challenge for zeptoproteomics with the combined effect of limited numbers of cells and minimal protein expression, resulting in extremely low levels of total protein available for analysis; i.e. only 27 ng of protein in a single mouse embryo.7

The embryonic proteome Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is at present the standard technique for separation of total protein. This technology separates proteins in the horizontal dimension by isoelectric focusing (a pH gradient range of typically 3–10), and in the vertical dimension by molecular weight in a polyacrylamide gel gradient.8 2D-PAGE is efficient at differential protein quantitation and detecting posttranslational modifications with starting amounts of total protein isolated from typically 106 cells. Limitations to this technology include a long processing time, weak detection of low concentration proteins, and the inability to capture or resolve very acidic or basic proteins, membrane proteins, as well as very small or large molecular weight proteins.8 Protein databases involving 2D-PAGE that represent the entire mouse preimplantation period from fertilization to the blastocyst stage have been constructed to provide a means of studying protein synthesis and characterizing protein changes.9,10 2D-PAGE was performed through the analysis of radiolabeled proteins after embryos were exposed to 1–3 hour incubation in a high concentration of radiolabeled amino acids. After protein resolution, spots were detected by fluorography and a software program assembled the images into protein databases.9 Comparison of the proteins between the 8-cell mouse embryo and the fully expanded mouse blastocyst database identified a total of 43 spots, approximately 3% of all total spots, which were only detected at the 8-cell stage, and 75 spots identified solely at the blastocyst stage.10 2D-PAGE in the field of embryology has been limited by the requirement for larger amounts of starting template as well as the lack of robustness and degree of

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labor intensity. Consequently, protein-based studies have concentrated on identifying and localizing individual proteins by Western blot analysis. Two insulinresponsive glucose transporter isoforms (GLUT4 and GLUT8) and the insulin receptor (IR) proteins were confirmed by Western blot analysis as being present in rabbit blastocysts.11 Another study observed the expression of stress-activated protein kinase/Jun kinase (SAPK/ JNK) phosphoproteins and p38 mitogen-activated protein kinases (MAPKs) by Western blotting from groups of over 100 mice embryos.12 A limitation of this approach is that proteins do not function individually, but within pathways; thus the analysis of the embryonic proteome as a whole is critical. Mass spectrometry (MS) has rapidly become the key technology in proteomics, facilitating the rapid identification and quantitation of proteins such as low-expression proteins. An array of templates can be applied, including tissues, cells, and biological fluids. MS involves an ion source for production of charged species in the gas phase, and the analyzer, which separates ions by their mass-to-charge (m/z) ratio. The commonly used ionization methods include electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI), and surface-enhanced laser desorption/ionization (SELDI), and are most commonly coupled to time-offlight (TOF), ion trap, or quadrupole analyzers. Posttranslational modifications are also identifiable since the modification will change the m/z ratio of a protein. Protein identification can then be performed by protease digestion to generate specific fragments from wellcharacterized cleavage products. These fragments are identifiable following tandem MS analysis and protein database searching.13 A comprehensive proteomics approach has been applied to study the mammalian oocyte; however, in these studies the starting template is still considerable. In one study, approximately 200 porcine oocytes were used to separate and visualize proteins of interest by 2D-PAGE, with an even larger starting template used for peptide profiling by MALDI-TOF MS and peptide sequencing by liquid chromatography–tandem MS.14 More recently, Vitale et al used 2D-PAGE and MS to identify differentially expressed proteins during murine oocyte maturation. A total of 500 germinal vesicle (GV) and meiosis II (MII) stage oocytes were extracted and resolved on 2D gels stained with silver; 12 proteins were observed to be differentially expressed between the GV and MII stages. These proteins were then characterized by MS, with the identification of nucleoplasmin 2 (Npm2), an oocyte-restricted protein.15 Another study investigated mature mouse cumulus–oocyte complexes and identified 156 individual proteins following 2D-PAGE and MS. Several protein families were discovered that may play important roles in ovarian follicular development.16 With further advances in proteomic technologies, the identification and quantitation of very small quantities of proteins has become more of a reality. SELDI-TOF

MS involves the application of small sample volumes (µl range) and enables detection of both the low- and high-molecular-weight proteins, with the optimal range for the technology at <20 kDa. The sensitivity is stated to be in the picomole to femtomole range, making proteomic profiling of diverse and limited biological samples possible. This technology is also capable of studying samples based on activated surfaces for preselection, including hydrophobic interaction, anion or cation exchange, and metal affinity capture.17 Bound proteins are laser activated, thereby liberating gaseous ions by desorption/ionization. The TOF tube is under a vacuum, which causes smaller ions to travel faster towards the detector, thereby allowing for a separation of these ions according to their m/z. The technology has been applied to a variety of biological sources, including serum18 and cell lysates,19 with specific focus on oncoproteomics and the early detection, metastatic ability, and therapeutic outcome of an assortment of different cancers.20 Pregnancy-related problems have also been the subject of SELDI-TOF MS studies, searching for early detection of conditions such as ectopic pregnancy21 and neonatal sepsis.22 Some concerns regarding this technology involve the dynamic and sensitive nature of the proteome to variables during sample collection, handling, processing, and storage, as well as peaks prejudiced by MS calibration and instrument drift.23 Overall, SELDI-TOF MS (Bio-Rad Technologies) is a highly sensitive, highthroughput, and cost-effective method that has the ability to identify changes in protein expression from varying biological and disease states. The development of a zeptoproteomics approach using SELDI-TOF MS has led to the detection of protein profiles associated with in vivo murine embryonic development.24 Owing to the multifactorial nature of mammalian embryonic development, panels of proteins specific to each of the individual stages have been successfully identified, allowing for the possibility of utilizing these panels to accurately gauge the level of perturbation of a biological system and effectively diagnose developmental competence (Fig 31.1).24 These profiles represent optimal embryonic cellular function and provide a potential diagnostic platform for improving IVF procedure, including the further improvement of in vitro culture conditions, stimulation protocols, and oocyte cryopreservation techniques.24,25 The data revealed that a specific panel of 10 proteins/biomarkers effectively discriminated in vitro mouse embryos cultured at low oxygen concentrations (5%) from in vitro mouse embryos cultured at high oxygen concentrations (20%) (p <0.05).24 Biomarkers can be defined as candidate proteins or peptides that are either down- or up-regulated in response to different physiological states. Confirming the pathological effects of oxygen during embryonic development,26 mouse embryos cultured in the presence of a reduced oxygen concentration exhibited a more in vivo-like profile (Fig 31.2).24

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12000 Da

Day 2

Day 3

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8000

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Fig 31.1 Protein profiling signatures across preimplantation embryonic development in the m/z range from 8000 to 12 000 Da. Data are shown as the original spectra and gel view. From ref. 24, with permission.

(a)

(b)

Fig 31.2 Two examples of differential expression between in vivo and in vitro embryos at both low and high oxygen concentrations. (a) A 12 150 Da protein (p <0.001). (b) A 3460 Da protein (p <0.01). Line represents the mean. From ref. 24, with permission.

SELDI-TOF MS analysis of the individual human embryonic proteome demonstrated that not all human blastocysts with similar morphology have identical protein signatures.27 Such data are consistent with the observations that human blastocysts from the same patient that have similar morphologies vary greatly in their metabolic fingerprint.28 Furthermore, specific

blastocyst developmental stages displayed differential protein expression profiles, as shown by the significant up- and down-regulation of biomarkers in expanded blastocysts compared to early blastocysts.27 Taken as a whole, human blastocyst morphology could be recognized according to specific individual protein signatures, with significant differences in

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Fig 31.3 A heat map segregating developing human blastocysts and degenerate human embryos according to protein signatures. Each column of squares represents an individual human embryo, while each row of squares represents an individual protein profile. Clustering analysis facilitates the grouping of similar protein expression profiles. Two major clusters were classified as shown on the left of the heat map. The top cluster identified proteins that are up-regulated (red) in degenerating embryos, while the bottom cluster identified proteins that are up-regulated (red) in developing blastocysts. Red = up-regulated proteins; green = down-regulated proteins. From ref. 27, with permission.

protein expression related to specific blastocyst developmental time points and/or degeneration (Fig 31.3).27 A panel of up-regulated biomarkers distinguished arrested embryos from developing blastocysts (Fig 31.4). Candidate identification implicated both apoptotic and growth-inhibiting pathways.27 Although preliminary, some of these potential IDs have been directly linked to embryogenesis. One example is a cystatin-like precursor for the m/z 14.2 kDa biomarker. Successful mammalian implantation involves controlled trophoblastic invasion of the maternal uterine epithelium. This controlled invasion involves the extracellular degradation of the uterine matrix by a variety of proteinases, including cysteine proteinases.29 Since cystatins are known to inhibit cysteine proteinases, up-regulation of this protein may contribute to failure of implantation of degenerating blastocysts. Further investigation of any potential biomarkers will be necessary to determine definitive cellular roles during human embryonic development. The identification and characterization of the embryonic proteome at all stages of preimplantation development is expanding our knowledge of embryo physiology. In addition, this information is providing mechanistic insight into the biological processes occurring at the cellular level, especially just prior to implantation. Considering the relative amount of any individual protein does not necessarily correlate with either biological function or significance, the ability to capture and characterize low expressed proteins is essential for future research.

Protocol for embryonic protein profiling Ion exchange chromatography Embryo lysis is performed in 9 mol/l urea/2% CHAPS. Protein chips are washed several times with appropriate binding buffer (Bio-Rad Technologies). Lysates are spotted onto protein chips and incubated at room temperature for 30–60 minutes, depending on the type of protein chip. Any unbound sample is then discarded and each spot washed a further three times with binding buffer and twice with MQ-H2O for the removal of salts and detergents. Sinapinic acid, an energy-absorbing molecule (Bio-Rad Technologies), prepared as a saturated solution in 50% acetonitrile/0.5% trifluoroacetic acid, is applied twice in 1-µl aliquots and allowed to air-dry prior to SELDI-TOF MS (Bio-Rad Technologies).

SELDI-TOF MS TOF data is collected for low-molecular-weight peptides and proteins <20 000 Da at a lower laser intensity, averaging 530 laser shots per spot. Highmolecular-weight proteins >20 000 Da are collected using a higher laser intensity, averaging 530 laser shots per spot. Spectra are generated by Bio-Rad Technologies Express Software. Mass accuracy is calibrated with the all-in-one peptide molecular mass

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Statistical analysis Protein profiles are normalized to the total ion current, and peaks with a signal-to-noise ratio >6 are selected for statistical analysis. Hierarchical clustering is performed on group samples with similar protein expression profiles. Univariate statistical analysis of the peak masses and relative intensity values is performed by the Mann–Whitney nonparametric test (Bio-Rad Technologies Express Software). To test reproducibility, one sample is analyzed in triplicate and the coefficient of variance of individual peaks in the replicate spectra is calculated by dividing the SD by the mean peak height × 100%.

The embryonic secretome With the knowledge that blastocyst morphology could be recognized according to specific protein expression,27 it is proposed that developmentally viable embryos will also possess a unique proteome profile with some of these expressed proteins secreted into the surrounding environment (secretome). Analysis of the embryonic proteome, as described in the above section, represents a destructive approach. On the other hand, analysis of the embryonic secretome is a noninvasive method suitable for clinical application. Currently, the selection of embryos for transfer is based on morphological indices.30 Although successful, the field of assisted reproductive technologies (ART) would benefit from more quantitative and non-invasive methods of viability determination to run alongside morphological assessment. These quantitative methods hold the promise of improving IVF success rates as well as optimizing single embryo transfer.31 There have been several studies that have shown the existence of soluble factors secreted by human embryos that could impact either or both

19.8 kDa

Fig 31.4 Negatively charged proteins showing significant differential expression related to blastocyst morphology. Open bars, degenerating embryos; solid bars, developing blastocysts. **Significantly different from developing blastocysts (p <0.01). From ref. 27, with permission.

developmental competence and implantation. For example, Sakkas et al demonstrated the existence of a soluble molecule secreted by human blastocysts that modulates regulation of HOXA10 expression in an epithelial endometrial cell line.32 This form of reciprocal embryo–endometrial interaction could transform the local uterine environment, impacting both embryo development and the implantation process. Another example is soluble human leukocyte antigen G (sHLA-G). Studies have revealed higher pregnancy rates when sHLA-G was detected in spent IVF medium of day 3 embryos;33,34 however, the results were not absolute with pregnancies established from sHLA-G-negative embryos. Furthermore, technical differences, including inability to measure sHLA-G production in some supernatants, highlight serious concerns as a marker of developmental potential.35 In addition, the end products of proteins, their metabolites that are produced by the embryo under certain physiological conditions, as well as the embryo’s amino acid utilization, appear to vary with outcome.36,37 Further investigation of the embryonic metabolome using Raman and near-infrared spectroscopy coupled with targeted bioinformatics to detect specific oxidative stress biomarkers in spent culture medium, has also highlighted apparent differences in algorithms generated for positive vs negative IVF outcomes.38 It is more than likely that the multifactorial nature of embryonic development will dictate a panel of molecules to assess developmental competence and/or implantation potential than just the single variable. The SELDI-TOF MS analysis of proteins in the secretome of mammalian embryos throughout preimplantation development highlighted distinctive protein signatures at each 24-hour developmental stage (Table 31.1).39 These signatures uniquely identified an embryo, independent of morphology. Comparison of mouse and human secretomes across preimplantation development revealed similar patterns and little differences between species.39 Subsequent analysis revealed protein expression only at specific 24-hour developmental time points,

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Table 31.1 Each stage of mouse embryonic development revealed a panel of significant biomarkers that were only detected in the drops of spent IVF culture media at specific stages of development (p <0.05) Stage of embryonic development

No. of proteins/biomarkers

Description of events

Day 1–2 Day 2–3 Day 3–4 Day 4–5

∼20 candidate peaks ∼12 candidate peaks ∼20 candidate peaks ∼20 candidate peaks

Maternal control of early embryonic development Embryonic genome activated Embryonic proteins translated Initiation of blastocyst implantation

From ref. 39 with permission.

(a)

(b)

(c)

whereas other proteins were observed through several embryonic stages, particularly occurring either before or after the activation of the embryonic genome.39 Examples are shown in Fig 31.5; the light gray boxes highlight the profile of several biomarkers only secreted during the first 24 hours from day 1 to day 2 of mouse embryonic development (p <0.05). Other biomarkers were observed to be secreted at all stages of embryonic development with increasing expression towards the blastocyst stage as shown in Fig 31.5 (dark gray box; p <0.05). In addition, the expression of numerous biomarkers was only observed after the activation of the embryonic genome (Fig 31.5; black boxes). The transition from maternal inherited transcripts and proteins to the activation of the embryonic genome and the expression of key embryonic proteins must occur for continued embryonic development.40 Thus, embryos with a

Fig 31.5 Protein profiles enhanced around the 7–10 kDa range for the secretome of each individual stage of mouse embryonic development. The bottom profiles for each 24 hours are from the control drops of media cultured under exact incubation conditions without embryos: (a), day 1–2; (b), day 2–3; (c), day 3–4; (d), day 4–5. Dark gray boxes display protein expression across all developmental stages. Light gray boxes highlight day 1–2 maternal protein expression, whereas black boxes indicate protein expression after the activation of the embryonic genome. From ref. 39, with permission.

correctly activated embryonic genome, and hence, a fully functional embryonic proteome, may have a higher potential of developmental competence. Secretome analysis on day 5 was directly correlated with continuing blastocyst development, including the identification of differentially expressed biomarkers. The profile of an 8.5 kDa protein that was secreted every 24 hours from day 2 to day 5 of human embryonic development, with significantly increasing intensity towards the blastocyst stage, was also directly associated with ongoing development (Fig 31.6). The near lack of expression of this 8.5 kDa protein/biomarker from degenerating embryos, in conjunction with its high expression from developing blastocysts, potentially indicates an association between this biomarker and ongoing blastocyst development (Fig 31.6; p <0.05).39 Initial identification of this 8.5 kDa biomarker

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Fig 31.6 The expression of this 8.5 kDa protein/biomarker appears to be directly associated with ongoing blastocyst development. The black box highlights differences in protein expression across morphologically different day 5 embryos. From ref. 39, with permission.

involving reverse phase chromatography, SDS-PAGE, trypsin digest, tandem MS, and database peptide sequence searching indicated that the best candidate was ubiquitin. Ubiquitin is a protein implicated in the implantation process of mammalian species, with suggestions that it may control the activities and turnover of key signaling molecules.41 Protein identification and correlation of this and other biomarkers with embryonic developmental competence, viability, and clinical pregnancy is ongoing. Such data will provide insight into the unique molecular events occurring during mammalian embryonic development: e.g. revealing some of the complex dialogue between the developing embryo and its maternal environment. SELDI-TOF MS technology facilitates fast, sensitive, and high-throughput secretome analysis that could contribute to the development of a noninvasive viability assay for use in ART. For example, samples of IVF spent culture media could be collected and sent to a core facility for secretome analysis using SELDI-TOF MS and/or other proteomic technologies. This could translate into a cost-effective and reproducible assay for assessment of human embryonic viability. From a clinical perspective, noninvasive quantification of human embryonic viability alongside morphological assessment may lead to an increase in single embryo transfers and IVF live births.

Conclusions and future perspectives Recent advances in zeptoproteomics have initiated new studies to characterize the proteins expressed, as well as secreted, by the mammalian embryo during all stages of preimplantation development. This information has the potential to provide insight into the cellular function and biological processes during embryonic development and the implantation process, as well as to assist in the optimization of IVF procedures. In addition, this technology could be of value in the development of noninvasive viability assays that, in conjunction with current morphologically based selection methods, may increase IVF success rates while reducing the number of embryos transferred. However, taking into account the complexity and diversity of the mammalian embryo, an in-depth prospective will most likely require a merging of the ‘omics’ – genomics, proteomics, and metabolomics.

Looking towards the future of zeptoproteomics and the diagnostic capabilities of detecting and quantifying low-abundant biomarkers in complex biological systems, several platforms show promise. The first is protein microarray technology, with the advantage of offering confirmatory and complementary information to mRNA expression studies. However, there are several hurdles to overcome, including the high cost of microarrays, dependence on the existence of antibodies, and problems with nonspecific interactions.42 Another platform is two-dimensional capillary electrophoresis (2D-CE). A recent proof of principle study demonstrated the ability to separate and detect proteins from single mouse zygotes. The protein signature was produced by separation according to molecular weight in the first-dimension capillary sieving electrophoresis, followed by separation by micellar electrokinetic capillary chromatography.43 The emergence of nanotechnologies will be fundamental to the detection of low-abundant biomarkers in complex biological systems. Advances incorporating nanofluidics, nanoparticles, and nanostructures promise microscopic volume liquid handling and the monitoring of concentration changes that have never before been detectable, as well as the potential for separation- and label-free protein analysis.44 The advent of the field of nanoproteomics will offer exciting opportunities to discover diagnostic biomarkers of preimplantation embryonic development and viability.

References 1. Kenyon GL, DeMarini DM, Fuchs E, et al. Defining the mandate of proteomics in the post-genomics era: Workshop Report. Mol Cell Proteomics 2002; 1: 763–80. 2. Gygi SP, Rochon Y, Franza BR, Aebersold R. Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 1999; 19: 1720–30. 3. Wulfkuhle JD, Liotta LA, Petricoin EF. Proteomic applications for the early detection of cancer. Nat Rev Cancer 2003; 3: 267–75. 4. Espina V, Dettloff KA, Cowherd S, Petricoin EF 3rd, Liotta LA. Use of proteomic analysis to monitor responses to biological therapies. Expert Opin Biol Ther 2004; 4: 83–93.

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5. Petricoin EF, Liotta LA. Clinical applications of proteomics. J Nutr 2003; 133: 2476–84S. 6. Hachey DL. Chaurand P. Proteomics in reproductive medicine: the technology for separation and identification of proteins. J Reprod Immunol 2004; 63: 61–73. 7. Brinster RL. Protein content of the mouse embryo during the first five days of development. J Reprod Fertil 1967; 13: 413–20. 8. Patton WF. Detection technologies in proteome analysis. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 771: 3–31. 9. Latham KE, Garrels JI, Chang C, Solter D. Analysis of embryonic mouse development: construction of a high-resolution, two-dimensional gel protein database. Appl Theor Electrophor 1992; 2: 163–70. 10. Shi CZ, Collins HW, Garside WT, et al. Protein databases for compacted eight-cell and blastocyst-stage mouse embryos. Mol Reprod Develop 1994; 37: 34–47. 11. Navarette Santos A, Tonack S, Kirstein M, Kietz S, Fischer B. Two insulin-responsive glucose transporter isoforms and the insulin receptor are developmentally expressed in rabbit preimplantation embryos. Reproduction 2004; 128: 503–16. 12. Wang HM, Zhang X, Qian D, et al. Effect of ubiquitinproteasome pathway on mouse blastocyst implantation and expression of matrix metalloproteinases-2 and -9. Biol Reprod 2004; 70: 481–7. 13. Liebler DC. Introduction to Proteomics, Tools for the New Biology. Totowa, NJ: Humana Press, 2002. 14. Ellederova Z, Halada P, Man P, et al. Protein patterns of pig oocytes during in vitro maturation. Biol Reprod 2004; 71: 1533–9. 15. Vitale AM, Calvert ME, Mallavarapu M, et al. Proteomic profiling of murine oocyte maturation. Mol Reprod Dev 2006; 74: 608–16. 16. Meng Y, Liu XH, Ma X, et al. The protein profile of mouse mature cumulus–oocyte complex. Biochim Biophys Acta 2007; 1774: 1477–90. 17. Seibert V, Wiesner A, Buschmann T, Meuer J. Surfaceenhanced laser desorption ionization time-of-flight mass spectrometery (SELDI TOF-MS) and ProteinChip technology in proteomics research. Pathol Res Pract 2004; 200: 83–94. 18. Cazares LH, Diaz JI, Drake RR, Semmes OJ. MALDI/SELDI protein profiling of serum for the identification of cancer biomarkers. Methods Mol Biol 2007; 428: 125–40. 19. Jansen C, Hebeda KM, Linkels M, et al. Protein profiling of B-cell lymphomas using tissue biopsies: a potential tool for small samples in pathology. Cell Oncol 2008; 30: 27–38. 20. Cho WC. Contribution of oncoproteomics to cancer biomarker discovery. Mol Cancer 2007; 6: 25. 21. Gerton GL, Fan XJ, Chittams J, et al. A serum proteomics approach to the diagnosis of ectopic pregnancy. Ann N Y Acad Sci 2004; 1022: 306–16. 22. Buhimschi CS, Bhandari V, Hamar BD, et al. Proteomic profiling of the amniotic fluid to detect inflammation, infection, and neonatal sepsis. PLoS Med 2007; 4: e18. 23. Poon TC. Opportunities and limitations of SELDITOF-MS in biomedical research: practical advices. Expert Rev Proteomics 2007; 4: 51–65.

24. Katz-Jaffe MG, Linck DW, Schoolcraft WB, Gardner DK. A proteomic analysis of mammalian preimplantation embryonic development. Reproduction 2005; 130: 899–905. 25. Gardner DK, Sheehan CB, Rienzi L, Katz-Jaffe MG, Larman MG. Analysis of oocyte physiology to improve cryopreservation procedures. Theriogenology 2007; 67: 64–72. 26. Gardner DK, Lane M. Alleviation of the ‘2-cell block’ and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters. Hum Reprod 1996; 11: 2703–12. 27. Katz-Jaffe MG, Gardner DK, Schoolcraft WB. Proteomic analysis of individual human embryos to identify novel biomarkers of development and viability. Fertil Steril 2006; 85: 101–7. 28. Gardner DK, Lane M, Stevens J, Schoolcraft WB. Noninvasive assessment of human embryo nutrient consumption as a measure of developmental potential. Fertil Steril 2001; 76: 1175–80. 29. Afonso S, Romagnano L, Babiarz B. The expression and function of cystatin C and cathepsin B and cathepsin L during mouse embryo implantation and placentation. Development 1997; 124: 3415–25. 30. Ebner T, Moser M, Sommergruber M, Tews G. Selection based on morphological assessment of oocytes and embryos at different stages of preimplantation development: a review. Hum Reprod Update 2003; 9: 251–62. 31. Sakkas D, Gardner DK. Noninvasive methods to assess embryo quality. Curr Opin Obstet Gynecol 2005; 17: 283–8. 32. Sakkas D, Lu C, Zulfikaroglu E, Neuber E, Taylor HS. A soluble molecule secreted by human blastocysts modulates regulation of HOXA10 expression in an epithelial endometrial cell line. Fertil Steril 2003; 80: 1169–74. 33. Noci I, Fuzzi B, Rizzo R, et al. Embryonic soluble HLA-G as a marker of developmental potential in embryos. Hum Reprod 2005; 20: 138–46. 34. Fisch JD, Keskintepe L, Ginsburg M, Adamowicz M, Sher G. Graduated embryo score and soluble human leukocyte antigen-G expression improve assisted reproductive technology outcomes and suggest a basis for elective single-embryo transfer. Fertil Steril 2007; 87: 757–63. 35. Sargent I, Swales A, Ledee N, et al. sHLA-G production by human IVF embryos: can it be measured reliably? J Reprod Immunol 2007; 75: 128–32. 36. Brison DR, Houghton FD, Falconer D, et al. Identification of viable embryos in IVF by non-invasive measurement of amino acid turnover. Hum Reprod 2004; 19: 2319–24. 37. Van den Bergh M, Devreker F. Glycolytic activity: a possible tool for human blastocyst selection. RBM Online 2001; 3: 8. 38. Seli E, Sakkas D, Scott R, et al. Noninvasive metabolomic profiling of embryo culture media using Raman and near-infrared spectroscopy correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril 2007; 88: 1350–7. 39. Katz-Jaffe MG, Schoolcraft WB, Gardner DK. Quantification of protein expression (secretome) by

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Proteomic analysis of the embryo human and mouse preimplantation embryos. Fertil Steril 2006; 86: 678–85. 40. Telford NA, Watson AJ, Schultz GA. Transition from maternal to embryonic control in early mammalian development: a comparison of several species. Mol Reprod Dev 1990; 26: 90–100. 41. Wang Y, Puscheck EE, Lewis JJ, et al. Increases in phosphorylation of SAPK/JNK and p38MAPK correlate negatively with mouse embryo development after culture in different media. Fertil Steril 2005; 83: 1144–54.

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42. Spisak S, Tulassay Z, Molnar B, Guttman A. Protein microchips in biomedicine and biomarker discovery. Electrophoresis 2007; 28: 4261–73. 43. Harwood MM, Christians ES, Fazal MA, Dovichi NJ. Single-cell protein analysis of a single mouse embryo by two-dimensional capillary electrophoresis. J Chromatogr A 2006; 1130: 190–4. 44. Marko NF, Weil RJ, Toms SA. Nanotechnology in proteomics. Expert Rev Proteomics 2007; 4: 617–26.

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32 Embryonic and maternal dialogue and the analysis of uterine receptivity Francisco Domínguez, Jose Antonio Horcajadas, Carlos Simón

Introduction Successful implantation requires a functionally normal embryo at the blastocyst stage and a receptive endometrium, while the communication between them is also essential. This embryonic–endometrial cross-communication, already described in rodents1 and non-human primates,2 is a highly regulated and complex mechanism. During apposition, the human blastocyst finds a location to attach to in a specific area of the maternal endometrium. In the adhesion phase, which occurs 6–7 days after ovulation within the ‘implantation window,’ direct contact occurs between the maternal endometrial epithelium (EE) and the embryonic trophoectoderm (TE). Finally, in the invasion phase, the embryonic trophoblast breaches the basement membrane and invades the endometrial stroma up to the uterine vessels. Initial adhesion of the trophectoderm of the embryo to the EE plasma membrane is the prerequisite for implantation and placental development. EE is a specialized hormone-regulated cell population that must undergo cyclical morphological and biochemical changes to maintain an environment suitable for preimplantation embryonic development. Acting as a modulator, it translates and controls the impact of the embryo on the stromal and vascular compartment, and converts hormonal signals into embryonic signals. In recent years, several works have been produced regarding the genomics of the human endometrium under different physiological and pathological conditions. These studies have generated a large amount of information about the regulation and dysregulation of the window of implantation (WOI) genes under fertile, subfertile, and refractory conditions. However, the key molecules/mechanisms in endometrial receptivity remain to be elucidated. In this chapter, we report the existing information concerning the embryo–endometrial crosstalk implicated in human implantation compared with a related process, which is the transendothelial migration of white cells.

Comparative model: embryonic implantation versus leukocyte transendothelial migration A parallelism between the different steps in human embryo–endometrial apposition/adhesion/invasion and leukocyte–endothelium rolling/adhesion/extravasation has been established in recent years.3–5 Cascades of events that take place during both processes show similarities, although some details with regard to the time scale, the size of cells, and the identity of involved molecules, among others, are obviously different. During the apposition phase in implantation and leukocyte adhesion, the blastocyst–endometrium and leukocyte–endothelium dialogue relies on a first wave of soluble mediators, such as cytokines, chemokines, and other factors, which are produced and act in a bidirectional fashion.6,7 These molecules regulate the expression and functional activity of adhesion molecules, such as L-selectin and integrins, that mediate both processes. The first step of the extravasation sequence in leukocytes corresponds to the interaction of selectins with their carbohydrate-based ligands.8 This interaction, known as tethering, allows the leukocyte to roll onto the endothelial cell wall. These selectin interactions are highly dynamic and short-lived, so they are able to slow down the leukocyte through transient contacts with the endothelial monolayer, thus facilitating their firm adhesion (Fig 32.1). Leukocytes express L-selectin, which is shed from their surface to allow the transmigration process to proceed. The L-selectin system is also critically involved in the embryonic apposition phase.4 Carbohydrate ligands that bind L-selectin are localized on the luminal epithelium at the time of implantation, while the trophoectoderm expresses L-selectin strongly after hatching. Trophoblast lineages use L-selectin to bind to uterine epithelial oligosaccharide ligands, and adhesion to the epithelium is impaired when L-selectin is blocked with specific antibodies.4

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LK EC

Embryonic implantation

Blastocyst

EEC

Fig 32.1 Comparison of the sequential adhesion steps involved in leukocyte transmigration and embryonic implantation and their molecular players. Leukocyte rolling, via the interaction of selectins with their carbohydrate ligands, slows down the leukocyte and facilitates the binding of chemokines to their 6-protein-coupled receptors (GPCRs). Chemokines induce the high-affinity conformation of leukocyte integrins, that bind to intercellular adhesion molecule 1 (ICAM-1; blue) and vascular cell adhesion molecule 1 (VCAM-1; green), included into tetraspanin microdomains and anchored to the cortical actin cytoskeleton on the apical surface of endothelial cells. Upon leukocyte adhesion, endothelial cells develop a three-dimensional docking structure that prevents the detachment of the adhered leukocyte, allowing it to proceed to diapedesis. During diapedesis, leukocyte integrins interact with endothelial junctional adhesion molecules (JAMs; pink), that reseal the junction by homophilic interactions once the leukocyte has traversed the monolayer.

Mucins also exist in the glycocalyx of human endometrial epithelial cells (EECs), such as mucin-1 (MUC-1), which increases its expression from the proliferative phase to the secretory phase in endometrial tissue9 and is also regulated by the human blastocyst.10 The possible substrate candidates for MUC-1 binding include L-selectins,9 or intercellular adhesion molecules. Nonetheless, its function as either an adhesion or antiadhesion molecule is still controversial. During leukocyte rolling, chemokines induce the activation in situ of leukocyte integrins,11 as well as the polarization of the cell in cooperation with integrin-dependent signals.12,13 Many authors have studied integrins in human implantation. A subset of epithelial endometrial integrin subunits may be relevant to the process of implantation based on spatiotemporal considerations. Therefore, the embryo could induce a favorable epithelial integrin pattern for its implantation. Integrin knockout studies reveal that embryos develop normally up to the blastocyst stage in β1-/- mice, but fail to implant.14 However, no implantation-related phenotypes have been observed in other integrin knockouts. In the diapedesis step, leukocytes have to squeeze into the endothelial cell-to-cell junctions. During this process, the permeability of the endothelial monolayer is not usually compromised. Leukocyte integrins interact

with tight junction molecules, such as junctional adhesion molecules (JAMs), to establish heterotypic connections that are replaced by JAM–JAM homotypic interactions once the leukocyte has traversed the monolayer, thus restoring the initial situation15,16 (see Fig 32.1). The size of the blastocyst prevents its migration between EEC and therefore another strategy is needed. A paracrine apoptotic reaction is induced in humans and mice when the blastocyst adheres to the EEC monolayer.17,18 This embryo-induced apoptotic mechanism is triggered by a direct contact between the blastocyst and EEC, and is mediated, at least in part, by the Fas–Fas ligand system. To achieve successful invasion, trophoblasts must induce a repertoire of genes involved in the degradation of the extracellular matrix. Matrix metalloproteinase-9 (MMP-9) is closely associated with the invasive phenotype of trophoblasts19 (see Fig 32.1).

Chemokines and chemokine receptors: crucial mediators in implantation Chemokines, a family of small polypeptides with molecular weights in the range of 8–12 kDa, attract specific leukocyte subsets by binding to cell-surface receptors. Two main subfamilies are distinguished by

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the arrangement of the first two of the four (2nd and 4th) conserved cysteine residues near the amino terminus. CXC chemokines attract neutrophils, and CC chemokines act upon monocytes, eosinophils, T lymphocytes, and natural killer (NK) cells. The other two subfamilies are CX3C (fractalkine or neurotactin), with three amino acids between the two cysteines, and the C subfamily, also named lymphotactins,20 with only a single cysteine near the N-terminal domain, eliciting potent lymphocyte chemoattractant capacity, which does not act upon monocytes. Chemokines act through cell-surface G-proteincoupled receptors (GPCRs).21 One receptor might bind one chemokine or more from the same subfamily, and chemokines can bind several different receptors.22 Consequently, the activity of chemokines is the outcome of a complex cascade that depends on the cell type, the ligand, on both the structure and configuration of the receptor, and also on the activation enzymes. In reproductive biology, these molecules have been implicated in crucial processes such as ovulation, menstruation, embryo implantation, and parturition, as well as in pathological processes such as preterm delivery, human immunodeficiency virus (HIV) infection, endometriosis, and ovarian hyperstimulation syndrome.23,24 Chemokines produced and incorporated by the endometrial epithelium and the human blastocyst are implicated in this molecular network. We know that different subsets of leukocytes are recruited into the endometrium during implantation. The regulation of the uterine tissue during this process is thought to be orchestrated by uterine epithelial cells, which release an array of chemokines in a precise temporal pattern that is mainly driven by ovarian steroids.25 Chemokines act on a range of leukocyte subsets, which, in turn, release a number of proteases and other mediators that facilitate embryo invasion.26 Chemokine receptors belong to the superfamily of GPCRs. These receptors display seven sequences of 20–25 hydrophobic residues that form an α-helix and span the plasma membrane, an extracellular N-terminus, three extracellular loops, three intracellular domains, and an intracellular C-terminal tail. These receptors transmit information to the cell about the presence of chemokine gradients in the extracellular environment. They are named in accordance with the structure of their ligand (CXC or CC). The chemokine expression has been found in EE cells, including regulated and normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein-1 (MIP-1α, MIP-1β), and the macrophage chemotactic protein-1 (MCP-1). Interleukin-8 (IL-8), a CXC chemokine with neutrophil chemotactic/activating and T-cell chemotactic activity, is produced by human endometrial stromal and glandular cells in culture. IL-8 is found in both the surface epithelium and glands throughout the menstrual cycle. It has been suggested that it might be implicated in the recruitment of neutrophils and lymphocytes

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into the endometrium.27 After ovulation, the number of large granular lymphocytes increases in the uterus,28 and this effect might be mediated by endometrial epithelial chemokines. There is a synergism between prostaglandin E (PGE) and IL-8 in the infiltration of neutrophils from the peripheral circulation.29 Cyclooxygenase-2 (COX-2), IL-8, and MCP-1 also have similar modulators; IL-1β up-regulates MCP-1, IL-8, and COX-2 production, and this induction can be inhibited by dexamethasone and progesterone (P). Moreover, endometrial explants in culture produce IL-8, which is inhibited by P.30 Estradiol (E2) has also been implicated in the control of endometrial leukocyte migration by regulating the production of the granulocyte–macrophage colony-stimulating factor (GM-CSF) by endometrial epithelial cells.31 In short, P and E2 withdrawal initiates a cascade of events involving EE chemokine production (IL-8, MCP-1, and GM-CSF), which plays a role in inducing the premenstrual influx of leukocytes. On day 1 of pregnancy in rodents, there is a high density of leukocytes in the luminal epithelium, where macrophages are the most predominant cell type. Granulocytes crawl across the epithelium into the lumen to phagocytose sperm debris, suggesting that semen may contain granulocyte-specific chemokines. On day 3 (when apposition occurs), macrophages decrease and are evenly distributed through the uterine tissue.32 On day 5 (when adhesion occurs), they become more closely associated with the epithelium. All these findings suggest that a preimplantation surge of chemokines occurs, including RANTES, MCP-1, or growth factors, and including GM-CSF produced by the EE in response to ovarian steroids that start the implantation process. Endometrial epithelial chemokines can also be regulated by the embryo. Examination of both the embryonic regulation of IL-8 mRNA33 and the production and secretion in human endometrial epithelial cells demonstrates no effect of the human blastocyst on EE IL-8 production and secretion. However, 4-8–cell embryos inhibit IL-8 secretion by EEC, suggesting that endometrial IL-8 might be relevant for the migration of the early preimplantation embryo. Interestingly, there is an up-regulation of the IL8 mRNA expression on EEC co-cultures with embryos, compared to those without embryos.33 Our group has analyzed the mRNA expression of four chemokine receptors (CXCR4, CXCR1, CCR5, and CCR2) throughout the human menstrual cycle using quantitative fluorescence polymerase chain reaction (QF-PCR). CXCR1 and CCR5 receptors showed a progesterone-dependent pattern in the early secretory phase that continued into the mid secretory phase and was maximal in the late secretory phase. CXCR4 (stromal-derived factor-1 [SDF-1] receptor) presented a more pronounced up-regulation in the mid luteal phase than in the early and late luteal phases. Therefore, this receptor, which is located in the endometrial epithelium, is specifically up-regulated within the WOI.34

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Fig 32.2 Graphs indicating median ratio across each experimental group for final gene list. Only genes identified as altered by the presence of an IUD (intrauterine device) are shown. The expression ratio for each gene was calculated relative to expression of the same gene in biopsy 1 from the corresponding patient; then, the median for that gene in all the patients was plotted at each time point. First line (biopsy 2 vs biopsy 1), line 2 (biopsy 3 vs biopsy 1), and line 3 (biopsy 4 vs biopsy 1). Each panel represents one slide, HMN1 and HMN2. All genes are, by definition, identified as altered in expression in biopsy 2 (IUD present) vs biopsy 1 (pre-IUD). The majority remain altered in biopsy 3 (2 months after IUD removal) vs biopsy 1. However, 1 year after biopsy removal, the expression of most genes has returned to similar levels as seen in biopsy 1.

To study the ‘in vivo’ hormonal regulation of the four chemokine receptors, endometrial samples were analyzed by immunohistochemistry. On day 13, when patients were treated solely with estradiol, a very weak staining of CCR2B, CCR5, and CXCR4 was localized in the luminal and glandular epithelium and endothelial cells. During the prereceptive and receptive periods (days 18 and 21, respectively), an increase of staining intensity for the CXCR1 receptor was noted in the glandular compartment. The CCR5 receptor was also immunolocalized, mainly at the luminal epithelium, but also in the stromal and perivascular cells, showing a slight increase compared to the nonreceptive phase. The CCR2B receptor showed a moderate increase of staining in the luminal epithelium on days 18 and 21, while no staining was observed in endothelial cells or stroma. The CXCR4 receptor showed the same staining as CCR5, and was mainly expressed in the epithelium on days 18 and 21. Endothelial and stromal cells were also positive.34 The embryonic impact on chemokine receptors CXCR1, CXCR4, CCR5, and CCR2B polarization in cultured EEC was investigated without our apposition model for human implantation. This model consists of culture human blastocysts in a monolayer of EEC cells. When the blastocyst was absent, chemokine receptors CXCR1, CXCR4, and CCR5 are barely detectable in a few cells of the EEC monolayer. When a human blastocyst was present, however, an increase in the number of stained cells for CXCR1, CXCR4, and

CCR5 was noted, and these receptors were polarized in one of the cell poles of the endometrial epithelium. Immunolocalization and polarization changes in the CCR2B receptor were not present in the EEC monolayer, and this receptor was not regulated by the presence of the human blastocyst.34 Finally, we detected immunoreactive CCR2B and CCR5 receptors in the human blastocyst. CCR2B staining was localized mainly at the inner cell mass, whereas CCR5 staining can be visualized across the trophoectoderm. In all cases (n = 3), CCR5 staining was more intense than that of the CCR2B receptor, while the zona pellucida was not stained in any case. Immunoreactive CXCR4 and CXCR1 were not detected in human blastocysts (Fig 32.2).34 Other clues as to the relevance of the chemokines in the implantation process are derived from the chemokine interferon-inducible protein 10 kDa (IP10). IP-10 has been implicated in the regulation of blastocyst migration, apposition, and initial adhesion in ruminants.35 More indirect evidence of the implication of chemokines in the attraction of the blastocyst arises from clinical trials, demonstrating that scar tissue (i.e. a persistent inflammatory focus) from previous cesarean sections or endometrial surgery became an attractive implantation site.36 Furthermore, some chemokines, such as CX3CL1 (fractalkine), CCL14 (HCC-1), and CCL4 (MIP-1β), seem to promote human trophoblast migration at the feto–maternal interface.37

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Human receptivity using wide genomic analysis The development of the microarray technology38 has made it possible to analyze the expression of thousands of genes at the same time. This technology allows us to globally investigate the genomics of endometrial development. Using a variety of in vitro and in vivo models, the gene expression profile of the human endometrium has been investigated either during decidualization39–41 or in response to progesterone.42 Nowadays, evidence exists about the identification of endometrial gene expression profiles that identify different phases of the menstrual cycle.43–45 Talbi and colleagues examined the biochemical molecular signatures of the human endometrium to prove their possible application as a molecular phenotyping tool.45 Principal component analysis (PCA) of their data revealed that samples were self-clustered into four groups consistent with the histological phenotypes of proliferative (PE), early-secretory (ESE), mid-secretory (MSE), and late-secretory (LSE) phases. Gene ontology analysis of the clusters demonstrated consistent cyclephase specific biological processes and molecular functions. New strategies have been incorporated given the complexity of the endometrial tissue, and the possible dilution factor of genes present in specific compartments that are underrepresented, such as the luminar or glandular epithelium. Laser capture microdissection has been used to separate both stromal and epithelial compartments to analyze the differences in gene expression between them in mice46 and humans.47 In our laboratory, the initial studies were performed using macroarrays that contained 375 genes.48 Immediately, researchers realized the powerful capacity of microarrays, and four papers using microarrays were published between 2002 and 2003.49–52 The differences observed in all these studies referring to design and methodology were reflected in the lack of a large list of consensus genes. Strikingly, only one gene, osteopontin, was consistently up-regulated in all five studies. This structural protein, involved in cell adhesion, is present in the endometrium, and can be localized in the glands and secreted into the lumen.53 Although it has assigned adhesive functions, its role in human embryonic implantation is still unknown.54 Kao’s work50 underlines the presence of members of the Wnt family in the list of up-regulated genes. The marked up-regulation of Dickkopf-1 in all four studies is of particular interest. Dickkopf-1 inhibits Wnt signaling by binding to LRP5/6.55 Wnt7A (-/-) null mice are infertile and present a complete absence of uterine glands and a reduction in mesenchymally derived uterine stroma.56 The role of the Wnt family in the human endometrium and implantation should be considered in future investigations. Uterine receptivity is diminished during controlled ovarian stimulation (COS), used for IVF compared to

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natural cycles.57 The endometrium suffers a morphological advancement in the early luteal phase, which is demonstrated by histological techniques,58 scanning electron microscopy,59 the down-regulation of the endometrial estrogen receptor and progesterone receptor,60 and by biochemical changes in the endometrial fluid.61 In 2004, Mirkin and colleagues62 compared the gene expression profile in the peri-implantatory endometrium in the natural cycle vs gonadotropin-stimulated cycle using recombinant follicle-stimulating hormone (rFSH) with either the gonadotropin-releasing hormone (GnRH) or GnRH antagonist, and with or without the P supplementation of the luteal phase. They concluded that although COS causes structural and functional changes compared to natural cycles, small changes were found when gene expression patterns were compared, and that it may not have a major impact on endometrial receptivity. Our group analyzed the endometrium in COS cycles with different results, assessing the endometrial impact of COS with a long protocol without progesterone supplementation. The endometrial profiles obtained at day hCG+7 (hCG = human chorionic gonadotropin) of COS were compared to LH+7 (LH = luteinizing hormone) of the previous natural cycle in the same patient. This design is important to avoid individual biological variability. In contrast to Mirkin et al 2004, we found that more than 200 genes were dysregulated between COS and natural cycles (hCG+7 vs LH+7).63 In conclusion, after a daily treatment with standard- or high-dose GnRH antagonist in women undergoing COS, the endometrial genomic profile mimics the natural cycle more closely when compared to the GnRH agonist. Nonetheless, the clinical relevance of these findings remains to be elucidated. Since the refractory endometrium represents the opposite part of the spectrum, researchers have investigated the gene expression profile under conditions that will render a receptive endometrium in refractory conditions, such as the presence of an inert IUD64 or endometrial explants treated in vitro with RU486.65 The effect of RU486 on the gene expression profile in endometrial explants was investigated using a cDNA array containing ~1000 verified gene targets. Only 12 genes displayed significant changes in expression; six up- and six down-regulated following the RU386 treatment.65 In this short list of genes, two important endometrial signaling pathways are included (i.e. the JACK/STAT and JNK pathways). With this approach, over 100 transcripts were identified as being acutely regulated or dysregulated on LH+7 by the administration of RU486 to women.66 Our approach to study refractoriness was the investigation into the effect of an intrauterine device (IUD) on the endometrium in fertile patients. Over a 16-month period, we analyzed the gene expression profile of receptive endometrium vs refractory endometrium in the same patient (n = 5) induced by the presence of an

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inert IUD. Surprisingly, the majority of genes that are dysregulated in the presence of the IUD remained dysregulated 2 months after removing the IUD, and recovered 1 year later64 (see Fig 32.2). Undoubtedly, all of these approaches have not only generated valuable information about the physiology of the human endometrium but have also provided a number of potential targets for the development of novel contraceptive or conceptive drugs.

Future prospects Normal hormone-regulated endometrium triggers molecular events, informing the blastocyst that it must produce a new set of molecules to communicate efficiently with, and to implant in, the endometrium. We have described how the human blastocyst behaves similarly to the leukocyte, and how it begins its way to the inflammation foci. The blastocyst, guided by chemokines and other cytokines, up-regulates adhesive endometrial epithelial molecules. Recent advances in molecular biology and gene technology, as well as the sequence of the human genome, invite us to reconsider the endometrial receptivity process from a genomic perspective. In the forthcoming years, however, we must focus not only on global genomic changes in the endometrium but also on proteomic and metabolomic changes to fully understand the complex mechanism of human implantation.

References 1. Shiotani M, Noda Y, Mori T. Embryo-dependent induction of uterine receptivity assessed by an in vitro model of implantation in mice. Biol Reprod 1993; 49: 794–801. 2. Cameo P, Srisuparp S, Strakova Z, et al. Chorionic gonadotropin and uterine dialogue in the primate. Reprod Biol Endocrinol 2004; 2: 50. 3. Dominguez F, Yanez-Mo M, Sanchez-Madrid F, et al. Embryonic implantation and leukocyte transendothelial migration: different processes with similar players? FASEB J 2005; 19: 1056–60. 4. Genbacev OD, Prakobphol A, Foulk RA, et al. Trophoblast L-selectin-mediated adhesion at the maternal–fetal interface. Science 2003; 299: 405–8. 5. Thie M, Denker HW. In vitro studies on endometrial adhesiveness for trophoblast: cellular dynamics in uterine epithelial cells. Cells Tissues Organs 2002; 172: 237–52. 6. Paria BC, Reese J, Das SK, et al. Deciphering the cross-talk of implantation: advances and challenges. Science 2002; 296: 2185–8. 7. Moser B, Loetscher P. Lymphocyte traffic control by chemokines. Nat Immunol 2001; 2: 123–8. 8. Ley K, Kansas GS. Selectins in T-cell recruitment to non-lymphoid tissues and sites of inflammation. Nat Rev Immunol 2004; 4: 1–11. 9. Hey NA, Graham RA, Seif MW, et al. The polymorphic epithelial mucin MUC1 in human endometrium is regulated with maximal expression in the implantation phase. J Clin Endocrinol Metab 1994; 78: 337–42.

10. Meseguer M, Aplin JD, Caballero-Campo P, et al. Human endometrial mucin MUC1 is up-regulated by progesterone and down-regulated in vitro by the human blastocyst. Biol Reprod 2001; 64: 590–601. 11. Vicente-Manzanares M, Sánchez-Madrid F. Role of the cytoskeleton during leukocyte responses. Nat Rev Immunol 2004; 4: 110–22. 12. Vicente-Manzanares M, Sancho D, Yáñez-Mó M, et al. The leukocyte cytoskeleton in cell migration and immune interactions. Int Rev Cytol 2002; 216: 233–89. 13. Sanchez-Madrid F, del Pozo MA. Leukocyte polarization in cell migration and immune interactions. EMBO J 1999; 18: 501–11. 14. Fässler R, Meyer M. Consequences of lack of β1 integrin gene expression in mice. Genes Dev 1995; 9: 1876–908. 15. Luscinskas FW, Ma S, Nusrat A, et al. The role of endothelial cell lateral junctions during leukocyte trafficking. Immunol Rev 2002; 186: 57–67. 16. Vestweber D. Regulation of endothelial cell contacts during leukocyte extravasation. Curr Opin Cell Biol 2002; 14: 587–93. 17. Galan A, Herrer R, Remohi J, et al. Embryonic regulation of endometrial epithelial apoptosis during human implantation. Hum Reprod 2000; 15 (Suppl 6): 74–80. 18. Kamijo T, Rajabi MR, Mizunuma H, et al. Biochemical evidence for autocrine/paracrine regulation of apoptosis in cultured uterine epithelial cells during mouse embryo implantation in vitro. Mol Hum Reprod 1998; 4: 990–8. 19. Bischof P, Meisser A, Campana A. Control of MMP-9 expression at the maternal–fetal interface. J Reprod Immunol 2002; 55: 3–10. 20. Kelner GS, Kennedy J, Bacon KB, et al. Lymphotactin: a novel cytokine which represents a new class of chemokine. Science 1994; 266: 1395–9. 21. Vaddi K, Keller M, Newton RC. The Chemokine Fact Book. London: Academic Press, 1997. 22. Horuk R, Peiper SC. Chemokines: molecular double agents. Curr Biol 1996; 6: 1581–2. 23. Cocchi F, DeVico AL, Garzino-Demo A, et al. Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells. Science 1995; 270: 1811–15. 24. Simón C, Caballero-Campo P, García-Velasco JA, et al. Potential implications of chemokines in the reproductive function: an attractive idea. J Reprod Immunol 1998; 38: 169–93. 25. Robertson SA, Mayrhofer G, Seamark RF. Ovarian steroid hormones regulate granulocyte-macrophage colony-stimulating factor synthesis by uterine epithelial cells in the mouse. Biol Reprod 1996; 54: 265–77. 26. Dudley DJ, Trantman MS, Mitchel MD. Inflammatory mediators regulate interleukin-8 production by cultured gestational tissues: evidence for a cytokine network at the chorio-decidual interface. J Clin Endocrinol Metab 1993; 76: 404–10. 27. Arici A, Seli E, Senturk LM, et al. Interleukin-8 in the human endometrium. J Clin Endocrinol Metab 1998; 83: 1783–7. 28. King A, Loke Y. Uterine large granular lymphocytes: a possible role in embryonic implantation. Am J Obstet Gynecol 1990; 162: 308–10.

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Embryonic and maternal dialogue and uterine receptivity 29. Colditz LG. Effects of exogenous prostaglandins E2 and actinomycin D on plasma leakage induced by neutrophil-activating peptide-l/interleukin-8. Immunol Cell Biol 1990; 68: 397–403. 30. Kelly RW, Illingworth P, Baldie G, et al. Progesterone control of IL-8 production in endometrium and chorio-decidual cells underlines the role of the neutrophil in menstruation and parturition. Hum Reprod 1994; 9: 253–8. 31. Robertson SA, Mau VJ, Tremellen KP, et al. Role of high molecular weight seminal vesicle proteins in eliciting the uterine inflammatory response to semen in mice. J Reprod Fertil 1996; 107: 265–77. 32. De Choudhuri R, Wood GW. Determination of the number and distribution of macrophages, lymphocytes and granulocytes in the mouse uterus from mating through implantation. J Leukoc Biol 1991; 50: 252–62. 33. Caballero-Campo P, Bernal A, Mercader A, et al. Embryonic regulation of IL-8 production and secretion in human endometrial cells. J Soc Gynecol Invest 1998; 5 (Suppl): 117A. 34. Dominguez F, Galan A, Martin JJ, et al. Hormonal and embryonic regulation of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in the human endometrium and the human blastocyst. Mol Hum Reprod 2003; 9: 189–98. 35. Nagaoka K, Nojima H, Watanabe F, et al. Regulation of blastocyst migration, apposition, and initial adhesion by a chemokine, interferon gamma-inducible protein 10 kDa (IP-10), during early gestation. J Biol Chem 2003; 278: 29048–56. 36. Shufaro Y, Nadjari M. Implantation of a gestational sac in a cesarean section scar. Fertil Steril 2001; 75: 1217. 37. Hannan NJ, Jones RL, White CA, et al. The chemokines, CX3CL1, CCL14, and CCL4, promote human trophoblast migration at the feto-maternal interface. Biol Reprod 2006; 74: 896–904. 38. Schena M, Shalon D, Davis RW, et al. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995; 270: 467–70. 39. Popovici RM, Kao LC, Giudice LC. Discovery of new inducible genes in in vitro decidualized human endometrial stromal cells using microarray technology. Endocrinology 2000; 141: 3510–13. 40. Brar AK, Handwerger S, Kessler CA, et al. Gene induction and categorical reprogramming during in vitro human endometrial fibroblast decidualization. Physiol Genomics 2001; 7: 135–48. 41. Tierney EP, Tulac S, Huang ST, et al. Activation of the protein kinase A pathway in human endometrial stromal cells reveals sequential categorical gene regulation. Physiol Genomics 2003; 16: 47–66. 42. Okada H, Nakajima T, Yoshimura T, et al. Microarray analysis of genes controlled by progesterone in human endometrial stromal cells in vitro. Gynecol Endocrinol 2003; 17: 271–80. 43. Ponnampalam AP, Weston GC, Trajstman AC, et al. Molecular classification of human endometrial cycle stages by transcriptional profiling. Mol Hum Reprod 2004; 10: 879–93. 44. Punyadeera C, Dassen H, Klopm J, et al. Oestrogenmodulated gene expression in the human endometrium. Cell Mol Life Sci 2005; 62: 239–50.

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45. Talbi S, Hamilton AE, Vo KC, et al. Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women. Endocrinology 2005; 147: 1097–121. 46. Niklaus AL, Pollard JW. Mining the mouse transcriptome of receptive endometrium reveals distinct molecular signatures for the luminal and glandular epithelium. Endocrinology 2006; 147: 3375–90. 47. Yanaihara A, Otsuka Y, Iwasaki S, et al. Differences in gene expression in the proliferative human endometrium. Fertil Steril 2005; 83 (Suppl 1): 1206–15. 48. Domínguez F, Avila S, Cervero A, et al. A combined approach for gene discovery identifies insulin-like growth factor-binding protein-related protein 1 as a new gene implicated in human endometrial receptivity. J Clin Endocrinol Metab 2003; 88: 1849–57. 49. Carson D, Lagow E, Thathiah A, et al. Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening. Mol Hum Reprod 2002; 8: 971–9. 50. Kao LC, Tulac S, Lobo S, et al. Global gene profiling in human endometrium during the window of implantation. Endocrinology 2002; 143: 2119–38. 51. Borthwick J, Charnock-Jones S, Tom BD, et al. Determination of the transcript profile of human endometrium. Mol Hum Reprod 2003; 9: 19–33. 52. Riesewijk A, Martin J, Horcajadas JA, et al. Gene expression profiling of human endometrial receptivity on days LH + 2 versus LH + 7 by microarray technology. Mol Hum Reprod 2003; 9: 253–64. 53. Johnson GA, Burghardt RC, Spencer TE, et al. Ovine osteopontin: II. Osteopontin and alpha(v)beta(3) integrin expression in the uterus and conceptus during the periimplantation period. Biol Reprod 1999; 61: 892–9. 54. Johnson GA, Burghardt RC, Bazer FW, et al. Osteopontin: roles in implantation and placentation. Biol Reprod 2003; 5: 1458–71. 55. Mao B, Wu W, Li Y, et al. LDL receptor-related protein 6 is a receptor for Dickkopf proteins. Nature 2001; 411: 321–5. 56. Miller C, Pavlova A, Sassoon DA. Differential expression patterns of Wnt genes in the murine female reproductive tract during development and the estrous cycle. Mech Dev 1998; 76: 91–9. 57. Paulson RJ, Sauer MV, Lobo RA. Embryo implantation after human in vitro fertilization: importance of endometrial receptivity. Fertil Steril 1990; 53: 870–4. 58. Kolibianakis EM, Bourgain C, Platteau P, et al. Abnormal endometrial development occurs during the luteal phase of nonsupplemented donor cycles treated with recombinant follicle-stimulating hormone and gonadotropin-releasing hormone antagonists. Fertil Steril 2003; 80: 464–6. 59. Nikas G. Endometrial receptivity: changes in cell-surface morphology. Semin Reprod Med 2000; 18: 229–35. 60. Develioglu OH, Hsiu JG, Nikas G, et al. Endometrial estrogen and progesterone receptor and pinopode expression in stimulated cycles of oocyte donors. Fertil Steril 1999; 71: 1040–7. 61. Simón C, Mercader A, Francés A, et al. Hormonal regulation of serum and endometrial IL-1a, IL-1b and IL-1ra: IL-1 endometrial microenvironment of

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the human embryo at the apposition phase under physiological and supraphysiological steroid level conditions. J Reprod Immunol 1996; 31: 165–84. 62. Mirkin S, Nikas G, Hsiu JG, et al. Gene expression profiles and structural/functional features of the peri-implantation endometrium in natural and gonadotropin-stimulated cycles. J Clin Endocrinol Metab 2004; 89: 5742–52. 63. Horcajadas JA, Riesewijk A, Polman J, et al. Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles. Mol Human Reprod 2005; 11: 195–205.

64. Horcajadas JA, Sharkey AM, Catalano RD, et al. Use of gene-expression profiling to identify human endometrial refractoriness. J Clin Endocrinol Metab 2006; 91: 3199–207. 65. Catalano RD, Yanaihara A, Evans AL, et al. The effect of RU486 on the gene expression profile in an endometrial explant model. Mol Human Reprod 2003; 9: 465–73. 66. Sharkey AM, Catalano R, Evans A, et al. Novel antiangiogenic agents for use in contraception. Contraception 2005; 71: 263–71.

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33 Quality management in reproductive medicine Christoph Keck, Cecilia Sjöblom, Robert Fischer, Vera Baukloh, Michael Alper

Introduction In the past quality management (QM) in the healthcare industry has not become a household term, although recent laws in some countries (for example, Germany) are changing this. The EU Tissue Directive clearly demands a quality management system for any institution handling human gametes/embryos. The primary concern of any healthcare system is, and will continue to be, medical performance. However, if we regard healthcare systems as ‘corporations’ dealing with patients, referring doctors, and employees, in addition to medical performance, then other qualities will have to be taken into consideration. More and more hospitals as well as independent medical practices will have to document the quality of their services to their patients and cost-bearers. This will mean that strict procedures for documentation of results will be needed and, furthermore, medical institutions will have to answer the question of whether or not they provide their services in a costeffective way. Many rules (such as the measures for limiting the spread of infections and the statute on protection against radiation) are set by law. However, beyond these rules, many medical institutions currently develop their own internal standards. These standards are often only informally documented and, most of the time, are fragmentary. Although it is not always obvious, these standards can affect and direct the internal workings of the organization and the interaction of various areas within the company. They may also affect the interaction of the company with external partners. With internal systems such as these, enormous differences can exist from one system to another with respect to the importance and validity of various sections and procedures. The Joint Commission on Accreditation of Health Care Organizations calls these elements of quality management ‘functions.’ One can show that these ‘functions’ differ from one institution to another, no matter whether or not they are applied in clinics or private practices, group or singleprovider practices, or government medical institutions. Essential elements are identifiable and applicable to every institution that aims at fulfilling the wishes and demands of its customers. It is not only patients who

are considered ‘customers,’ but all communication partners, including the referring physicians, the company’s suppliers, and the company’s own employees. The individual elements of a quality management system are developed to different degrees, always according to the tasks and the orientation of the particular institution. They exist in varied, yet always definable relationships, to one another. All of these elements and their interconnection as a whole enable a clinic or private practice to reach the expected and agreed results with the customer on a timely basis, and with an appropriate use of resources. The sum of directive elements and elements that transcend or relate to the process is called the ‘quality management system’ of a clinic or a private practice. Of all the medical fields, reproductive medicine has led the way (in Europe) with the introduction of quality management systems over the past several years. In this chapter, different QM systems are described, the instruments of these systems are discussed, and the question of how QM systems contribute to success in reproductive medicine is addressed.

Different quality management systems In the past, a series of specific QM systems for various industries have come into existence worldwide. In 1964, the Good Production Practice (World Health Organization [WHO] directive) was developed for the pharmaceutical and food industries. The Good Laboratory Practice (Organization for Economic Cooperation and Development [OECD] directive) followed in 1978 and the Hazard Analysis of Critical Control Points (National Advisory Committee on Microbiological Criteria for Foods directive) in 1992. The European Union with its ‘Global Concept’ (1985) strongly promoted the development of a QM systems and expanded them to production and services, which therefore covered the documentation of ecologically justifiable dealings in energy, material, and waste.

ISO 9001 standards The systems that followed – the manuals of the International Standardization Organization (ISO 9000

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Table 33.1

Elements/criteria of the ISO standard

Number Quality element according to ISO 9000 ff.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Responsibility Quality management system Contract control Design management Document and data management Measures Management of products provided by customers Designating and retrospective observation Process management Revision Control of the revision resources Evidence of revisions Defective product management Corrections and preventive measures Handling, storage, packaging, conservation, distribution Quality report management Internal quality audits Training Maintenance Statistical methods

series) – became the most widespread, worldwide standard. In the 1980s, the ISO created regulations for QM systems with the standard series 9001 through 9004 developed for the production of goods and services. These manuals described the basic elements of the QM system in a relatively abstract manner. Medical institutions were required to adapt these standards to suit them and this required some interpretation and modification. The introduction of ISO 9000 states: The demands of the organizations differ from each other; during the creation of quality management systems and putting them into practice, the special goals of the organization, its products and procedures and specific methods of acting must be taken into consideration unconditionally. This means that, for medical applications, the standards state which elements should be considered in the QM system, but the manner in which these elements should be realized in the specific medical organization have to be defined individually. The ISO standards have now been adapted to medical institutions, which, since there is no QM system specifically designed for hospitals or medical practices, is fortunate. ISO 9001 through 9003 standards contain the elements important for a quality system (Table 33.1). The criteria according to which QM systems are applied vary with the type of enterprise. For example, the 9001 standard is applicable to the manufacturing and complicated service companies including hospitals. On the other hand, the 9002 standard is more suitable for rehabilitation and foster-care institutions.1 The

application of a certified QM system for hospitals can be performed on the basis of ISO 9001 or ISO 9004.2 As mentioned earlier, in vitro fertilization (IVF) units occupy a special place within clinical medicine. It is a highly specialized area involving the interaction of staff in various areas, including the laboratory, ultrasound, and administration, and the optimal collaboration between physicians and nurses. Treatment can only be successful when a structured interaction exists between the clinical and laboratory departments. ISO 9001:20003 is very much concentrated on a process approach and directed to the outcome of the process; i.e. that the products or services meet the previously determined requirements. Since this does not necessarily assure that a laboratory will be successful, or that it will achieve the highest level of care for the patients that it serves, assisted reproductive technologies (ART) laboratories may also want to consider additional requirements, including standards concerning qualification and competence. Relevant standards are provided by the ISO/IEC 17025:19994 (IEC being the International Electrotechnical Commission). This standard, entitled ‘General requirements for the competence of testing and calibration laboratories,’ replaces both the ISO/IEC Guide 255 and the European standard EN 45001.6 Compliance with the ISO 17025 standard can lead to accreditation (‘A procedure by which an authoritative body gives formal recognition that a body or person is competent to carry out specific tasks’), which exceeds certification (‘A procedure by which a third party gives written assurance that a product, process or service conforms to specific requirements’). ART laboratories should consider ISO 17025 accreditation (see Chapter 3). However, one should realize that both ISO/IEC Guide 25 and EN 45001 are focused more on the technical aspects of competence, and do not cover all areas within clinical laboratories. It has already been stated that, although the ISO standards are the most widely accepted standards in the world, there is no appropriate international standard for laboratories in the healthcare sector. To fill this ‘vacuum,’ several professional associations and laboratory organizations have also framed and published standards and guidelines, most of which are confined to a specific clinical laboratory discipline. Some specific and relevant examples of guidelines for 7–10 ART laboratories commonly available are: 1. 2.

3. 4.

Guidelines for human embryology and andrology laboratories, the American Fertility Society, 1992. Guidelines for good practice in IVF laboratories, the European Society of Human Reproduction and Embryology (ESHRE), 2000. Reproductive laboratory accreditation standards, College of American Pathology, 2002. Accreditation standards and guidelines for IVF laboratories, the Association of Clinical Embryologists, 2000.

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Quality management in reproductive medicine Plan for improvement

4

1 Plan Continuation or adjustment

Act

Do

Performance of plan

Check 3 Assessment of realization

2

AUDITS

Fig 33.1

Total Quality Management (TQM): the Deming cycle.

The above-mentioned guidelines and standards describe the specific requirements for reproductive laboratories, and include various aspects of the implementation of a QM system. These well-defined standards describe the minimum conditions which should be met by laboratories/clinics. Recently, the EU Tissue Directive11 has been released which demands a quality QM system for every medical institution dealing with human gametes or embryos.

TQM and EFQM There is a wide range of quality management models and strategies based on continuous improvement. Two of the best-documented models/strategies are Total Quality Management (TQM) and the Excellence Model of the European Foundation for Quality Management (EFQM). Total Quality Management is an all-encompassing concept that integrates quality control, assurance, and improvement. It is more a philosophy than a model. The basics of this concept were developed after World War II by Deming. Both the TQM and the EFQM models incorporate the objective of continuously striving to improve every aspect of a service, and require continuous scrutiny of all components of the quality management system of an organization. Measurement and feedback are crucial elements in quality management. This can be illustrated by the so-called Deming cycle (‘Plan–Do–Check–Act’ cycle) (Fig 33.1). Important elements of a TQM program are: 1. Appropriately educated and trained personnel with training records. 2. Complete listing of all technical procedures performed. 3. Housekeeping procedures: cleaning and decontamination procedures. 4. Correct operation, calibration, and maintenance of all instruments with manuals and logbook records. 5. Proper procedure policy and safety manuals. 6. Consistent and proper execution of appropriate techniques and methods. 7. Proper documentation, record-keeping, and reporting of results.

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8. Thorough description of specimen collection and handling, including verification procedures for patient identification and chain of custody. 9. Safety procedures, including appropriate storage of materials. 10. Infection control measures. 11. Documentation of suppliers and sources of chemicals and supplies, with dates of receipt/expiry. 12. System for appraisal of test performance correction of deficiencies and implementation of advances and improvements. 13. Quality materials, tested with bioassays when appropriate. 14. Quality assurance programs.

Quality policy One of the first steps for the implementation of a QM system in medical institutions is to define the quality policy. Quality policies are a group of principles according to which the medical institution works. This, by necessity, includes the best treatment practices, but also must by far exceed this goal. Although successful treatment of an existing disease or reduction of discomfort is certainly the highest priority for most medical institutions, it might be an important goal to achieve this in the most efficient manner possible. This means that structure is needed to assure that diagnostic and therapeutic procedures are performed using as little financial, organizational, personnel, or time resources as possible, while still striving for a high quality of treatment. After all, optimum quality is achieved by the ‘right’ balance of cost with quality achievement. The quality policy of a medical institution cannot be defined by a single person (e.g. the owner or medical director), but should be developed as a consensus between management and employees. Only in this way will personnel identify with the quality policy of the institution. A quality policy should be formulated in an active manner and the formulation should also be short and simple so that every employee can repeat the quality policy at any time. The most important aspects of the quality policy should be posted in suitable and accessible areas of the institution for employees, patients, and visitors, to strengthen the employees’ knowledge of common goals, improve their identification with their own fields of competence, and communicate these principles to others. It is important to state that the quality policies should be reviewed periodically to make sure that the principles are still valid and that management and employees still agree with them. As an organization’s perspectives and goals change, the quality policy needs to be modified accordingly.

Management’s responsibility In spite of the fact that the responsibility of management (or the governing structure) can be defined

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Nursing care

Reception

Patient care

Blood-drawing, intraoperational, and hospital care

Department head

Staff physicians

Gynecologic office visits, andrologic office visits

Laboratory

Administration

Research group

IVF laboratory, andrologic laboratory

differently in various medical institutions, according to ISO standards, certain generally valid aspects can be defined. The hierarchy of the institution has to be defined and outlined clearly. While, in most cases, hospitals are administered by an appointed director, the structure might be more difficult in private centers with multiple partners in equal positions. In such cases, an agreement that describes the division of responsibilities for particular fields among the partners must be in place and in these cases several possibilities are available. For, example, it is possible to place specific responsibilities permanently under the authority of one of the partners (e.g. personnel development/ accounting/billing, etc.). However, for many privately held practices, a model of dividing these tasks on a rotational basis has been successful; i.e. dividing the responsibility for various fields equally among all of the partners (in leadership positions) so that all partners are familiar with the different responsibilities. The picture becomes far more complex if there are many administrative layers to the organization and it is here that clear descriptions of authority for all positions within the organization are required and must be available to everyone within the organization. The more complex the hierarchic structures within a medical institution are, the more precisely these structures have to be defined for the system to work effectively and robustly at all times and under all (extraordinary) conditions. The ‘decision’ of the head of the organization must be available at any time, even if he or she is absent. Therefore, this must be absolutely clear to everyone within the organization who has the competence and authority to make decisions. It is also important for all partners outside of the company to be aware of who the decision-makers are for various tasks. There are various ways of making these structures as transparent as possible. One easy way is the development of an organizational diagram (Fig 33.2).

Administration and billing

Secretaries, billing, medical records

Fig 33.2 Organizational diagram. IVF, in vitro fertilization.

This organizational diagram can be placed in a suitable and accessible location, helping employees to understand everyone’s roles and responsibilities. Furthermore, making the organizational diagram available to everyone strengthens trust, cooperation, and professionalism within the company. It is also important in communication with patients, interested parties, or cooperating departments and, therefore, the organizational diagram should be updated frequently. Management should strongly support the quality policies for the company, and should take an active part in its development and implementation. It is important to lead by example.

Management of processes Processes are all the procedures that are necessary for the completion of tasks. For medical facilities, the most important processes are those of diagnostic and therapeutic procedures. In addition, many other processes are involved in the care of patients, such as the scheduling of patients for tests, communication, and anything else that may greatly affect the patient’s (= customer’s) perspective. Sometimes poor communication can ruin a patient’s experience, despite the best diagnostic procedures within the organization. In fact, it is our observation that it is more likely that a patient will leave a medical facility because of an organizational problem such as a substandard secretarial or administrative problem than in the case of a medical deficiency. Even with properly working medical treatment, poor communication with colleagues can endanger or directly destroy the positive result of the treatment. When starting to establish a QM system, it is necessary to define and describe precisely all relevant processes and to structure them according to QM guidelines. These descriptions are often best realized by flow diagrams that can overlap in various places (Fig 33.3). These areas of contact between two flow

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IVF

439

Process 4

SS 1 Internal sperm sample

+ Egg cell

External sperm sample SS 4

Evaluation Result −

No culture

+

With given indication

Sperm preparation, centrifugation

Egg cell culture SS 2

Process 2

Insemination after 2–4 hours

Swim-up

PN exam (16 h after insemination)

ICSI

Process 1 Result −

+

No 2 PN

Up to # × 2 PN

Culture

Culture

PN exam

Process 3

More than 3 × 2 PN

+ SS 3

− Egg cell destruction

+

Cryopreservation

Egg cell destruction

Embryo transfer 48−72 h after OPU

diagrams are called ‘boundaries, interferences, joints or areas of juncture.’

Documentation in a QM system In addition to defining the processes relevant for the system, it is important for everything to be documented. The different levels of documentation are shown in Fig 33.4. One of the most important documents in a QM system is the quality manual. The main purpose of the quality manual is to outline the structure of the documentation used in the quality system.12 It should also include or refer to the standard operating procedures (SOPs). There should be clear definitions of the management’s areas of responsibility, including its responsibility for ensuring compliance with the international standards on which the system is built. A simple overview of the quality system requirements and the position of the quality manual are shown in Fig 33.4. A good-quality manual

Fig. 33.3 Flow diagram of processes. SS, boundary interface; IVF, in vitro fertilization; PN, pronuclei; OPU, oocyte pick-up; ICSI, intracytoplasmic sperm injection.

should be precise and brief; it should be an easily navigable handbook for the whole quality system. The most important procedures are preferably included in the manual itself, but deeper descriptions should be referred to in underlying documentation. An easy way to start building a system is to make up a table of contents for the quality manual and to decide which processes should be described in the manual and which should rather be described in the underlying documentation (e.g. SOPs). Whereas the quality manual contains more general information, the individual processes and procedures are described in a more detailed way in handbooks/job instructions or SOPs. These SOPs go through the processes step-by-step and describe the materials and methods used and the way the process is performed precisely. Standard operating procedure manuals should be available to all personnel and every single procedure in these manuals must be fully documented with signature, date, and regular review.

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Level 1 Quality manual

Defines approach and responsibility Level 2

Procedures

Job instruction SOPs Results and other documentation

Defines Who? What? When? Level 3 Answers How? Level 4 Shows the system is operating

Document control According to ISO 9001:2000 4.2.3 the clinic should establish and maintain procedures to control all documents that form part of its quality documentation. This includes both internally generated documentation such as SOPs and protocol sheets, and externally generated documentation such as law texts, standards, and instruction manuals for equipment. Document handling and control are an important part of the quality system and, if not designed properly, can become a heavy load for a smooth running system. Since it is something that touches every part of the system, it is important to sit down and think through how this system of paperwork shall be handled in your clinic and to ensure that the system you choose covers the demands of the standards. The identification of the documents should be logical, and it is a good suggestion to use numbers as unique identifiers. The same identification number could then be used for the file name within the computerized version. The issue number in parentheses or after a dash could follow this number. Pagination, is important. If you choose not to use pagination, you must clearly mark where the document starts and ends. The dates of issue together with information on who wrote the document and who approved it (signature) are usually included in the document header. Questions that should have an answer in your document control system: 1. Is all documentation in the laboratory or clinic covered by your document control system? 2. Who writes or changes the document? 3. Who approves and has the authority to issue documents? 4. Does the document have: a. b. c. d.

A unique identification? Issue number and current revision status? Date of latest issue? Pagination?

Table 33.2 1. 2. 3. 4. 5. 6. 7.

Fig. 33.4 Levels of documentation. SOP, standard operating procedure.

The Seven Tools

Ishikawa diagram Pareto diagram Histogram Statistical process control Correlative diagram General graphic depiction Checklists

5. Where can I find the document: physical location, level in the system, and on computer file? 6. Who assures that only the latest issue of the document is present in the system, removes outdated issues, and files them? 7. Are amendments to documents clearly marked, initialled, and dated? 8. How are changes in a document implemented with the personnel?

Documentation of results A very important level of documentation is that of the ‘results.’ This includes not only the results of treatment such as pregnancy rate per treatment cycle but also all documents referring to: 1. 2. 3. 4.

Control of quality records. Internal audits. Control of nonconformity. Corrective and preventive action.

The control of nonconformity has to be done for all elements, especially system checks (Table 33.2). As an example for control of nonconformity, the control cards for incubators in the IVF laboratory should be mentioned (Fig 33.5). Incubators are one of the most important pieces of equipment in the IVF laboratory and need to be controlled properly. Two markers of incubator performance are the temperature

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37.4°C OG = upper action limit

5.2% CO2 37.2°C 5.0% CO2 37.0°C

UG = lower action limit

4.8% CO2 36.8°C

Problem

Monday

1. .............

IIII IIIII IIII

2. .............

IIIII

3. ............. Totals

Tuesday

Wednesday

Thursday

Friday

Totals

IIIII IIIII

IIIII

II

37

III

II

III III

I

17

IIII IIIII

IIIII

IIIII

IIII IIIII

II

30

27

15

17

20

5

IIIII

II

and the CO2 level. These two parameters are documented on the control cards, and upper and lower limits of tolerance are defined to determine when corrective actions are needed. It is useful to plot results of system checks on a graph, so that there is a clear visual image that can monitor: 1. Dispersion: increased frequency of both high and low numbers. 2. Trend: progressive drift of reported values from a prior mean. 3. Shift: an abrupt change from the established mean. If nonconformity to the standard is diagnosed, it is important to collect data (Fig 36.6) on: 1. 2. 3.

Fig. 33.5 Monitoring temperature and CO2 levels in an incubator.

25 26 27 28 29 30 31

37.4°C

1 2 3 4 5 6 7 8 9 10

5.2% CO2 36.4°C

When the problem was realized. How often the problem could be identified. How conformity to the standards could be reassured.

Audits and management reviews Audits are essential in ensuring that a quality system is working. Audits can be internal, initiated by the organization itself, or external, initiated by a governing body, certification, or accreditation body. ISO 9001:2000 8.8.2 lays out the rules for internal audits and demands that the clinic undertakes internal audits at planned intervals in order to determine how well the system is functioning and if it is effectively implemented and maintained. Audits are tools for improving and keeping your system up to date

Fig. 33.6

Tally of problems.

with the standards and it is the quality manual that should include specific instructions covering both how and how often they shall be performed. The management usually chooses internal auditors, and they should be familiar with both the standards and the activities performed in the clinic. The manual should include a document describing the approach and the areas of responsibility for the internal auditors and have well-documented procedures for how internal auditors are trained. To achieve a certification according to ISO 9001:2000, the clinic needs to be audited externally by a certification body. Many organizations believe that having an audit and not finding any nonconformities is a proof of an outstanding performance; however, it could very well be a proof of an inadequate audit procedure. If an audit is properly conducted, even in organizations with outstanding performance, areas for improvement will be found; therefore, people should put in a lot of effort to find the right certification body to undertake the audits. Some questions that might help to identify a good certification body are: • • • •

Are they accredited to certify medical institutions? Have they previously certified IVF clinics and how many? Do they have IVF experts on their audit team? How much time do they allocate to the audit?

While to some it may seem obvious, it is important to mention, especially with respect to the factors above, that the cheapest certifying body is not necessarily the best.

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Influence magnitudes

Medical area Gynecology, urology, anesthesiology

Personnel requirement plan

Administration and billing

Nonmedical area Medical tech., biologists, IVF lab techs.

Assistants, receptionists, etc.

Obtaining patients

Offered services

Market development (national healthcare system)

Calculation base/reference parameters

Key indicator = the task = the treatment of a (‘Key volume indicator’)

Together with the audits, the management review is important for improvement of the system and for the long-term correction of errors and incidents that might occur. According to ISO 9007:2000 5.6, the management of the clinic with executive responsibility shall periodically conduct a review of the quality system and testing activities. The quality manual shall include a written agenda for these reviews, which should fulfill the demands in the standard.

Incidents and complaints All clinics should have a policy and procedure for the resolution of incidents and complaints received from patients, clients, and/or other parties. The routines of how these are filed and how corrective actions are taken should be documented in a clinic’s quality manual. When applying a quality system it is important not to hide these incidents and complaints, but to use them as resources to improve the system. The management reviews should ensure that the incidents and complaints lead to long-term corrections and improvements of the quality of work.

Staff management High-quality treatment can only be realized with qualified staff. Therefore the recruitment, training, and motivation of highly qualified people is one of the most important tasks for the management team of an organization. To make sure that a sufficient number of qualified people are working within the respective areas of the institution, a staff requirement plan should be developed. This can be organized in different ways:

Fig. 33.7 Example of a staff requirement plan. tech, technician, IVF, in vitro fertilization.

1. Allocating people according to their abilities. 2. Allocating people according to different responsibility levels. 3. Allocating people according to the type of work that has to be done. In most medical institutions it is recommended to define the levels for which the number of staff should be planned. Thus, the leading level (management) and other levels (which can be further divided according to qualification) are defined. The number of employees should be determined, for particular fields, according to their tasks and the range of treatments. This is why a regulation for the equalization of staff must be created. This system makes planning easier, and emphasizes the qualifications and, for instance, the procedure of substitution. The development of work descriptions is crucial for this system. They must be created for particular posts, and must state, among other things, at which post a given employee works, what his or her qualification is, and which qualification attributes are required. In addition to this formal information, the work description should also contain information about the employee’s personal attributes. For various posts, different qualities are important: 1. Social competence. 2. Organizing abilities. 3. Communication abilities, etc. The staff requirement plan (Fig 33.7) must be set up so that it is possible to react sufficiently to unexpected situations. Furthermore, it must consider staff absenteeism caused by holidays, illness, and further

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education. A minimal presence of employees must be determined for certain fields, which does not depend on the actual workload. For the development of a staff requirement plan for an IVF center, the medical as well as the nonmedical areas have to be defined and considered. The question of how many people are needed to do the job properly can be answered on the basis of calculating the ‘influence magnitudes.’ The type of services offered strongly influences the number of people required. Thus, the staff requirements are different in a center in which predominantly conservative treatments and intrauterine inseminations are performed, compared with a center in which predominantly IVF/intracytoplasmic sperm injection (ICSI) and cryopreservation cycles are performed.

Training of employees One of the most important principles for the management of a medical institution is: ‘Give your employees the chance to be the best.’ This means that if you expect your employees to do their work at the highest-quality level possible, you should give them proper training. In principle, there are two different types of educational event: 1. Internal events of further education. 2. External events of further education (i.e. conventions, conferences, workshops, etc.). The advantage of internal events of further education is that they can be offered on a regular basis and are usually ‘low-budget-projects,’ whereas external events need more organizational and financial input. However, when carefully planned, external educational events sometimes have a higher motivational aspect. So the management should take care to offer a balanced program of internal and external educational events. In order to make it possible to use the clinic’s resources adequately and to estimate and plan the potential of development with regard to the individual abilities of particular employees or with regard to the abilities of the entire institution, educational activities should be evaluated and analyzed at regular intervals. For example, at the beginning of each year, the employee should decide which educational events he or she would like to visit or take part in. This helps the management to introduce new fields of activity, and also allows them to perform advance planning of the specialization. It is striking to see that, in most ART centers, detailed and prospective plans have been developed for the training of medical doctors, but that far less attention has been paid to the training of nurses, technicians, etc. However, a well-trained nurse can significantly reduce the workload for the doctor and, furthermore, tremendously increase the patient’s trust in the institution while also improving the referring doctor’s satisfaction. Therefore, besides training activities for the doctors, adequate educational events for nurses and technicians, etc., should be considered.

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Interaction between management and employees Success in reproductive medicine clearly depends on an optimal interaction between different professional groups: i.e. only if doctors communicate and work together with staff in the laboratory, nurses, receptionists, etc. can success be achieved. The same is true for the interaction between management and employees. Communication and collaboration between different professional groups of the same hierarchic rank is called ‘horizontal’ communication, whereas communication and collaboration between professional groups of different hierarchic ranks is called ‘vertical’ communication. One of the most important instruments to optimize vertical communication is the ‘staff interview.’ The staff interview is a course that runs periodically during which the employee and his or her direct superior think (independently of their everyday communication) about their collaboration. The interview should take place in a structured way and a protocol should be written and signed by both sides, so that the content of the interview is assigned some kind of formal character. However, details of the interview can never be communicated with others without mutual consent. For the employee, the goals/opportunities of the interview are: 1. To become familiar with the goals of the department. 2. To realize weaknesses and strengths. 3. To be able to discuss own experiences/opinions on the management style. 4. To discuss further strategies for professional development. 5. To participate in planning goals/strategies for the future. For the superior, the goals/opportunities of the interview are: 1. To discuss the co-worker’s performance. 2. To focus the activities of the employee on future goals of the institution. 3. To increase mutual understanding in the event of problems. 4. To increase the employee’s responsibility. 5. To get feedback on his/her management skills. For the above-mentioned reasons, the staff interview is one of the most important and powerful tools in staff development, and should be widely used in the process of continuous improvement.

Conclusions No internationally accepted standards exist for quality in the IVF laboratory and the IVF center as a whole. In order to assure high quality and continual improvement, it is recommended that all IVF centers striving for excellence should consider a QM system. Furthermore, legal

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guidelines and the recently released EU Tissue Directive clearly demand a QM system for medical institutions. A QM system allows the organization to gain control of its documents and procedures and to monitor the clinical and nonclinical outcomes. Furthermore, the issues of staff recruitment and staff development can be addressed systematically and thereby, again, the overall outcome will be improved. The ISO standards offer the medical facility access to an internationally endorsed and proven QM system. ART practitioners in particular have the unique opportunity to set the standard in medicine for quality management principles.

References 1. Pinter E, Vitt KD. Umfassendes Qualitätsmanagement für das Krankenhaus – Perspektiven und Beispiele. Frankfurt: pmi-Verlag, 1996. 2. Viethen G. Qualität im Krankenhaus. Grundbegriffe und Modelle des Qualitätsmanagements. Stuttgart: Schattauer-Verlag, 1995. 3. ISO 9001:2000. Quality management systems – Requirements. Geneva: International Standardization Organization, 1987. 4. ISO/IEC 17025:1999. General requirements for the competence of testing and calibration laboratories. Geneva: International Standardization Organization, 1999. 5. ISO/IEC Guide 25:1990. General requirements for the competence of testing and calibration laboratories. Geneva: International Standardization Organization, 1990. 6. EN 45001:1991. General criteria for the operation of testing laboratories. 7. Fertil Steril 1992; 58 (Suppl 1). 8. Gianaroli L, Plachot M, Van Kooij R, et al. and Committee of the Special Interest Group on Embryology. ESHRE guidelines for good practice in IVF laboratories. Hum Reprod 2000; 15: 2241–6. 9. http://www.cap.org/lap/rlap.html 10. Association of Clinical Embryologists UK. Accreditation standards and guidelines for IVF laboratories. http://www.ivf.net/ace/accred1.html (2001) 11. Directive 2004/23/EC of the European Parliament and of the Council of 31 March, 2004 on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells. Official Journal of the European Union, L 102, 7.4.2004, pp. 48–58 http://europa.eu.int/eur-lex/en/oj/. 12. Huismam W. Quality system in the medical laboratory: the role of a quality manual. Ann Biol Clin (Paris) 1994; 52: 457–61.

Suggested reading Alper MM, Brinsden PR, Fischer R, Wikland M. Is your IVF programme good? Hum Reprod 2002; 17: 8–10. American Society for Reproductive Medicine. Revised minimum standards for in vitro fertilization, gamete intrafallopian transfer and related procedures. http:// www.asrm.com/media/practice/revised.html (1998) Bloor G. Organisational culture, organisational learning and total quality management: a literature review and synthesis. Aust Health Rev 1999; 22: 162–79.

Bron MS, Salmon JW. Infertility services and managed care. Am J Manag Care 1998; 4: 715–20. Brown RW. Errors in medicine. J Qual Clin Pract 1997; 17: 21–5. Clancy C. AHRQ: coordinating a quantity of quality. Healthplan 2003; 44: 42–6. Collings J. An international survey of the health economics of IVF and ICSI. Hum Reprod Update 2002; 8: 265–77. Colton D. The design of evaluations for continuous quality improvement. Eval Health Prof 1997; 20: 265–85. Darr K. Risk management and quality improvement: together at last – Part. Hosp Top 1999; 77: 29–35. Garceau L, Henderson J, Davis LJ et al. Economic implications of assisted reproductive techniques: a systematic review. Hum Reprod 2002; 17: 3090–109. Geraedts HP, Montenarie R, Van Rijk PP. The benefits of total quality management. Comput Med Imaging Graph 2001; 25: 217–20. Glattacker M, Jackel WH. [Evaluation of quality assurance – current data and consequences for research] Gesundheitswesen 2007; 69(5): 277–83. Gondringer NS. Benchmarking: friend or foe. AANAJ 1997; 65: 335–6. Greenberg L. Accreditation strengthens the disease management bridge over the quality chasm. Dis Manag 2003; 6: 3–8. ISO/DIS 15189:2:2002. Medical Laboratories – Particular requirements for quality and competence. Geneva: International Standardization Organization, 2002. Matson PL. Internal quality control and external quality assurance in the IVF laboratory. Hum Reprod 1998; 13(Suppl 4): 156–65. Minkman M, Ahaus K, Huijsman R. Performance improvement based on integrated quality management models: what evidence do we have? A systematic literature review. Int J Qual Health Care 2007; 19(2): 90–104. Sackett DL, Rosenberg WMC, Gray JAM, et al. Evidencebased medicine: what it is and what it isn’t. Br Med J 1996; 312: 71–6. Sandle LN. The management of external quality assurance. J Clin Pathol 2005; 58(2): 141–4. Sciacovelli L, Secchiero S, Zardo L, et al. Risk management in laboratory medicine: quality assurance programs and professional competence. Clin Chem Lab Med 2007; 45(6): 756–65. Shaw CD. External quality mechanisms for health care: summary of the ExPeRT project on visitatie, accreditations, EFQM and ISO assessment in European Union countries. External Peer Review Techniques. European Foundation for Quality Management. International Organization for Standardization. Int J Qual Health Care 2000; 12: 169–75. Varkey P, Reller MK, Resar RK. Basics of quality improvement in health care. Mayo Clin Proc 2007; 82(6): 735–9. Vogelsang J. Quantitative research versus quality assurance, quality improvement total quality management and continuous quality improvement. J Perianesth Nurs 1999; 14: 78–81. Warnes GM, Norman RJ. Quality management systems in ART: are they really needed? An Australian clinic’s experience. Best Pract Res Clin Obstet Gynaecol 2007; 21(1): 41–55.

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Quality management in reproductive medicine Yasin MM, Meacham KA, Alavi J. The status of TQM in healthcare. Health Mark Q 1998; 15: 61–84.

Relevant internet addresses http://www.agrbm.de http://www.asrm.com

http://www.eshre.com http://www.ferti.net http://www.guideline.gov http://www.iso.ch http://www.isoeasy.org http://www.ivf.net/ace http://www.praxion.com

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34 Indications for IVF treatment: from diagnosis to prognosis Nick S Macklon, Frank J Broekmans, Bart CJM Fauser

Introduction Since the birth of the first IVF baby almost 30 years ago, dramatic developments have occurred in in vitro fertilization (IVF). IVF was initially designed to overcome the problem of tubal infertility but is now widely held to represent the treatment of choice for unexplained infertility, male factor, endometriosis, and ovarian dysfunction resistant to ovulation induction.1,2 The introduction of intracytoplasmic sperm injection (ICSI) has rendered severe forms of male infertility amenable to treatment and further widened the scope of IVF.3 High-profile publicity given to the latest achievements with IVF has led to its perception as a panacea for all those having difficulty in conceiving a pregnancy. This has been reflected in the rapid expansion of indications for IVF and an estimated current annual number of IVF cycles worldwide approaching 500 000.4 The degree to which IVF merits this growth in application remains unclear, however, since prospective randomized trials comparing the effectiveness of IVF with simpler fertility treatments remain scarce. In recent years, increasing attention has been given to the balance between benefits, burdens, and risks of IVF treatment, and the concept of achieving pregnancy at all costs is being increasingly rejected.5 The level of provision of IVF treatment varies greatly from country to country, and few provide access to IVF treatment to all those who may benefit.6 The challenge is therefore two-fold: first to identify those couples for whom the potential benefits of IVF treatment merit the associated risks and costs, and secondly to improve the risk/benefit balance in favor of the latter. In recent years, progress has been made on both counts. New studies focusing on IVF outcomes have further clarified those factors which determine outcome and offer the prospect of individualizing ovarian stimulation protocols and embryo transfer policies. The concept of considering indications for IVF has become more sophisticated than simply identifying a cause for infertility which might be amenable to IVF.

Conventional approach: diagnosis as the indication for IVF The original indication for IVF, tubal disease, remains an important medical indication for IVF, but in terms of numbers of patients treated, other indications have become more important. National guidelines for IVF continue to focus primarily on underlying diagnoses when determining indications for IVF (Table 34.1). Over the years, a consensus has grown as to what constitute the primary medical indications (Fig 34.1). This is reflected in the similar frequency of indications revealed by independent databases. Variations between databases may simply reflect differences in definition or population. Patients with low-grade endometriosis may, for instance, be considered as having either a tubal or an idiopathic indication. Depending on inclusion and exclusion criteria, infertility is categorized as idiopathic in 10% to more than 30% of cases. The extent to which the underlying pathology itself can impact on the chance of success has been the subject of considerable study. Initial reports indicated certain causes of infertility to be associated with a lower chance of success than others. However, large published studies on the effect of the cause of female infertility have shown no significant effect on outcome of IVF2,7,8 (Table 34.2, Fig 34.2). Instead, pregnancy chances were again determined by female age, duration of infertility, and previous pregnancy.2 In recent years, the impact of certain underlying causes of infertility on IVF outcome has become clearer.

Endometriosis Early reports from major IVF centers indicated that IVF success rates in women were not adversely affected by endometriosis.9,10 These were followed by a number of studies which reported a significant decrease in the fertilization rate in vitro in women with endometriosis.11,12 More recent studies2,13 cast further doubt on the true impact of endometriosis on IVF outcome. In a meta-analysis studying the effect of

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Table 34.1

IVF indications as recommended by the Dutch Society of Obstetrics and Gynecology

1. Tubal pathology • If tubal surgery is not a realistic option, IVF is method of choice. • In case of impaired tubal function but no occlusion is present, or following tubal surgery, IVF is method of choice after an infertility duration of 2 years or longer. Depending on the female age IVF can be done after a shorter duration of infertility. 2. Unexplained infertility (idiopathic)* • In case of idiopathic infertility IVF is indicated if the duration is 3 years or longer. If the woman is older than 36 years, IVF may be considered earlier. 3. Male infertility • Total motile sperm count (TMC) < 1 million: first treatment of choice is ICSI • TMC > 1 and < 10 million: IVF can be performed if infertility duration is 2 years or longer* • TMC > 10 million: treat as unexplained infertility 4. Endometriosis • In case of mild or moderate endometriosis treat as unexplained infertility. • In case of severe endometriosis policy treat as tubal pathology 5. Cervical factor / immunological infertility∗ • After an infertility duration of 2 years, IVF is indicated. This may be considered sooner if the woman is over 36 years of age. 6. Hormonal disturbances* • Anovulatory cycle abnormalities are an indication for IVF if 12 cycles of treatment with ovulation induction have been unsuccessful ∗

In these situations intrauterine insemination treatment merits consideration before proceeding to IVF.

Ovulatory dysfunction 6.0% Endometriosis 6.0%

Uterine factor 1.3% Male factor 18.5%

Other cause 7.0% Diminished ovarian reserve 7.9% Multiple factors, female+male 18.4%

Tubal factor 11.3% Multiple factors, female only 11.7%

Unexplained cause 12.0%

*Total does not equal 100% due to rounding

Table 34.2

Fig 34.1 Relative frequency of indication for assisted reproductive technologies reported by US IVF centers (CDC 2004).8

Impact of cause of infertility on live birth rate from IVF Live birth rate (%) (95% CI)

Cause of infertility

Number of cycles

Tubal disease Endometriosis Unexplained Cervical

19096 4117 12340 4232

Per treatment cycle

Per egg collection

Per embryo transfer

13.6 (13.0–14.0) 14.2 (13.2–15.3) 13.4 (12.9–14.1) 14.2 (13.2–15.3)

15.0 (14.5–15.6) 15.9 (14.7–17.0) 15.2 (14.6–15.9) 16.2 (15.1–17.4)

16.5 (15.9–17.1) 17.9 (16.6–19.3) 19.7 (18.8–20.5) 18.8 (17.5–20.2)

Adapted from Templeton et al.2

endometriosis on IVF, significantly lower fertilization, implantation, and pregnancy rates were observed in endometriosis when compared to tubal factor controls.14 Moreover, stronger negative associations were consistently observed in women with severe disease.

However, none of the studies included were randomized controlled trials, limiting the conclusions that could be drawn. Furthermore, insufficient data are available on the true value of treating endometriosis prior to IVF treatment.

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Live births per cycle (%)

50 40 34.7 30

33.3

32.0

29.9

29.3 25.1

24.2

26.1 23.2

20 13.9 10

0 s, or ct fa ale le tip +m , ul le M ma tors fe fac le nly se tip o ul le au M ma d c fe ine la xp ne e U us ca er th O or ct fa e al or M ct fa e s r in si te U r io n et ia m ar do ov d En n he io is ct in e un im v D ser ysf re y d r to la vu r O to ac lf ba Tu

Diagnosis

Tubal dysfunction No randomized controlled studies have been performed comparing tubal surgery and IVF in patients with tubal damage or dysfunction. The decision to carry out IVF rather than tubal surgery has therefore a large subjective element, and tends to be based on a clinical assessment of the severity of tubal damage, the age of the patient, and the availability of specialized surgical services and IVF.15 The impact of tubal dysfunction on IVF outcome is similarly controversial.16,17 Although tubal disease in general is not associated with poor outcome from IVF, there is increasing evidence that distal tubal disease associated with hydrosalpinx may affect the chances of success from IVF treatment. Several retrospective studies have indicated that hydrosalpinges negatively influence the chance of success with IVF by decreasing implantation rates.18–20 In a meta-analysis evaluating differences in pregnancy rates after IVF in tubal infertility with and without hydrosalpinx, pregnancy rates of 31.2% were observed in the absence of hydrosalpinx and 19.7% in the presence of hydrosalpinx (odds ratio [OR] = 0.64, 95% confidence interval [CI] 0.56–0.74).21 It has been less clear whether surgical intervention for tubal disease prior to IVF is effective in improving the likelihood of successful outcome, since most data are retrospective or poorly controlled. In a meta-analysis of three randomized controlled trials, the odds of pregnancy (OR = 1.75, 95% CI 1.07–2.86) and of ongoing pregnancy and live birth (OR = 2.13, 95% CI 1.24–3.65) were increased with laparoscopic salpingectomy for hydrosalpinges prior to IVF.22 The major study addressed in this metaanalysis demonstrated that this effect was due to the positive effect among those with a hydrosalpinx visible on ultrasound.23,24 In a recent randomized study, proximal tube occlusion was shown to be as effective as salpingectomy in improving implantation rates

Fig 34.2 The impact of cause of infertility on IVF outcomes in the USA, given as live births per cycle (CDC 2004).8

when compared to no intervention.25 Any discussion of the potential risks and benefits should also highlight the potential effect of delaying IVF treatment, especially in older patients where other factors may play the determining role.

Anovulation Chronic anovulation is a common cause of infertility. Most anovulatory women have irregular menstrual cycles and normal serum follicle-stimulating hormone (FSH) concentrations (World Health Organization [WHO] group 2).26,27 Depending on the criteria used, polycystic ovary syndrome (PCOS) is diagnosed in approximately 60–70% of these women.28,29 Cumulative singleton live birth rates of up to 71% in 2 years can be achieved in this group of patients with classical induction of ovulation, applying clomiphene citrate as firstline and exogenous gonadotropins as second-line treatment.30 Alternative treatment options such as IVF should therefore be avoided as first-line therapy in these patients, except for subgroups with a poor prognosis. Those women who may benefit from IVF as first-line therapy can be identified by older age, longer duration of infertility, and higher insulin:glucose ratio.30 When classical ovulation induction fails, IVF is a feasible therapeutic option.31 The outcome of IVF in women with PCOS has been the subject of a number of studies, and conflicting data have been published. Early studies showed that in PCOS, more oocytes could be retrieved, but fewer fertilized than in control patients, suggesting that an increased number of immature oocytes are recruited. Moreover, both mature and immature oocytes of PCOS patients show reduced fertilization rates, presumably due to endogenous hormonal imbalance.32,33 Despite reduced overall fertilization, IVF pregnancy rates in PCOS patients appeared to be comparable to normo-ovulatory women.32–34 The outcomes of studies in this field should be interpreted with caution, however,

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Table 34.3 • • • • •

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Indications for ICSI

Total motile sperm count (TMC) < 1 million < 4% normal morphology and TMC < 5 million No or poor fertilization in the first IVF cycle when TMC < 10 million No or poor fertilization in two IVF cycles when TMC > 10 million Epididymal or testicular spermatozoa

due to differences in stimulation protocols, the notorious heterogeneity of patients diagnosed with PCOS, and differences in the presence of additional factors such as tubal infertility.35 In a study in which IVF outcomes were compared between a carefully defined group of women with WHO group 2 anovulatory infertility and a matched control group of women with tubal infertility,36 obese women suffering from WHO group 2 anovulatory infertility were at an increased risk of having their IVF cycle cancelled due to insufficient response. However, once oocyte retrieval was achieved, live birth rates were comparable with controls. These findings have been confirmed in a recent meta-analysis of IVF outcomes in women diagnosed with PCOS on the basis of the Rotterdam criteria.37 Although PCOS subjects produced more oocytes, a lower fertilization rate was observed.31

Male factor infertility Only a small proportion of subfertile males are amenable to treatment. Fortunately, high female fecundity can often compensate for the presence of low sperm concentrations.38 In those couples presenting with male factor infertility, intrauterine insemination (IUI) with washed and prepared sperm can be an effective treatment.39 The additional value of ovarian stimulation to IUI in this context remains a topic of debate.40–42 Whereas ovarian stimulation with clomiphene citrate does not appear to increase the efficacy of IUI,43,44 the addition of gonadotropin ovarian hyperstimulation does appear to increase pregnancy rates, but at the expense of higher incidence of multiple pregnancy.40 The risk/benefit balance of ovarian stimulation for IUI is yet to be demonstrated in prospective randomized studies. If fewer than 1–2 million motile sperm are present after sperm preparation, IVF is normally indicated. The results of IVF in the treatment of male factor infertility are determined primarily by the age of the woman,45 the degree of sperm motility, and sperm morphology.46–48 Many studies have reported a strong correlation between impaired semen parameters and fertilization capacity in IVF, and when severe male factor infertility is present, total fertilization failure (TFF) may occur. In many centers, a post-wash total motile sperm count of less than 500 000 is considered to indicate ICSI treatment,49 while others apply a cutoff value of 1 million (Table 34.3). These values remain largely arbitrary, since few reliable data are

available which enable the prediction of the chance of TFF in a given couple.48 Although ICSI has transformed the fertility prognosis for couples with severe male factor infertility (including those where TFF occurs during IVF), the appropriate indications for ICSI remain controversial.50 While in some countries ICSI tends to be restricted to treating severe oligoasthenospermy and total fertilization failure, other European and US centers apply a more liberal policy to the use of ICSI, reflecting primarily differences in national or local funding policy. However, absolute indications for ICSI are agreed to include the use of microsurgical (epididymal or testicular) aspirated spermatozoa (Table 34.3). While many clinics have a lower clinical threshold for applying ICSI, and some apply it to all cases of IVF, this approach is not supported by well-designed prospective studies. In one study comparing IVF to ICSI in couples with tubal infertility but with normozoospermic semen, no differences in fertilization rates were observed.51 There is some evidence that ICSI may have detrimental effects, leading to poorer embryo development compared to IVF.52,53 In a multicenter randomized study comparing ICSI to IVF in the treatment of unexplained infertility, no benefit of ICSI was demonstrated.54 Indeed, the implantation rate was higher in the IVF group (30%) than in the ICSI group (22%); relative risk = 1.35 (95% CI 1.04–1.76). The pregnancy rate per cycle was also higher after IVF (33% vs 26%). These data indicate that ICSI offers no advantage over IVF in terms of pregnancy rates in cases of nonmale-factor infertility. However, data relating to live birth and miscarriage rates are still required.55

Unexplained infertility In a large number of couples attending a physician for fertility problems, a clear diagnosis explaining their decreased or absent fertility cannot be found.56 The relative value of IVF compared to expectant management or IUI in unexplained infertile couples remains uncertain. Spontaneous pregnancy chances in these untreated couples vary from 30 to 70% within 2 years.57 In general, IUI has been shown to result in pregnancy rates varying between 5 and 15% per cycle. However, when combined with vigorous ovarian stimulation, complication rates (especially higherorder multiple pregnancies) are unacceptably high.58 Conventional infertility management has been

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demonstrated to be more cost-effective than IVF as first-line therapy59 in the treatment of unexplained infertility. In a randomized comparison of 250 couples between a single IVF cycle and 6 months’ expectant management, no difference in pregnancy rates was observed when bilateral tubal occlusion was excluded.60 For the group of patients with more subtle abnormalities (such as endometriosis, minor tubal disease, oligospermia, or unexplained), proper management should focus on prognosis rather than diagnosis. The prognosis of a given couple for spontaneous pregnancy should be weighed against pregnancy chances after more invasive treatment strategies such as IUI (with or without ovarian stimulation) or IVF. In a Cochrane systematic review addressing this issue, only four randomized trials were deemed suitable for analysis.61 No difference in clinical pregnancy rates between IVF and expectant management was reported. No evidence for a difference in live birth rates between IVF and IUI either without (OR = 1.96, 95% CI 0.88–4.4) or with (OR = 1.15, 95% CI 0.55–2.4) ovarian stimulation was observed. There was no evidence of a difference in the multiple pregnancy rates between IVF and IUI with ovarian stimulation (OR = 0.63, 95% CI 0.27–1.5). The small sample sizes mean that differences in the effect of IVF relative to expectant management or IUI with or without ovarian stimulation in terms of live birth rates may have been hidden. On the basis of a randomized comparison between IUI or IVF in idiopathic and male subfertility, it was concluded that IUI was more cost-effective, despite higher pregnancy rates per cycle for IVF.62 In conclusion, a true cause for the infertility cannot be found in many couples presenting with fertility problems. Therefore, causal therapy is only possible in a small proportion of patients. For the remaining couples, a pragmatic prognosis-oriented approach should be applied. Most important, chances for spontaneous pregnancy should be assessed for each given couple. Evidence is accumulating that female age is by far the most crucial factor in determining chances for pregnancy, either spontaneously or after fertility therapy. This becomes even more predominant over the years, since women in the Western world tend to delay their wish to conceive. Increasing attention is now focusing on the identification of prognostic factors capable of determining the chance of spontaneous conception and of successful outcome to infertility treatment in individual couples.

From diagnosis to prognosis Infertility is defined as the inability of a couple to conceive within 1 year of regular intercourse. These infertile couples can be separated into two groups; those who are unable to conceive without therapy (i.e. absolute infertility), and those with reduced fertility chances who still have a considerable chance to conceive spontaneously

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with time. Disease states underlying the inability to conceive spontaneously include anovulation, complete tubal occlusion, and azoospermia. Hence, an underlying cause for the infertility can be diagnosed conclusively in these conditions. A regular fertility work-up – including tests to evaluate ovulation, sperm analysis, and tests for tubal patency – can easily identify these problems. In couples with decreased fertility, conditions such as endometriosis, oligozoospermia, or luteal phase insufficiency may be found, but it remains uncertain to what extent they contribute to the reduced fertility. Hence, in a large number of couples attending a physician for fertility problems, a clear diagnosis explaining their decreased or absent fertility cannot be found (also referred to as unexplained subfertility). Indeed, success rates per cycle of a given treatment should be weighed against costs, side effects, and inconvenience for the patient, and the chances of complications for mother and child. Risks for financedriven overtreatment remain substantial. Many endogenous factors play a role in determining how an individual woman will respond to IVF treatment. However, any individual approach to infertility treatment must begin with an assessment of a given couple’s chance of conceiving spontaneously. The chance of achieving a spontaneous pregnancy is frequently underestimated by couples and their physicians.63 The increasing tendency to delay childbearing for career, social, or other reasons is putting physicians under greater pressure to intervene when spontaneous conception does not occur quickly. Time is increasingly an issue for couples seeking to conceive. Yet patience can pay dividends for many who are now subject to premature and unnecessary intervention. Most couples seeking help will present with subfertility rather than absolute infertility. On the basis of a modest range of investigations and certain individual characteristics, the chances of an individual couple conceiving spontaneously over a given period of time can be calculated. In recent years a number of prediction models for calculating individual chances of spontaneous conception in subfertile couples have been published.38,64,65 On the basis of the results of a number of fertility investigations and patient parameters such as age and duration of infertility, the chance of conception over a given time frame can be calculated. For instance, after 3 years of failure to conceive, the residual likelihood of spontaneous pregnancy in untreated couples with unexplained infertility falls to 40% and after 5 years to 20%.63,66 Hunault et al combined original data from three previously published models to construct a synthesis model which was observed to perform better.67 When considering the appropriate moment for therapeutic intervention for couples with unexplained infertility, prognostic models may aid the clinician. Caution is, however, required when applying a prediction model developed elsewhere to one’s own

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patient population. Before a prediction model can be introduced into everyday clinical practice, prospective external validation is required. Furthermore, knowledge of the development cohort is important when selecting a model for application in one’s own setting. Few prediction models have been subject to validation on a different population to that on which the model was developed.68 The reliability of these models when applied in other, similar, clinics is in most cases not known. In a recent study, the discriminative ability and reliability of the Eimers model for predicting spontaneous pregnancy among subfertile women was measured on an independent Canadian data set.69 The model, which was developed in a Dutch population in 1994, was found to have a moderate predictive power in the Canadian population in which the birth rate was generally lower. With adjustment for the average live birth rate, the Eimers model gave reliable spontaneous pregnancy predictions. In a prospective evaluation of the performance of the Eimers model in a tertiary care center, the expected and observed incidences of spontaneous pregnancy in the different risk groups correlated well.70 More recently, the Hunault synthesis model67 was shown in a prospective study to accurately predict spontaneous pregnancy in subfertile couples.71 In those women with a poor chance of conceiving spontaneously, or with other fertility treatments, consideration of a number of factors will aid in assessing the likely outcome of IVF. While duration of infertility has been shown to be associated with the chance of spontaneous pregnancy,35 its impact on the chance of success with IVF treatment has been less clear.62 In a large retrospective analysis of factors affecting outcomes in IVF, there was a significant decrease in ageadjusted life birth rates with increasing duration of infertility.2 Previous pregnancy had a significantly positive impact on the chance of success with IVF, with the effect being stronger for pregnancies resulting in a live birth. This positive association with previous live birth was even stronger if it had followed an IVF pregnancy. The same authors calculated a previous live birth to be associated with a live birth rate per IVF treatment cycle of 23.2%, compared with 12.5% when no previous pregnancy had occurred. This association with previous pregnancy and successful outcome has since been confirmed by other studies.9,72

Ovarian aging The most prominent determining factor for IVF outcome is the individual variability in ovarian response to stimulation. Rather than exhibiting the desired response, women can present with either a hypo-response or a hyper-response to stimulation. While hyper-response to gonadotropin stimulation can usually be prevented by modification of the stimulation regimen, a poor response to ovarian stimulation is highly resistant to therapeutic intervention.73 Strategies for stimulating ‘low

responders’ include varying the dose or day of the cycle for initiating stimulation with gonadotropins. Studies undertaken so far have been unable to demonstrate a beneficial effect of gonadotropin dose increase in patients who exhibit a poor response to standard-dose regimens.73,74 Alternative approaches include early cessation or microdose gonadotropin-releasing hormone (GnRH) agonist protocols, and the adjunctive use of aromatase inhibitors, growth hormone, and GnRH antagonists.75 Initial small studies focusing on surrogate outcomes such as number of cancelled cycles rather than ongoing pregnancy may produce encouraging results. However, at present, no therapeutic intervention has been shown in large randomized studies to offer a solution to poor response to ovarian stimulation in IVF. It might indeed be argued that therapeutic interventions aimed at increasing the chance of meeting criteria for oocyte pick-up are unethical unless ongoing pregnancy rates can also be shown to improve. Poor response to ovarian stimulation for IVF is clearly associated with chronological aging. An agerelated decline in response to stimulation with gonadotropins and a reduction in the number of oocytes,76 oocyte quality,77 fertilization rates,78,79 and ultimately embryos80–82 have been well documented. Many studies point to 40 years of age as being a significant cut-off line for effectiveness of IVF.83–86 This agerelated effect on pregnancy rates is similar to that reported in donor sperm programs87 and chances for spontaneous pregnancy. A multiple regression analysis of factors influencing IVF outcomes revealed a predicted live birth rate of 17% per cycle at age 30, falling to just 7% at 40 years, and 2% at 45 years of age.2 Although age is an important predictor of IVF outcome,88 chronological age is poorly correlated with ovarian aging. The association between cycle cancellation and poor success rates and poor ovarian response due to diminished ovarian reserve is well established.89,90 A major individual variability exists in follicle pool depletion within the normal range of menopausal age, as complete follicle pool exhaustion may occur between 40 and 60 years. The quantity and quality of the primordial follicle pool diminishes with age, reducing ovarian reserve.91 This results in a decline in both therapy-induced and spontaneous pregnancies.92 However, while some women above 40 years of age will show a good response to ovarian stimulation, and subsequently conceive with IVF, other women under 40 may fail to respond, as a result of accelerated ovarian aging.93 The concept of poor response as a feature of chronological and ovarian aging has been further supported by recent studies linking poor response to ovarian hyperstimulation to subsequent early menopause.94–96 Indeed, the response of a woman to ovarian hyperstimulation for IVF can be considered as an extended challenge test of ovarian function. In recent years, attention has been given to the identification of sensitive and specific markers of ovarian aging which may enable prediction of poor or

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good response to ovarian hyperstimulation. This would open the way to improved counseling and patient selection for IVF. The value of FSH and other prognostic markers in predicting ovarian response to hyperstimulation in IVF treatment is dealt with further in Chapter 54. Clearly related to the ovarian response to stimulation, the number of embryos available for transfer appears to be a crucial factor in determining the chance of success with IVF,97 and this is of equal importance in older women.98 In a study of the factors influencing the chance of success with IVF in women of 40 years and above, Widra et al85 observed that if four or more embryos were transferred, pregnancy rates per embryo transfer were similar to those observed in younger women. Similarly, Alrayyes et al99 found that when more than three embryos were available for transfer there was no significant difference in pregnancy rates between women under or over 37 years of age. These data suggest uterine senescence to be less important than embryo quality in determining IVF outcome in older women. Further support for this comes from the observed success of oocyte donation programs in women over the age of 40.100

Lifestyle and concurrent medical conditions There is now a substantial amount of evidence showing that environmental and lifestyle factors influence the success rates of assisted reproductive technologies (ART),101–103 and it is therefore important that serious attempts are made to provide adequate preconceptional screening counseling and interventions in order to optimize health prior to starting IVF. The importance of full medical assessment prior to IVF treatment is increasing as the average age of our patients continues to rise. A greater proportion of infertility patients may now also present with concurrent medical conditions that may impact on the safety and management of the IVF treatment as well as pregnancy. The appropriate management of the medically complicated patient presenting for IVF can be complex and often requires an interdisciplinary approach. For further information in this field, the reader is referred to Macklon.104 The most important lifestyle factor impacting on fertility outcomes is tobacco smoking. Smoking during pregnancy has long been known to increase the risk of a number of adverse obstetric and fetal outcomes such as miscarriage, placenta previa, preterm birth, and low birth weight.102 In recent years the association between smoking and infertility in women has become clear. Chemicals present in cigarette smoke can reach the developing egg in vivo, as both cotinine, the metabolite of nicotine, and cadmium and heavy metals in cigarette smoke are increased in the follicular fluid surrounding the egg.105 It has also been

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demonstrated that active smoking increases oxidative stress in the growing follicle and cytotoxity in the egg and surrounding granulosa cells.106 Reports have appeared linking smoking to damage of the meiotic spindle in oocytes, increasing the risk of chromosomal errors.107 In men who smoke, all parameters of sperm quality are reduced.102 Smoking in men and passive smoking in women has been associated with a longer time to achieve a pregnancy.102 The effects of smoking on live birth rate among women who undergo IVF is similar in magnitude to the effect of an increase in female age of more than 10 years.101 As a result, smokers require twice as many IVF cycles to become pregnant as nonsmokers.101 A recent American Society for Reproductive Medicine (ASRM) Practice Committee publication on smoking and infertility has highlighted the considerable contribution of smoking to infertility and treatment outcomes and the need for a more proactive approach to stop smoking prior to fertility treatment.108 Epidemiological evidence clearly shows that being overweight contributes to menstrual disorders, infertility, miscarriage, poor pregnancy outcome, impaired fetal well-being, and diabetes mellitus.109 Overweight women (body mass index [BMI] >27 kg/m²) have been shown to have a 33% reduced chance of a live birth after their first IVF cycle compared to women with a BMI of 20–27 kg/m². The association was strongest in women with unexplained infertility.101 In men, a BMI <20 or >25 kg/m² is associated with reduced sperm quality.102 A number of studies have shown that weight loss can improve fecundity in overweight women, and many centers include weight-loss programs as part of their fertility treatment. However, few data are available regarding the impact of type of diet on IVF outcomes. Recent studies have highlighted the importance of certain nutritional factors for healthy gamete development, and hence embryo quality. Folic acid supplementation was shown to alter the vitamin microenvironment of the oocyte,110 whereas seminal plasma cobalamin levels were demonstrated to affect sperm concentration.111 Concerns that folate supplementation may increase twinning rates in IVF are better addressed by practicing single embryo transfer, rather than withholding folate supplementation.112 It is becoming clear that preconceptional care aimed at optimizing medical, lifestyle, and nutritional factors should be an integral part of fertility therapies; and in our center, all IVF patients attend a preconceptional clinic before commencing treatment.

Defining success in IVF The approach of maximizing pregnancy rates per cycle has led to very complex and costly ovarian hyperstimulation protocols with considerable risk of side effects and complications. In fact, many couples do not consider a second IVF attempt, even if they can afford one,

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because of the stress associated with the first treatment cycle.113 Research on less-complex, more patientfriendly stimulation protocols, along with transfer of a reduced number (preferably one) of embryos, will only prosper in an environment in which singleton healthy birth is regarded as the most appropriate endpoint of infertility treatment. This primary outcome should be judged in the context of the risk of adverse effects, complications, and costs per treatment (which might include multiple cycles) or during a given treatment period. In a recent randomized study, the cumulative live singleton birth rate achieved at 1 year after commencing treatment was measured after two treatment strategies.113 The ‘conventional’ strategy consisted of three cycles in which the conventional ‘long protocol’ was applied and two embryos per cycle transferred. The mild strategy comprised four cycles in which a mild stimulation protocol was combined with the transfer of just one embryo. After 1 year of treatment, cumulative singleton rates were equivalent, but those treated with the mild strategy had incurred lower costs, far fewer multiple pregnancies, and lower drop-out rates113 (Fig 34.3). If IVF outcomes are expressed in terms of live singleton births per period of treatment, milder regimens with fewer risks and complications will be more readily adopted into clinical practice, improving the prognosis of a complication-free successful IVF treatment.114

The future As our knowledge of factors influencing outcome following fertility therapies increases, treatment will become more individualized, maximizing costeffectiveness and minimizing inconvenience and risk for the patient. Prognostic models based on individual factors are likely to predominate over population costeffectiveness considerations when deciding, for instance, who receives IUI rather than IVF for the treatment of unexplained infertility. In addition, the developments of mild-hyperstimulation IVF and the prospect of improving implantation rates by optimizing embryo culture conditions and the provision of preimplantation genetic screening will demand continuing reassessment of the cost–benefit issues. This degree of individualization requires the development and application of sophisticated, accurate, and prospectively validated prediction models. An individual approach to IVF may impact on one of the major problems still facing IVF, that of multiple pregnancy. Although a trend toward the transfer of fewer embryos is now clear, the ability to identify those treatment cycles in which single embryo transfer would avoid the risk of twin pregnancy without reducing the chance of achieving a singleton pregnancy would encourage the adoption of single embryo transfer into clinical practice, and progress is now being made in this area. A major limit to the indications for IVF is the process of ovarian aging. Apart from donation, there appears to be little sign of a therapeutic intervention capable of circumventing this phenomenon. While

60 Proportion of pregnancies leading to term live birth(%)

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0

0

3 6 9 Time since randomization (months) Standard

12

Mild

Number of patients Standard Mild

199 205

152 174

123 149

106 130

97 109

Fig 34.3 Proportions of pregnancies leading to cumulative term live birth within 12 months after starting IVF. Mild: mild ovarian stimulation with gonodotropin-releasing hormone (GnRH) antagonist and single embryo transfer. Standard: standard ovarian stimulation with GnRH antagonist and dual embryo transfer. The shaded area represents the singleton live birth rate after 12 months. Reprinted from The Lancet, vol. 369, Heijen EM, Eijkemans MJ, De Klerk C, et al. A mild treatment strategy for invitro fertilisation: a randomised non-inferiority trial. pp. 743–9, Copyright 2007, with permission from Elsevier

the ongoing tendency to delay childbirth will increase the need for assisted conception services, the negative impact of aging on IVF outcome is likely to increase. In the future, IVF will be increasingly applied for indications other than infertility. The growing applications for preimplantation genetic diagnosis are producing a new range of indications for IVF. IVF is becoming simply a tool to enable preimplantation diagnosis, and thus prevention of hereditary disorders in normally fertile couples at risk of having children with serious medical conditions. In addition, IVF allows the creation of ‘designer babies’ capable of donating human leukocyte antigen (HLA)-matching tissue to treat a sick sibling. While these indications for IVF remain under close scrutiny by national regulators of IVF such as the Human Fertilisation and Embryology Authority (HFEA), they are likely to become established in the near future. The theoretical possibilities for medical therapies based on the in vitro culture and selective differentiation of embryonic stem cells are likely to be translated into therapeutic reality before long. The treatment of infertility may very soon be but a minor indication for IVF.

Summary At the time of its introduction into clinical practice, the principal indication for IVF was tubal infertility. Since then the indications have multiplied, and IVF now has

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a central place in the treatment of female and male factor infertility, as well as the infertile couple with no clear underlying cause. The underlying indication for treatment has a limited impact on the probability of success. More important determining factors are patient age and duration of infertility. With increasing knowledge of the factors that influence a given couple’s chance of conceiving either spontaneously or following fertility treatment, the emphasis is shifting from diagnosis to prognosis. The most important variable with respect to IVF is the response of the patient to ovarian stimulation. In recent years, the link between poor response to ovarian stimulation and ovarian aging has become clear, but effective remedial therapies remain elusive. Certain lifestyle factors such as smoking and obesity have also been shown to impact negatively on fertility and IVF outcomes. These factors are amenable to intervention, and due attention should be given by both clinicians and their patients to optimizing preconceptional conditions for a successful treatment and pregnancy.

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12. Simon C, Gutierrez A, Vidal A. Outcome of patients with endometriosis in assisted reproduction: results from in-vitro fertilization and oocyte donation. Hum Reprod 1994; 9: 725–9. 13. Dmowski WP, Rana N, Michalowska J, et al. The effect of endometriosis, its stage and activity, and of autoantibodies on in vitro fertilization and embryo transfer success rates. Fertil Steril 1995; 63: 555–62. 14. Barnhart K, Dunsmoor-Su R, Coutifaris C. Effect of endometriosis on in vitro fertilization. Fertil Steril 2002; 77: 1148–55. 15. Strandell A. Evidence based treatment of tubal pathology. Hum Reprod 2003; 18(Suppl 1): O–248. 16. Check JH, Lurie D, Callan C, Baker A, Benfer K. Comparison of the cumulative probability of pregnancy after in-vitro fertilization–embryo transfer by infertility factor and age. Fertil Steril 1994; 61: 257–61. 17. Dor J, Seidman DS, Ben-Shlomo I, et al. Cumulative pregnancy rate following in-vitro fertilization: the significance of age and infertility aetiology. Hum Reprod 1996; 11: 425–8. 18. Barmat LI, Rauch E, Spandorfer S, et al. The effect of hydrosalpinges on IVF-ET outcome. J Assist Reprod Genet 1999; 16: 350–4. 19. Cohen MA, Lindheim SR, Sauer MV. Hydrosalpinges adversely affect implantation in donor oocyte cycles. Hum Reprod 1999; 14: 1087–9. 20. De Wit W, Gowrising CJ, Kuik DJ, Lens JW, Schats R. Only hydrosalpinges visible on ultrasound are associated with reduced implantation and pregnancy rates after in-vivo fertilization. Hum Reprod 1998; 13: 1696–701. 21. Aboulghar MA, Mansour RT, Serour GI. Controversies in the modern management of hydrosalpinx. Hum Reprod Update 1998; 4: 882–90. 22. Johnson NP, Mak W, Sowter MC. Surgical treatment for luteal disease in women due to undergo in vitro fertilization. Cochrane Database Syst Rev 2004; 3: CD002125. 23. Strandell A, Lindhard A, Waldenstrom U, Thorburn J. Hydrosalpinx and IVF outcome: cumulative results after salpingectomy in a randomized controlled trial. Hum Reprod 2001; 16: 2403–10. 24. Strandell A. The patient with hydrosalpinx. In: Macklon NS, ed. IVF in the Medically Complicated Patient. London: Informa, 2005. 25. Kontoravdis A, Makrakis E, Pantos K, et al. Proximal tubal occlusion and salpingectomy result in similar improvement in in vitro fertilization outcome in patients with hydrosalpinx. Fertil Steril 2006; 86: 1642–9. 26. The ESHRE Capri Workshop Group. Anovulatory infertility. Hum Reprod 1995; 10: 1549–53. 27. Rowe PJ, Comhaire FH, Hargreave TB, Mellows H. Female partner. In: Rowe PJ, Comhaire FH, Hargreave TB, Mellows H, eds. WHO Manual for the Standardized Investigation and Diagnosis of the Infertile Couple. Cambridge: Cambridge University Press, 2000: 40–67. 28. van Santbrink EJ, Hop WC, Fauser BC. Classification of normogonadotropic infertility: polycystic ovaries diagnosed by ultrasound versus

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Textbook of Assisted Reproductive Technologies endocrine characteristics of polycystic ovary syndrome. Fertil Steril 1997; 67: 452–8. Laven JS, Imani B, Eijkemans MJ, Fauser BC. New approach to polycystic ovary syndrome and other forms of anovulatory infertility. Obstet Gynecol Surv 2002; 57: 755–67. Eijkemans MJC, Imani B, Mulders AGMGJ, Habbema JDF, Fauser BCJM. High singleton live birth rate following classical ovulation induction in normogonadotrophic anovulatory infertility (WHO 2). Hum Reprod 2003; 18: 1–6. Heijnen EM, Eijkemans MJ, Hughes EG, et al. A meta-analysis of outcomes of conventional IVF in women with polycystic ovary syndrome. Hum Reprod Update 2006; 12: 13–21. Dor J, Shulman A, Levran D, et al. The treatment of patients with polycystic ovarian syndrome by invitro fertilization and embryo transfer: a comparison of results with those of patients with tubal infertility. Hum Reprod 1990; 5: 816–18. Urman B, Fluker MR, Yuen BH, et al. The outcome of in vitro fertilization and embryo transfer in women with polycystic ovary syndrome failing to conceive after ovulation induction with exogenous gonadotropins. Fertil Steril 1992; 57: 1269–73. Homburg R, Berkowitz D, Levy T, et al. In vitro fertilization and embryo transfer for the treatment of infertility associated with polycystic ovary syndrome. Fertil Steril 1993; 60: 858–63. Salat-Baroux J, Alvarez S, Antoine JM, et al. Results of IVF in the treatment of polycystic ovary disease. Hum Reprod 1988; 3: 331–5. Mulders AGMGJ, Laven JSE, Imani B, Eijkemans MJC, Fauser BCJM. IVF outcome in anovulatory infertility (WHO group 2) – including polycystic ovary syndrome – following previous unsuccessful ovulation induction. Reprod Biomed Online 2003; 7: 50–8. The Rotterdam ESHRE/ASRM-sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril 2004; 81: 19–25. Collins JA, Burrows EA, Willan AR. The prognosis for live birth among untreated infertile couples. Fertil Steril 1995; 64: 22–8. Cohlen BJ, Vandekerckhove P, te Velde ER, Habbema JD. Timed intercourse versus intra-uterine insemination with or without ovarian hyperstimulation for subfertility. Cochrane Database Syst Rev 2000; 2. Fauser BCJM, Devroey P, Macklon NS. Multiple birth resulting from ovarian stimulation for subfertility treatment. Lancet 2005; 365: 1807–16. Cohlen B, Cantineau A, D’Hooghe T, te Velde E. Multiple pregnancy after assisted reproduction. Lancet 2005; 366: 452–3. Macklon NS, Devroey P, Fauser BCJM. Multiple pregnancy after assisted reproduction. Lancet

2005; 366: 453–4. 43. Martinez AR, Bernardus RE, Voorhorst FJ, Vermeiden JP, Schoemaker J. Intrauterine insemination does and clomiphene citrate does not improve fecundity in couples with infertility due to male or idiopathic factors: a prospective, randomized, controlled study. Fertil Steril 1990; 53: 847–53.

44. Arici A, Byrd W, Bradshaw K, et al. Evaluation of clomiphene citrate and human chorionic gonadotropin treatment: a prospective, randomized, crossover study during intrauterine insemination cycles. Fertil Steril 1994; 2: 314–18. 45. Forti G, Krausz C. Evaluation and treatment of the infertile couple. J Clin Endocrinol Metab 1998; 83: 4177–88. 46. Van Uem JF, Acosta AA, Swanson RJ, et al. Male factor evaluation in in vitro fertilization: Norfolk experience. Fertil Steril 1985; 44: 375–83. 47. Donnelly ET, Lewis SEM, McNally JA, Thompson W. In vitro fertilization and pregnancy rates: the influence of sperm motility and morphology on IVF outcome. Fertil Steril 1998; 70: 305–14 48. Repping S, van Weert JM, Mol BWJ, de Vries JWA, van der Veen F. Use of the total motile sperm count to predict total fertilization failure in in vitro fertilization. Fertil Steril 2002; 78: 22–8. 49. Devroey P, Vandervorst M, Nagy P, Van Steirteghem A. Do we treat the male of the gamete? Hum Reprod 1998; 13(Suppl 1): 178–85. 50. Devroey P, Van Steirthegem A. A review of ten years experience of ICSI. Hum Reprod Update 2004; 10: 19–28. 51. Staessen C, Camus M, Clasen K, De Vos A, Van Steirteghem A. Conventional in-vitro fertilization versus intracytoplasmic sperm injection in sibling oocytes from couples with tubal infertility and normozoospermic semen. Hum Reprod 1999; 14: 2474–9. 52. Ola B, Afnan M, Sharif K, et al. Should ICSI be the treatment of choice for all cases of in vitro conception? Considerations of fertilization and embryo development, cost effectiveness and safety. Hum Reprod 2001; 16: 2485–90. 53. Verpoest W, Tournaye H. ICSI: hype or hazard? Hum Fertil (Camb) 2006; 9: 81–2. 54. Bhattacharya S, Hamilton MP, Shaaban M, et al. Conventional in vitro fertilisation versus intracytoplasmic sperm injection for the treatment of nonmale-factor infertility: a randomised controlled trial. Lancet 2001; 357: 2075–9. 55. Van Rumste MM, Evers JL, Farquhar CM. Intracytoplasmic sperm injection versus conventional techniques for oocyte insemination during in vitro fertilization in patients with non-male subfertility. Cochrane Database Syst Rev 2003; 2. 56. Aboulghar MA, Mansour RT, Serour GI, Al-Inany HG. Diagnosis and management of unexplained infertility: an update. Arch Gynecol Obstet 2003; 267: 177–88. 57. Smeenk JM, Braat DD, Stolwijk AM, Kremer JA, Stolwijk AM. Pregnancy is predictable: a largescale prospective external validation of the prediction of spontaneous pregnancy in subfertile couples. Hum Reprod 2007; 22: 2344–5. 58. Guzick DS, Carson SA, Coutifaris C, et al. Efficacy of superovulation and intrauterine insemination in the treatment of infertility. National Cooperative Reproductive Medicine Network. N Engl J Med 1999; 340: 177–83. 59. Karande VC, Korn A, Morris R, et al. Prospective randomized trial comparing the outcome and cost of in vitro fertilization with that of a traditional

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treatment algorithm as first-line therapy for couples with infertility. Fertil Steril 1999; 71: 468–75. Soliman S, Daya S, Collins J, Jarrell J. A randomized trial of in vitro fertilization versus conventional treatment for infertility. Fertil Steril 1993; 59: 1239–44. Pandian Z, Bhattacharya S, Nikolaou D, Vale L, Templeton A. In vitro fertilisation for unexplained subfertility. Cochrane Database Syst Rev 2002; 2. Goverde AJ, McDonnell J, Vermeiden JP, et al. Intrauterine insemination or in-vitro fertilisation in idiopathic subfertility and male subfertility: a randomised trial and cost-effectiveness analysis. Lancet 2000; 355: 13–18. Evers JL. Female subfertility. Lancet 2002; 360: 151–9. Eimers JM, te Velde ER, Gerritse R, et al. The prediction of the chance to conceive in subfertile couples. Fertil Steril 1994; 61: 44–52. Snick HK, Snick TS, Evers JL, Collins JA. The spontaneous pregnancy prognosis in untreated subfertile couples: the Walcheren primary care study. Hum Reprod 1997; 12: 1582–8. Collins JA, Rowe TC. Age of the female partner is a prognostic factor in prolonged unexplained infertility: a multicenter study. Fertil Steril 1989; 52: 15–20. Hunault CC, Habbema JDF, Eijkemans MJC. Two prediction rules for spontaneous pregnancy leading to live birth among subfertile couples, based on the synthesis of three previous models. Human Reprod 2004; 19: 2019–26. Stolwijk AM, Straatman H, Zielhuis GA, et al. External validation of prognostic models for ongoing pregnancy after in-vitro fertilization. Hum Reprod 1998; 13: 3542–9. Hunault CC, Eijkemans MJ, te Velde ER, Collins JA, Habbema JD. Validation of a model predicting spontaneous pregnancy among subfertile untreated couples. Fertil Steril 2002; 78: 500–6. Collins JA, Milner RA, Rowe TC. The effect of treatment on pregnancy among couples with unexplained infertility. Int J Fertil 1991; 36: 145–52. Van der Steeg JW, Steures P, Eijkemans MJC, et al. Pregnancy is predictable: a large-scale prospective external validation of the prediction of spontaneous pregnancy in subfertile couples. Hum Reprod 2007; 22: 536–42. Croucher CA, Lass A, Margara R, Winston RM. Predictive value of the results of a first in-vitro fertilization cycle on the outcome of subsequent cycles. Hum Reprod 1998; 13: 403–8. Tarlatzis BC, Zepiridis L, Grimbizis G, Bontis J. Clinical management of low ovarian response to stimulation for IVF: a systematic review. Hum Reprod Update 2003; 9: 61–76. van Hooff MH, Alberda AT, Huisman GJ, Zeilmaker GH, Leerentveld RA. Doubling the human menopausal gonadotrophin dose in the course of an in-vitro fertilization treatment cycle in low responders: a randomized study. Hum Reprod 1993; 8: 369–73. Karande VC. Managing and predicting low reponse to standard in vitro fertilization therapy. A review of the options. Treat Endocrinol 2003; 2: 257–72. Piette C, de Mouzon J, Bachelot A, Spira A. In-vitro fertilization: influence of women’s age on pregnancy rates. Hum Reprod 1990; 5: 56–9.

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77. Tucker MJ, Morton PC, Wright G, et al. Factors affecting success with intracytoplasmic sperm injection. Reprod Fertil Dev 1995; 7: 229–36. 78. Ashkenazi J, Orvieto R, Gold-Deutch R, et al. The impact of woman’s age and sperm parameters on fertilization rates in IVF cycles. Eur J Obstet Gynecol Reprod Biol 1996; 66: 155–9. 79. Yie SM, Collins JA, Daya S, et al. Polyploidy and failed fertilization in in-vitro fertilization are related to patients age and gamete quality. Hum Reprod 1996; 11: 614–17. 80. Cordiero I, Calhaz-Jorge C, Barata M, et al. Repercussao da idade de mulher, de taxa de clivagem e da qualidade embrionaria, na obtencao de graviez por fertilizacao in-vitro. Acta Med Port 1995; 8: 145–50. 81. Hull MG, Fleming CF, Hughes AO, McDermott A. The age-related decline in female fecundity: a quantitative controlled study of implanting capacity and survival of individual embryos after in vitro fertilization. Fertil Steril 1996; 65: 783–90. 82. Sharif K, Elgendy M, Lashen H, Afnan M. Age and basal follicle stimulating hormone as predictors of in vitro fertilisation outcome. Br J Obstet Gynaecol 1998; 105: 107–12. 83. Yaron Y, Botchan A, Amit A, et al. Endometrial receptivity: the age-related decline in pregnancy rates and the effect of ovarian function. Fertil Steril 1993; 60: 314–18. 84. Roest J, Van Heusden AM, Mous H, Zeilmaker GH, Verhoeff A. The ovarian response as a predictor for successful in vitro fertilization treatment after the age of 40 years. Fertil Steril 1996; 66: 969–73. 85. Widra EA, Botchan A, Amit A, et al. Endometrial receptivity: the age-related decline in pregnancy rates and the effect of ovarian function. Fertil Steril 1996; 65: 103–8. 86. Legro RS, Shakleworth DP, Moessner JM, Gnatuk CL, Dodson WC. ART in women 40 and over. Is the cost worth it? J Reprod Med 1997; 42: 76–82. 87. van Noord-Zaadstra BM, Looman CW, Alsbach H, et al. Delaying childbearing: effect of age on fecundity and outcome of pregnancy. BMJ 1991; 302: 1361–5. 88. Chuang CC, Chen CD, Chao KH, et al. Age is a better predictor of pregnancy potential than basal follicle-stimulating hormone levels in women undergoing in vitro fertilization. Fertil Steril 2003; 79: 63–8. 89. Pellicer A, Lightman A, Diamond MP, Russell JB, DeCherney AH. Outcome of in vitro fertilization in women with low response to ovarian stimulation. Fertil Steril 1987; 47: 812–15. 90. Jenkins JM, Davies DW, Devonport H, et al. Comparison of ‘poor’ responders with ‘good’ responders using a standard buserelin/human menopausal gonadotrophin regime for in-vitro fertilization. Hum Reprod 1991; 6: 918–21. 91. te Velde ER, Pearson PL. The variability of female reproductive aging. Hum Reprod Update 2002; 8: 141–54. 92. Scott RT, Opsahl MS, Leonardi MR, et al. Life table analysis of pregnancy rates in a general infertility population relative to ovarian reserve and patient age. Hum Reprod 1995; 10: 1706–10.

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93. Beckers NG, Macklon NS, Eijkemans MJ, Fauser BC. Women with regular menstrual cycles and a poor response to ovarian hyperstimulation for in vitro fertilization exhibit follicular phase characteristics suggestive of ovarian aging. Fertil Steril 2002; 78: 291–7. 94. de Boer EJ, den Tonkelaar I, te Velde ER, et al. A low number of retrieved oocytes at in vitro fertilization treatment is predictive of early menopause. Fertil Steril 2002; 77: 978–85. 95. Nikolaou D, Lavery S, Turner C, Margara R, Trew G. Is there a link between an extremely poor response to ovarian hyperstimulation and early ovarian failure? Hum Reprod 2002; 17: 1106–11. 96. Lawson R, El-Toukhy T, Kassab A, et al. Poor response to ovulation induction is a stronger predictor of early menopause than elevated basal FSH: a life table analysis. Hum Reprod 2003; 18: 527–33. 97. Templeton A, Morris JK. Reducing the risk of multiple births of two embryos after in-vitro fertilization. N Engl J Med 1998; 339: 573–7. 98. Van Kooij RJ, Looman CW, Habbema JD, Dorland M, te Velde ER. Age dependent decrease in embryo implantation rate after in-vitro fertilization. Fertil Steril 1996; 66: 769–75. 99. Alrayyes S, Fakih H, Khan I. Effect of age and cycle responsiveness in patients undergoing intracytoplasmic sperm injection. Fertil Steril 1997; 68: 123–7. 100. Abdalla HI, Burton G, Kirkland A, et al. Age, pregnancy and miscarriage: uterine versus ovarian factors. Hum Reprod 1993; 8: 1512–17. 101. Lintsen AM, Pasker-de Jong PC, de Boer EJ, et al. Effects of subfertility cause, smoking and body weight on the success rate of IVF. Hum Reprod 2005; 20: 1867–75. 102. Younglai EV, Holloway AC, Foster WG. Environmental and occupational factors affecting fertility and IVF success. Hum Reprod Update 2005; 11: 43–57. 103. Homan GF, Davies M, Norman R. The impact of lifestyle factors on reproductive performance in

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the general population and those undergoing infertility treatment: a review. Hum Reprod Update 2007; 13(3): 209–23. Macklon NS, ed. IVF in the Medically Complicated Patient. London: Informa, 2005. Zenzes MT, Krishnan S, Krishnan B, Zhang H, Casper RF. Cadmium accumulation in follicular fluid of women in in vitro fertilization–embryo transfer is higher in smokers. Fertil Steril 1995; 64: 599–603. Paszkowski T, Clarke RN, Hornstein MD. Smoking induces oxidative stress inside the Graafian follicle. Hum Reprod 2002; 17: 921–5. Zenzes MT, Wang P, Casper RF. Cigarette smoking may affect meiotic maturation of human oocytes. Hum Reprod 1995; 10: 3213–17. The Practice Committee of the American Society for Reproductive Medicine. Smoking and infertility. Fertil Steril 2006; 86: S172–7. Norman RJ, Clark AM. Obesity and reproductive disorders: a review. Reprod Fertil Dev 1998; 10: 55–63. Boxmeer JC, Brouns, RM, Lindemans J, et al. Preconception folic acid treatment affects the microenvironment of the maturing oocyte in the human. Fertil Steril 2007 Oct 6 [Epub ahead of print]. Boxmeer JC, Smit M, Weber RF, et al. Seminal plasma cobalamin significantly correlates with sperm concentration in males undergoing IVF or ICSI procedures. J Androl 2007; 28: 521–7. Boxmeer JC, Fauser BC, Macklon NS. Effect of B vitamins and genetics on success of in-vitro fertilisation. Lancet 2006; 368(9531): 200. Heijnen EM, Eijkemans MJ, De Klerk C, et al. A mild treatment strategy for in-vitro fertilisation: a randomised non-inferiority trial. Lancet 2007; 369: 743–9. Heijnen EM, Macklon NS, Fauser BC. What is the most relevant standard of success in assisted reproduction? The next step to improving outcomes of IVF: consider the whole treatment. Hum Reprod 2004; 19: 1936–8.

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35 Initial investigation of the patient (female and male) Bulent Gulekli, Tim J Child, Seang Lin Tan

Introduction Infertility affects one in six or seven couples, and more couples are seeking help than previously. As in other fields of medicine, management of the patient(s) can only be appropriately provided once the causes(s) of the problem are discovered, which in turn requires a proper history, physical examination, and appropriate investigations to be undertaken. Since investigations may be expensive and occasionally invasive, unnecessary testing will only serve to increase the already high (financial and in terms of time commitment) cost to the couple and to the healthcare system. Couples wish to know why they have not been able to conceive and, depending on the etiology, to be provided with the most appropriate options available to them for treatment. Critical evaluation of various investigations for infertility was undertaken at a recent workshop of the European Society of Human Reproduction and Embryology (ESHRE).1 The classical criteria used to evaluate the usefulness of any diagnostic test include sensitivity (to minimize false negatives), specificity (to minimize false positives), usefulness (does knowing the result alter the management), positive and negative predictive values, safety, and cost. The aim of this review is to provide an overview of the approach to the investigation of the infertile couple. Traditionally, infertility investigations are generally begun after a year of involuntary infertility. However, individual circumstances differ, and if there are factors such as increased female age, irregular menstrual cycles, or a history of previous pelvic surgery, earlier investigation may be warranted.

History Female We ask all couples to complete a self-assessment form before attending their first visit at the McGill Reproductive Center. We are particularly interested in the length of infertility, menstrual history, and details of any previous pregnancies, illnesses, surgery, and previous fertility investigations and/or treatment.

Endometriosis may be suggested by a history of pelvic pain, and polycystic ovarian syndrome (PCOS) by oligomenorrhea or hirsutism. Previous abdominopelvic surgery or pelvic infection may suggest peritubular adhesions or tubal obstruction. The female body mass index (BMI) should be calculated by dividing the weight in kilograms by the height in meters squared (kg/m2). The normal range is considered to be 18.5–25 kg/m2. The association between obesity and ovulatory disturbances is well documented,2,3 and there is a correlation between the amount of gonadotropins needed to stimulate the ovaries and the weight of women.4,5 We encourage our patients with increased BMI to reduce weight, because this may allow resumption of ovulation itself or increase the patient’s responsiveness to stimulation. Similarly, women who are grossly underweight should be asked to increase their BMI to the normal range before any induction of ovulation is attempted, to minimize the risk of low-birth-weight babies. Women complaining of infertility should be advised to stop smoking to enhance their fecundity and reduce the risk of miscarriage.6 On the other hand, the evidence concerning alcohol use and infertility in women is conflicting. In a recent study, a statistically significant risk of ovulatory infertility with increasing alcohol consumption was observed, but the risk of infertility with alcohol intake was not increased if the primary diagnosis of infertility was cervical factor, tubal disease, or unexplained infertility.7 Similarly, Zaadstra et al found no correlation between moderate alcohol intake and the probability of conception per cycle or the cumulative pregnancy rate.8 Notwithstanding the above, because the detrimental effect of alcohol on fetal development is well recognized,9 we advise our patients not to drink more than one or two units of alcohol once or twice a week when trying to become pregnant.

Male A detailed history should be obtained and all previous pregnancies fathered recorded. Problems with sexual functioning, such as impotence or ejaculatory

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disturbances, or a history of genitourinary infection, may not be volunteered, and should be specifically inquired about. Maldescent of one or both testes is common in boys, and is associated with an increased risk of testicular failure, while herniorrhaphy as a child may result in inadvertent and unrecognized damage to the vas deferens. Almost one-third of diabetic men sustain ejaculatory dysfunction, most commonly retrograde ejaculation owing to peripheral neuropathy involving the sympathetic nerves. Other neurologic conditions such as multiple sclerosis can also disrupt the ejaculatory reflex. Patients with retrograde ejaculation often note a cloudy quality to their urine. A postejaculatory urine sample will demonstrate large quantities of sperm. A history of postpubertal mumps with associated orchitis (which is unilateral in 67%) is followed by testicular atrophy in 36% of men.10 Any recent febrile illness should be noted as it may have interfered with semen production, causing an abnormal semen analysis result. Some prescription drugs affect male fertility through either sperm production or ejaculatory function. Sulfasalazine and cimetidine may be gonadotoxic, but their effects should be reversed on drug withdrawal. Antihypertensives, antipsychotics, and antidepressants can all cause ejaculatory dysfunction. Anabolic steroids, used by sportsmen, can cause depression in the gonadotropin drive to the testes and a subsequent reduction in spermatogenesis. Vasectomy may lead to the production of antisperm antibodies, which can interfere with sperm function. A reversal is more likely to be successful the sooner it is performed after the vasectomy. A history of infertility among male members of the patient’s family may suggest an inherited disorder.

Examination Female A full physical examination should be routinely undertaken. Severe acne or increased facial hair which may be a result of androgen excess should be noted. The thyroid gland should be palpated, and any signs of acanthosis nigricans associated with insulin resistance or ‘buffalo neck’ as a result of Cushing’s syndrome noted. Turner’s syndrome may be indicated by short stature, webbed neck, shield chest, undeveloped breasts, and cubitus valgus. At abdominal examination any surgical scars, pelvic masses, and the striae associated with Cushing’s syndrome should be noted. Secondary sexual characteristics should be staged by using Tanner’s pubertal development scale. Scanty or absent axillary or pubic hair may indicate either gonadotropin deficiency (for example, androgen insensitivity syndrome, or Kallmann’s syndrome, which is usually associated with anosmia) or impairment of sex steroid production (for example, Turner’s syndrome).

Pelvic examination should be performed during the initial visit. Congenital absence of the vagina (Rokitansky–Küster–Mayer syndrome – the most frequent anatomic cause of primary amenorrhea), imperforate hymen, vaginal septa (either transverse or longitudinal), or double cervices can easily be detected. On speculum examination, the appearance of the cervix should be noted. A microbiologic culture of abnormal vaginal discharge should be taken. We routinely perform a cervical smear and chlamydial cervical culture yearly. The importance of cervical chlamydia in the pathogenesis of pelvic inflammatory disease (PID) is well recognized.11 PID in women can lead to tubal infertility, increased risk of ectopic pregnancy, and chronic pelvic pain.12 Maternal rubella infection in the first 8–10 weeks of gestation results in severe fetal abnormalities in up to 90% of cases; we therefore routinely check the rubella status, and, if seronegative, vaccination is offered. The patient should be advised not to become pregnant within 1 month of immunization. All women presenting with infertility in our clinic are advised to take 0.4 mg folic acid as a supplement to prevent neural tube defects.13 However, supplementation started >30 days after conception has no protective effect.14 On bimanual palpation the size, shape, position, and mobility of the uterus should be noted as well as whether or not there is discomfort. The adnexal and parametrial structures should then be examined for the presence of large ovarian masses. Nodularity in the uterosacral ligaments on bimanual palpation or rectovaginal examination may indicate endometriosis.

Male Examination of the male partner is often neglected during the work-up of an infertile couple. Although the examination should be mainly focused on the urogenital system, disproportionate limb length and height, along with gynecomastia, which may suggest Klinefelter’s syndrome, a reduction in body hair consistent with hypoandrogenism, and a ‘bodybuilder’ physique, which may indicate the use of steroids, should be noted. The scrotal contents are best examined with the man standing. First the presence of a visible varicocele is noted. The size (volume) of the testes is best measured with the aid of a graded Prader orchidometer. Since most testicular tissue is composed of seminiferous tubules, small testes may be indicative of reduced spermatogenesis. The epididymis is palpated for the presence of cysts or nodularity which may arise secondary to infection. The presence or absence of the vasa deferentia should be particularly noted, since bilateral absence will obviously explain a finding of azoospermia and also indicate the need for cystic fibrosis screening. The groin is next examined for surgical scars. The inguinal canal should be palpated for hernia or maldescended testicles, and the penis examined for hypospadias or phimosis.

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Investigations The basic aim of investigations is to determine if ovulation occurs, if the fallopian tubes are patent, and if the man has a normal semen analysis. Although many diagnostic tests have been recommended for the evaluation of the infertile couple, our main criterion before deciding to perform a test is whether the results might be of value in the management. As the treatment of mild endometriosis without tubal adhesions, luteal insufficiency, antisperm antibodies, hyperprolactinemia, or thyroid dysfunction in the presence of normal ovulation has not been shown to result in improved conception rates, diagnostic tests for them are not routinely performed.1,15 The postcoital test is a clinical method of determining the interaction between cervical mucus and spermatozoa. The main problem with this test is poor timing, since cervical mucus is thick and viscous except during the periovulatory period. Consequently, the postcoital test should be performed as close as possible to the time of ovulation, which is tedious since the mucus may be receptive only for a day or two. Because of this, we have largely abandoned the use of this test in our center. Currently, there are no standard protocols for investigation of subfertile couples that are universally accepted, although there are guidelines produced by the World Health Organization (WHO)16 and the Royal College of Obstetricians and Gynaecologists.17 Instead of giving a stepwise protocol of investigation, the diagnostic tests are discussed briefly.

Female Tests for ovulation A woman with regular menstrual cycles every 21–35 days is most likely to be ovulating. However, this should be confirmed, albeit indirectly, by a midluteal serum progesterone measurement, because in a small percentage of cases (<10%), there may still be anovulation.18 It is important that the sample be timed in relation to the subsequent onset of menses, otherwise interpretation is difficult. Blood for serum progesterone measurement should be taken a week before the onset of expected menses (for example, day 21 if the woman has a 28-day cycle or day 28 in a regular 35-day cycle), and then retrospectively confirmed as a midluteal sample by recording the date of the next cycle. If a woman has long and unpredictable cycles, the sample may need to be repeated weekly until the next cycle starts. The precise level of serum progesterone above which ovulation is assumed to have occurred is not universally agreed. The WHO uses a level of 18 nmol/l to confirm ovulation, as this represents the 2.5th centile in their large population study,10 whereas values >16 nmol/l for a minimum of 5 days or a single value exceeding 32 nmol/l is advocated by ESHRE.19 If the results are equivocal, the test

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should be repeated. The basal body temperature (BBT) chart has largely been abandoned in many centers (including ours), because it is cumbersome and is not reliable as a predictor of the time of ovulation.20,21 Serial ovarian ultrasound scans during the follicular phase provide information about follicular development, while a disappearance of the preovulatory follicle/follicles together with the appearance of free fluid in the pouch of Douglas confirms that ovulation has occurred. Indirect methods for predicting ovulation generally involve measuring luteinizing hormone (LH) levels in the blood or urine to detect the LH surge. According to the WHO, regular use of urinary LH kits should be discouraged because of the psychological pressure of timing intercourse and the expense of the kits.16 Endometrial biopsy is performed as an outpatient procedure, and is preferably done between days 21 and 24 of the luteal phase of the cycle. A secretory type of endometrium, due to progesterone secreted by the corpus luteum, supports ovulation. The results of the biopsy are interpreted according to Noyes criteria,22 and a discrepancy of two or more days behind the menstrual dating defines luteal-phase deficiency (LPD). However, there are wide variations in results, which may be due to interobserver variation. A short or inadequate luteal phase may occasionally be observed even in fertile women. Finally, there is no validated treatment for LPD per se.23 Therefore, we do not perform an endometrial biopsy as a part of our routine investigation of the infertile couple.

Pelvic ultrasonography Recent advances in ultrasound technology have made accurate noninvasive assessment of the pelvic organs feasible. Transvaginal color and pulsed Doppler ultrasonography has become an important tool in the evaluation of utero-ovarian perfusion during both the menstrual cycle and in vitro fertilization (IVF) treatment.24,25 A baseline ultrasound scan is able to diagnose congenital anomalies, uterine fibroids, hydrosalpinges (Fig 35.1), ovarian cysts, endometriomas, and polycystic ovaries (Fig 35.2). Polycystic ovaries (PCO) are generally larger, due to increased stromal volume, than normal ovaries.26 Criteria for diagnosis vary, depending on the requisite number of follicles or cysts identified. One of the first ultrasonographic definitions of PCO required the presence of at least 10 small cysts visualized in one sonographic plane arranged around a dense stroma, or scattered throughout an increased amount of stroma.27 Later studies suggested that with the improvement in ultrasound technology and resolution, particularly when utilizing a transvaginal rather than transabdominal approach, the definition should require at least 15, and usually more than 20, cysts.28 PCO is commonly found in apparently normal women, with a prevalence in one study of 22%; in fertility-patient populations the

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Fig 35.1 Hydrosalpinx adjacent to ovary. Color Doppler ultrasonography demonstrates the absence of vascularity within the structure.

Fig 35.2 Polycystic ovary. Increased stromal volume and numerous small circumferential cysts.

prevalence is increased to around 33%.29,30 Though many women are asymptomatic, PCO is the most common cause of anovulatory infertility. Polycystic ovarian syndrome (PCOS) is diagnosed when ovaries of polycystic morphology are present, along with hyperandrogenism, chronic anovulation, and an elevated LH/FSH (folliclestimulating hormone) ratio. Women with PCOS are recognized to have an exaggerated response to gonadotropins, resulting in an increased risk of the ovarian hyperstimulation syndrome (OHSS).30 Indeed, women with polycystic ovarian morphology in the absence of the clinical manifestations of PCOS have recently been shown to exhibit an excessive response to ovarian stimulation, and also to be at increased risk of OHSS.31 Knowledge of the presence of PCO prior to fertility treatment is vital to allow reduction in gonadotropin dose and increased monitoring to reduce the risk of OHSS. An excessively thick endometrium at baseline scan may suggest the presence of a polyp or submucosal fibroid. Uterine cavity distention with saline instillation during vaginal ultrasonography assists in the differentiation of these pathologies (Fig 35.3). Lack of distention in the presence of a thin endometrium can be consistent with intrauterine adhesions (Asherman’s syndrome).

Color and pulsed Doppler ultrasonography may be used to assist in the diagnosis of PCO, to help predict ovarian responsiveness to gonadotropin stimulation, and to aid in the prediction of embryo implantation during IVF treatment. Women with PCO have a higher ovarian stromal blood flow velocity not only at the baseline scan but also during the entire menstrual cycle.24,25 Thus, peak stromal velocity may be an additional marker for PCO.32 The increased blood flow may mean that in response to the same dose of gonadotropin a larger amount of hormone is delivered to the target cells, which could explain the increased ovarian response and associated risk of OHSS. We have shown a positive independent relation between ovarian stromal blood flow velocity both in the early follicular phase,33 and after pituitary suppression,34 with subsequent ovarian follicular response, even in women with normal ovaries. Serum FSH concentrations have been suggested as a suitable guide to ovarian response. However, normal serum FSH levels do not always predict optimal ovarian response, and the presence of wide intercycle variation in basal FSH levels affects the predictive value of the test.35 Therefore, measurement of the maximum ovarian stromal blood flow velocity in the early follicular phase is

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Fig 35.3 Endometrial cavity polyp diagnosed during saline instillation. The patient was noted to have a thickened endometrium at baseline scan.

Fig 35.4 Color and pulsed Doppler ultrasonography of the uterine artery.

useful to predict ovarian responsiveness during IVF treatment, and should be considered for routine use to help determine the appropriate gonadotropin starting dose. Studies have confirmed the predictive value of uterine artery impedance indices (Fig 35.4) on implantation rates, measured after pituitary suppression,36 on the day of human chorionic gonadotropin (hCG) administration,37 and on the day of embryo transfer.38 However, other studies have found no such predictive value.39 Differences may be accounted for by the observation of a circadian variation40 in uterine artery blood flow during the menstrual cycle, and also a variation in measurements depending on the position of the patient.41 When the uterine artery pulsatility index is raised (consistent with increased resistance to blood flow and reduced implantation and pregnancy rates), options include delaying oocyte collection by a few days in the hope that pelvic conditions improve, or freezing all embryos for replacement in a later cycle. Alternatively, it has been suggested that a drug such as

nitroglycerin42 or sildenafil (Viagra)43 may be administered to improve pelvic perfusion. Conventional two-dimensional (2D) ultrasound allows observation of the transverse and longitudinal aspects of pelvic organs of interest. In order to be able to examine the third orthagonal plane (necessary for a frontal view of the uterine cavity), reconstruction of the 2D images by a 3D system is required (Fig 35.5). The views obtained allow detailed inspection of uterine morphology and accurate calculation of volumes. Jurkovic et al were able to diagnose all uterine abnormalities, differentiating between bicornuate and subseptate uteri in a single 3D examination, without the need for a hysterosalpingogram (HSG).44 Previously, a laparoscopy would be required before considering resection of an HSG-diagnosed uterine septum, to ensure that the malformation was not in fact a bicornuate uterus. With the 3D frontal view of the uterus, the presence or absence of a septum can be seen (Fig 35.6). The frontal uterine view also allows for accurate

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Fig 35.5 3D multiplanar view of a normal uterus. The frontal view, demonstrating uterine cavity shape, is at the lower left.

Fig 35.6 3D frontal view of a bicornuate uterus. The fundal myometrial indentation differentiates this from a septate uterus.

examination of fibroids and polyps, and their degree of interference with the endometrial cavity. We previously showed that 3D calculation of endometrial and ovarian volumes was associated with a low intra- and interobserver variability.45 Low (<2 ml) endometrial volumes prior to embryo transfer during IVF are associated with a significantly lower implantation and pregnancy rate.46 Subendometrial spiral artery blood flow measured using power Doppler and 3D scanning on the first day of stimulation in an IVF program has been correlated with treatment outcome.47 Recently, the use of power Doppler 3D ultrasound in association with an echogenic contrast medium to test tubal patency has been reported.48 Power Doppler is able to detect the slow movement of contrast media through a

patent tube, captured in a 3D reconstructed volume. A power Doppler 3D image of the media can be reconstructed to demonstrate tubal filling and shape and fimbrial spill.

Tests of tubal function Assessment of fallopian tube patency is important, as tubal problems affect approximately 30% of infertility patients.49 The most widely used test of tubal patency is hysterosalpingography (HSG), which has the added advantage of assessing the uterine cavity. Uterine cavity defects such as fibroids, polyps, and synechiae can be diagnosed, and an incompetent cervix can sometimes be visualized. The diagnostic value of HSG and

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hysteroscopy in infertility investigation was studied in 400 infertile patients.50 There was a good correlation between HSG and hysteroscopy findings except in the case of uterine synechiae, where HSG tended to overdiagnose the problem. HSGs are performed after cessation of menses but before ovulation, to avoid interfering with an early pregnancy. Prostaglandin inhibitors taken 1–2 hours prior to the HSG are helpful, since many women find the procedure uncomfortable. The disadvantages of HSG include a limited ability to assess peritubal adhesions and the risk of infection. The use of oil-based contrast media has been claimed to result in higher pregnancy rates compared with aqueous dye.51 However, water-soluble media reduce inflammatory reactions, especially granulomatous inflammation, and the risk of oil embolism.52 In a meta-analysis, it was demonstrated that HSG has 65% sensitivity and 83% specificity for diagnosing tubal obstruction.53 A prospective Canadian study attempted to determine whether early laparoscopy in women who had had a normal HSG made a difference to the live birth rate, and suggested that using HSG as a screening test in a low-risk infertile population and deferring laparoscopy do not adversly affect outcome.54 Hysterosalpingo-contrast sonography (HyCoSy) is increasingly used as a test of tubal function. Tubal patency is assessed using transvaginal ultrasound and an injection of a solution containing gas microtubules stabilized on galactose microparticles. The main advantage of this procedure compared with HSG is the lack of radiation exposure and the ability to image the ovaries at the same time.49 However, it remains to be confirmed whether the results of HyCoSy are equivalent to those of HSG in large-scale studies. Salpingoscopy and falloposcopy are new techniques introduced to assess tubal function. Difficulty with passing probes via the uterine cavity into the fallopian tube has been overcome with hysteroscopically guided falloposcopy. By using this method, it is possible to examine the entire length of the tubal lumen.55 Alternatively, the tube can be examined starting from the fimbrial end, guided by laparoscopy, a technique called salpingoscopy.56 Both approaches are essentially only of research interest at present. Complete assessment of the pelvis requires laparoscopy. Visualization of the pelvic cavity by laparoscopy is necessary not only to ascertain tubal patency but also to determine whether there are adnexial adhesions that could interfere with ovum retrieval by the oviducts. In our practice, laparoscopy is generally performed if the results of the HSG are abnormal, unless there is a previous history of ruptured appendix, tubo-ovarian disease, or abdominopelvic surgery. Laparoscopy performed under general anesthesia, with tubal patency checked by the transcervical injection of methylene blue dye, remains the ‘gold standard’ for the accurate assessment of tubal patency.16 However, it involves hospital admission, general anesthesia, a 1–2% complication rate including

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postoperative infection and injury to bowel or blood vessels, and mortality of eight per 100 000.57 In addition to tubal patency, laparoscopy can provide information about other pelvic pathologies such as endometriosis or peritubular adhesions. Some abnormalities detected at the time of diagnostic laparoscopy can be treated during the same procedure (for example, lysis of adhesions, salpingotomy, ovarian cystectomy, or cauterization or vaporization of endometriotic implants). Although it has not been definitively demonstrated that mild pelvic endometriosis without tubal adhesion is a cause of infertility,58 one study suggests that ablative therapy of mild endometriosis increases pregnancy rates.59 In some centers, laparoscopy is also combined with hysteroscopy. Hysteroscopy can allow the diagnosis and treatment of intrauterine adhesions and differentiate submucosal fibroids from endometrial polyps. Hysteroscopy is assumed to be the best method for the detection of intrauterine abnormalities, as it is the only test that directly visualizes intrauterine abnormalities. However, there is no evidence as yet to suggest that all infertile women need a hysteroscopy, as there are no prospective, controlled studies that evaluate fertility outcome after treating uterine abnormalities. Therefore, we reserve hysteroscopy for cases where there is a high suspicion of uterine or cervical pathology, including those with a history of having had repeated difficult intrauterine insemination (IUI) or embryo transfer (ET).

Male The primary investigation is a semen analysis performed after 2–5 days’ abstinence. The WHO criteria for normal semen values are the ones generally used.16 Because of the fluctuation of semen parameters, we repeat semen analyses twice if the first is found to be abnormal (using WHO criteria).60 If no spermatozoa at all are found, the ejaculate should be centrifuged, as very low numbers of sperm suitable for intracytoplasmic sperm injection (ICSI) may be discovered without needing to proceed to testicular biopsy for sperm retrieval. The seminal vesicles contribute approximately 70%, the vasa deferentia 10%, and the prostate 20% of the ejaculate volume. A low-volume (<1 ml), acidic (pH<7.5), azoospermic ejaculate may be associated with the absence or blockage of the seminal vesicles or blockage of the ejaculatory ducts. The level of fructose (produced by the seminal vesicles) will be low. Men with normal-volume azoospermia (>1 ml, pH>7.5) are likely to have spermatogenic failure or vasal/epididymal occlusion. White blood cells are present in all ejaculates, and may have important roles in immune surveillance and clearance of abnormal sperm. Increased numbers of leukocytes in semen may be associated with reduced sperm function, and warrant further investigation.61 If the basic semen analysis is abnormal on repeated testing, further tests are indicated. Vitality stains are

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used to distinguish live from dead sperm. Viable sperm have a plasma membrane that is able to exclude dye. The test is useful for determining the percentage of live sperm in the sample. However, once a sperm has been dyed it cannot be used for ICSI. Vitality staining is therefore used in association with the hypo-osmotic swelling (HOS) test to help determine the chance of finding viable sperm in a subsequent sample for use in an IVF–ICSI cycle. The HOS test works on the principle that viable spermatozoa have plasma membranes which are able to set up an osmotic gradient. In a hypo-osmotic solution viable sperm will absorb fluid, resulting in curling of the tail that can be easily detected. The viable sperm can then be selected for ICSI. Vitality staining and HOS testing can be combined during a semen analysis prior to an IVF–ICSI cycle to confirm that HOS testing is able to identify viable sperm. Other tests include computerassisted semen analysis (CASA), acrosome reaction, sperm penetration assays, and tests of fertilizing ability – for example, the hamster egg penetration test. However, none of these are in widespread clinical use because of a lack of reproducibility, and no widely accepted standards for evaluation and interpretation.62 Antisperm antibodies (ASAs) of immunoglobulin class A (IgA) and IgG have been implicated in subfertility by reducing the progression of sperm through cervical mucus and/or interfering with sperm binding at the zona pellucida. Levels can be measured in the seminal plasma or serum of either the male or female, using either the immunobead or direct and indirect mixed agglutination reaction (MAR) tests. Again, there is some debate as to the role of ASAs in infertility (and in particular the relevance of IgG), the most appropriate method of testing for them, the levels of ASAs that are clinically important, and the most suitable management of affected couples.63,64 In our unit, we test for ASAs in seminal plasma only when there is sperm clumping or abnormal sperm movements (for example, ‘shaking’) on semen analysis. Men with azoospermia or severe oligospermia should have their serum levels of FSH, LH, testosterone, and prolactin measured. On the basis of these results, the men can be classified into a few broad categories. Patients with testicular failure have raised concentrations of FSH, normal or raised concentrations of LH, and either normal or low concentrations of testosterone. If testicular failure is diagnosed, then chromosome analysis should be performed to exclude Klinefelter’s syndrome and other genetic abnormalities. Hypogonadotropic hypogonadism, indicated by low levels of FSH, LH, and testosterone, is uncommon, and may be a result of Kallmann’s syndrome, hyperprolactinemia, or other acquired causes. If the hormonal levels and testicular size are normal, then the man is likely to have obstructive azoospermia.

Technology, in the form of IVF with ICSI, is now able to achieve fertilization when nature intended otherwise. It is therefore vitally important to test for the possibility of a genetic cause of azoospermia or severe oligospermia. Not to do so risks the transmission of a genetic abnormality to the offspring. Nonobstructive azoospermia or severe oligospermia may be due to Y-chromosome microdeletions of the azoospermia factor region (AZF) in Yq11 or karyotypic abnormalities such as sex chromosome aberrations (Klinefelter’s syndrome) or translocations.65 The most common genetic cause of obstructive azoospermia is inheritance of cystic fibrosis (CF) genes resulting in vasal aplasia.65 Transmission is autosomal recessive, and the carrier frequency in white people of Northern European descent is 1 in 25. The phenotypic expression varies, depending on the combination of mutations inherited. At its most severe, men will manifest the full picture of CF along with bilateral vasal agenesis. A less severe manifestation is congenital bilateral absence of the vas deferens (CBAVD), in which men have no other phenotypic expression of CF. Of the 10% of men with CBAVD but no CF gene abnormality found, up to 40% will have unilateral renal agenesis or renal ectopy. Men with CF or CF gene-associated CBAVD have normal renal anatomy. It is imperative to test the partner of a patient carrying the CF gene to define their risks for transmitting CF or CBAVD to their offspring. Ejaculatory duct obstruction is suggested by a lowvolume azoospermia or oligoasthenospermia in the absence of testicular atrophy or raised FSH indicative of primary testicular failure.62 If suspected, these men should be referred to the urologist. Testicular biopsy, before the advent of ICSI, was used diagnostically for differentiating obstruction and testicular failure (and whether the type of failure was Sertoli cell-only syndrome, maturation arrest, or hypospermatogenesis) as a cause of azoospermia. Today, testicular biopsy has an additional therapeutic role in sperm retrieval for use in ICSI.62 Biopsies can be performed prior to a planned IVF–ICSI cycle and sperm, if present, cryopreserved for later ICSI. Alternatively, multiple small testicular biopsies can be performed on the day of egg retrieval during an IVF cycle. In this case, the couple must understand the risk of no sperm being found, and may be advised to have back-up donor sperm available for use if necessary. Biopsies may be performed via an open or percutaneous route. Sperm suitable for ICSI may be found even when the FSH level is increased, indicative of testicular failure. After complete investigation, a proportion of infertile couples will be labeled as ‘unexplained infertility.’ These couples either have a subtle cause of infertility not diagnosed by conventional infertility investigations, or are not very fertile as a couple. The treatment options in both cases are the same, and, depending on individual factors such as the age of the women and the length of infertility, either superovulation and intrauterine insemination or IVF should be

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offered. For women with PCO, an alternative treatment would be in vitro maturation of oocytes.66,67

References 1. The European Society of Human Reproduction and Embryology Capri Workshop Group. Optimal use of infertility diagnostic tests and treatments. Hum Reprod 2000; 15: 723–32. 2. Green BB, Weiss NS, Daling JR. Risk of ovulatory infertility in relation to body weight. Fertil Steril 1988; 50: 721–6. 3. Friedman Cl, Kim MH. Obesity and its effect on reproductive function. Clin Obstet Gynecol 1985; 28: 645–63. 4. Chong AP, Rafael RW, Forte CC. Influence of weight in the induction of ovulation with human menopausal gonadotropin and human chorionic gonadotropin. Fertil Steril 1986; 46: 599. 5. Halme J, Hammond MG, Talbert LM, et al. Positive correlation between body weight, length of human menopausal stimulation and oocyte fertilization rate. Fertil Steril 1986; 45: 372–3. 6. Hughes EG, Brennan BG. Does cigarette smoking impair natural or assisted fecundity? Fertil Steril 1996; 66: 679–89. 7. Grodstein F, Goldman MB, Cramer DW. Infertility in women with moderate alcohol use. Am J Public Health 1994; 84: 1429–32. 8. Zaadstra BM, Looman CWN, te Velde ER, et al. Moderate drinking: no impact on female fecundity. Fertil Steril 1994; 62: 948–54. 9. Royal College of Obstetricians and Gynaecologists. Alcohol consumption in pregnancy. London: RCOG, 1996. 10. Beard CM, Benson RC Jr, Kelalis PP, et al. The incidence and outcome of mumps orchitis in Rochester, Minnesota, 1935 to 1974. Mayo Clin Proc 1977; 52: 3–7. 11. Westrom L, Wolner-Hanssen P. Pathogenesis of pelvic inflammatory disease. Genitourin Med 1993; 69: 9–17. 12. Stacey C, Munday P, Taylor-Robinson D. A longitudinal study of pelvic inflammatory disease. Br J Obstet Gynaecol 1992; 99: 994–9. 13. MRC Vitamin Study Research Group. Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. Lancet 1991; 338: 131–7. 14. Sheppard S, Nevin NC, Seller MJ, et al. Neural tube defect recurrence after “partial” vitamin supplementation. J Med Genet 1989; 26: 326–9. 15. Conway DI, Glazener CMA, Kelly NJ, et al. Routine measurements of thyroid hormones and FSH in infertility not worthwhile. Lancet 1985; 1: 977–8. 16. World Health Organization. WHO manual for standardized investigation and diagnosis of the infertile couple. Cambridge: Cambridge University Press, 1993. 17. Royal College of Obstetricians and Gynaecologists. The initial investigation and management of the infertile couple. London: RCOG, 1998. 18. Landgren BM, Unden AL, Diczfalusy E. Hormonal profile of the cycle in 68 normally menstruating women. Acta Endocrinol 1980; 94: 89–98.

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19. European Society of Human Reproduction and Embryology. Guidelines to the prevalence, diagnosis, treatment and management of infertility. Hum Reprod 1996; 11: 1775–807. 20. Bauman JE. Basal body temperature: unreliable method of ovulation detection. Fertil Steril 1981; 36: 729–33. 21. Templeton AA, Penney GC, Lees MM. Relation between the luteinizing hormone peak, the nadir of the basal body temperature and the cervical mucus score. Br J Obstet Gynaecol 1992; 89: 985–8. 22. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1950; 1: 3–25. 23. Karamardian LM, Grimes DA. Luteal phase deficiency: effect of treatment on pregnancy rates. Am J Obstet Gynecol 1992; 167: 1391–8. 24. Tan SL, Zaidi J, Campbell S, et al. Blood flow changes in the ovarian and uterine arteries during the normal menstrual cycle. Am J Obstet Gynecol 1996; 175: 623–31. 25. Zaidi J, Jacobs H, Campbell S, et al. Blood flow changes in the ovarian and uterine arteries in women with polycystic ovary syndrome who respond to clomiphene citrate: correlation with serum hormone concentrations. Ultrasound Obstet Gynecol 1998; 12: 188–96. 26. Al-Took S, Watkin K, Tulandi T, et al. Ovarian stromal echogenicity in women with clomiphene citratesensitive and clomiphene citrate-resistant polycystic ovary syndrome. Fertil Steril 1999; 71: 952–4. 27. Adams J, Franks S, Polson D, et al. Multifollicular ovaries: clinical and endocrine features and response to pulsatile gonadotropin-releasing hormone. Lancet 1985; 2: 1375–8. 28. Fox R, Corrigan E, Thomas PA, et al. The diagnosis of polycystic ovaries in women with oligo-amenorrhoea: predictive power of endocrine tests. Clin Endocrinol 1991; 34: 127–31. 29. Polson DW, Wadsworth J, Adams J, et al. Polycystic ovaries: a common finding in normal women. Lancet 1988; 2: 870–2. 30. MacDougall MJ, Tan SL, Balen A, Jacobs HS. A controlled study comparing patients with or without polycystic ovaries undergoing in vitro fertilization. Hum Reprod 1993; 8: 233–7. 31. Engmann L, Maconochie N, Sladkevicius P, et al. The outcome of in vitro fertilization treatment in women with sonographic evidence of polycystic ovarian morphology. Hum Reprod 1999; 14: 167–71. 32. Zaidi J, Campbell S, Pittrof R, et al. Ovarian stromal blood flow in women with polycystic ovaries – a possible new marker for diagnosis? Hum Reprod 1995; 10: 1992–6. 33. Zaidi J, Barber J, Kyei-Mensah A, et al. Relationship of ovarian stromal blood flow at baseline ultrasound to subsequent follicular response in an in vitro fertilization program. Obstet Gynecol 1996; 88: 779–84. 34. Engmann L, Sladkevicius P, Agrawal R, et al. The value of ovarian stromal blood flow velocity measurement after pituitary suppression in the prediction of ovarian responsiveness and outcome of IVF treatment. Fertil Steril 1999; 71: 22–9. 35. Scott RT, Hoffman GE, Oeninger S, Muasher SJ. Intercycle variability of day 3 follicle-stimulating

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Textbook of Assisted Reproductive Technologies hormone levels and its effect on stimulation quality in in vitro fertilization. Fertil Steril 1990; 54: 297–302. Bloechle M, Schreiner T, Kuchler I, et al. Colour Doppler assessment of ascendant uterine artery perfusion in an in vitro fertilization–embryo transfer programme after pituitary desensitization and ovarian stimulation with recombinant follicle stimulating hormone. Hum Reprod 1997; 12: 1772–7. Zaidi J, Pittrof R, Shaker A, et al. Assessment of uterine artery blood flow on the day of human chorionic gonadotropin administration by transvaginal colour Doppler ultrasound in an in vitro fertilization program. Fertil Steril 1996; 65: 377–81. Steer CV, Campbell S, Tan SL, et al. The use of transvaginal color flow imaging after in vitro fertilization to identify optimum uterine conditions before embryo transfer. Fertil Steril 1992; 57: 372–6. Teckay A, Martikainen H, Jouppila P. Blood flow changes in uterine and ovarian vasculature, and predictive value of transvaginal pulsed colour Doppler ultrasonography in an in vitro fertilization program. Hum Reprod 1995; 10: 688–93. Zaidi J, Jurkovic D, Campbell S, et al. Circadian variation in uterine artery blood flow during the follicular phase of the menstrual cycle. Ultrasound Obstet Gynecol 1995; 5: 406–10. Dickey RP, Hower JF, Matulich EM, Brown GT. Effect of standing on non-pregnant uterine blood flow. Ultrasound Obstet Gynecol 1994; 4: 480–7. Cacciatore B, Tiitinen A. Transdermal nitroglycerin administration improves uterine blood flow in infertile women. J Assist Reprod Genet 1997; 14 (suppl): 20–451. Sher G, Fisch JD. Vaginal sildenafil (Viagra): a preliminary report of a novel method to improve uterine artery blood flow and endometrial development in patients undergoing IVF. Hum Reprod 2000; 15: 806–9. Jurkovic D, Geipel A, Grubeck N, et al. Three dimensional ultrasound for the assessment of uterine anatomy and detection of congenital uterine anomalies. A comparison with hysterosalpingography and two dimensional ultrasound. Ultrasound Obstet Gynecol 1995; 5: 228–32. Kyei-Mensah A, Maconochie N, Zaidi J, et al. Transvaginal three-dimensional ultrasound: reproducibility of ovarian and endometrial volume measurements. Fertil Steril 1996; 66: 718–22. Raga F, Bonilla-Musoles F, Casan EM, et al. Assessment of endometrial volume by three-dimensional ultrasound prior to embryo transfer: clues to endometrial receptivity. Hum Reprod 1999; 14: 2851–4. Schild RL, Holthaus S, D-Alquen J, et al. Quantitative assessment of subendometrial blood flow by threedimensional ultrasound is an important predictive factor of implantation in an in vitro fertilization programme. Hum Reprod 2000; 15: 89–94. Sladkevicius P. Three-dimensional power Doppler imaging of the Fallopian tube. Ultrasound Obstet Gynecol 1999; 13: 287. Campbell S, Bourne TH, Tan SL. Hysterosalpingo contrast sonography (HyCoSy) and its future within the investigation of infertility in Europe. Ultrasound Obstet Gynecol 1994; 4: 245–53.

50. Fayez JA, Mutie G, Schneider PJ. The diagnostic value of hysterosalpingography and hysteroscopy in infertility investigation. Am J Obstet Gynecol 1987; 156: 558–60. 51. Rasmussen F, Lindequist S, Larsen C, Justesen P. Therapeutic effect of hysterosalpingography: oil versus water soluble contrast media – a randomized prospective study. Radiology 1991; 179: 75–8. 52. Rowe TC, Gomel V, McComb P. Investigations of tuboperitoneal causes of female infertility. In: Insler V, Lunenfeld B, eds. Infertility: Male and Female, 2nd edn. Edinburgh: Churchill Livingstone, 1993: 253–82. 53. Swart P, Mol BWJ, van der Veen F, et al. The accuracy of hysterosalpingography and the diagnosis of tubal pathology: a meta-analysis. Fertil Steril 1995; 64: 486–91. 54. Belisle S, Collins JA, Burrows EA, et al. The value of laparoscopy among infertile women with tubal patency. J Soc Obstet Gynecol Can 1996; 18: 326–36. 55. Kerin JF, Williams DB, San Roman GA, et al. Falloposcopic classification and treatment of fallopian tube lumen diseases. Fertil Steril 1992; 57: 731–41. 56. Brosens I, Boeckx W, Delathin P, et al. Salpingoscopy: a new preoperative diagnostic tool in tubal infertility. Br J Obstet Gynaecol 1987; 94: 768–73. 57. Chamberlain G, Brown JC. Gynaecological laparoscopy – the report of the working party of the confidential enquiry into gynaecological laparoscopy. London: Royal College of Obstetricians and Gynaecologists, 1978. 58. Inoue M, Kobayashi Y, Honda I, et al. The impact of endometriosis on the reproductive outcome of infertile patients. Am J Obstet Gynecol 1992; 157: 278–82. 59. Marcoux S, Maheux R, Berube S, et al. Laparoscopic surgery in infertile women with minimal or mild endometriosis. N Engl J Med 1997; 337: 217–22. 60. Schwartz D, Laplanche A, Jouannet P, David G. Within-subject variability of human semen in regard to sperm count, volume, total number of spermatozoa and length of abstinence. J Reprod Fertil 1979; 57: 391–5. 61. Turek PJ. Infections, immunology, and male infertility. Infertil Med Reprod Clin North Am 1999; 10: 435–70. 62. Kim ED, Lipshultz LI. Evaluation and imaging of the infertile male. Infertil Med Reprod Clin North Am 1999; 10: 377–409. 63. Hjort T. Antisperm antibodies and infertility: an unsolvable question? Hum Reprod 1999; 14: 2423–6. 64. Kutteh WH. Do antisperm antibodies bound to spermatozoa alter normal reproductive function? Hum Reprod 1999; 14: 2426–9. 65. Oates RD. The genetics of male reproduction. Infertil Med Reprod Clin North Am 1999; 10: 411–26. 66. Chian RC, Gulekli B, Buckett WM, Tan SL. Priming with human chorionic gonadotropin before retrieval of immature oocytes in women with infertility due to the polycystic ovary syndrome. N Engl J Med 1999; 341: 1624–6. 67. Chian RC, Buckett WM, Tulandi T, Tan SL. Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Hum Reprod 2000; 15: 165–70.

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36 Drugs used for ovarian stimulation: clomiphene citrate, aromatase inhibitors, metformin, gonadotropins, gonadotropin-releasing hormone analogs, and recombinant gonadotropins Zeev Shoham, Colin M Howles

Introduction Infertility treatment became available owing to developments in the characterization and purification of hormones. Treatment with gonadotropins and clomiphene citrate (CC) became available in 1961. It is the purpose of this chapter to overview their development, structure, and mode of action.

Clomiphene citrate Drug description Clomiphene citrate was synthesized in 1956, and an indisputable therapeutic breakthrough occurred in 1961 when Greenblatt and his group discovered that CC, a nonsteroidal analog of estradiol, exerts a stimulatory effect on ovarian function in women with anovulatory infertility.1 The drug was approved for infertility treatment by the United States Food and Drug Administration (FDA) in 1967. Clomiphene citrate is a triphenylchloroethylene derivative in which the four hydrogen atoms of the ethylene core have been substituted with three phenyl rings and a chloride anion. One of the three phenyl rings bears an aminoalkoxy (OCH2-CH2-N(C2 K2)2) side-chain, but the importance of its action on CC remains uncertain. The dihydrogen citrate moiety (C6H8O7) accounts for the fact that commercially available preparations represent the dihydrogen citrate salt form of CC. Clomiphene citrate is a white or pale yellow, odorless powder, unstable in air and light, with a melting point of 116–118°C. It is a triarylethylene compound (1-p-diethyl aminoethoxyphenyl-1,2-diphenyl2-chloroethylene citrate, with a molecular weight of 598.09) chemically related to chlorotrianisene (TACE),

which is a weak estrogen. Structurally, CC is related to diethylstilbestrol, a potent synthetic estrogen. Although this compound is not a steroid, but a triphenylchloroethylene, its steroic configuration bears a remarkable structural similarity to estradiol, and consequently facilitates binding to estrogen receptors (ERs). Clomiphene citrate is available as a racemic mixture of two stereochemical isomers referred to as (cis) Zu-clomiphene or the (trans) En-clomiphene configuration (Figs 36.1a, 36.1b), the former being significantly more potent. In the commercially available preparations, the isomers are in the ratio of 38% Zuand 62% En-clomiphene. Limited experience suggests that the clinical utility of CC may indeed be due to its cis isomer.2,3 However, it remains uncertain whether cis-CC is more effective than CC proper in terms of ovulation and conception rates.4–7 Following the development of a reverse-phase high-performance liquid chromatography (HPLC) assay8 it was apparent that each isomer exhibited its own characteristic pharmacokinetic profile, the En isomer being absorbed faster and eliminated more completely than the Zu isomer. Although CC tablets contain 62% En isomer and 38% Zu isomer, the observed plasma concentrations of the Zu isomer were much higher than those of the En isomer. Because the Zu isomer is considered more estrogenic than the En isomer, response of the target tissues should vary according to both the relative affinity and the concentrations of each isomer interacting with the relevant ER. Tracer studies of CC with radioactive carbon labeling have shown that the main route of excretion is via the feces, although small amounts are also excreted in the urine. After administration of CC for 5 consecutive days at a dose of 100 mg daily, the drug could be detected in serum for up to 30 days.

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Textbook of Assisted Reproductive Technologies En-clomiphene for authentic transisomer formerly ‘cis clomiphene’ (C2H5)NCH2CH2O

OCH2–CH2–N(C2H5)2 C

C

Cl C6H8O7

Zu-clomiphene for authentic cisisomer formerly ‘trans clomiphene’ Cl (C2H5)NCH2CH2O

Clomiphene citrate Cl C

C

Fig 36.1a Clomiphene citrate is available as a racemic mixture of two stereochemical isomers referred to as (cis) Zu-clomiphene or the (trans) En-clomiphene configuration, the former being significantly more potent. In the preparations commercially available, the isomers are in the ratio of 38% Zu- and 62% En-clomiphene.

Cis-clomiphene

Trans-clomiphene

about some impairment of follicular maturation, resulting in delayed ovulation. Shortly after discontinuation of CC, both gonadotropins gradually decline to the preovulatory nadir, only to surge again at midcycle. The drug interacts with ER-binding proteins similar to native estrogens and behaves as a competitive ER antagonist.14,15 Of importance, CC does not display progestational, corticotropic, androgenic, or antiandrogenic properties.

Indications and contraindications for treatment Fig 36.1b

The isomeric models in a different configuration.

Mechanism of action Administration of CC is followed in short sequence by enhanced release of pituitary gonadotropins, resulting in follicular recruitment, selection, assertion of dominance, and rupture. The principal mechanism of CC action is a reduction in the negative feedback of endogenous estrogens due to prolonged depletion of hypothalamic and pituitary ER.9,10 This action consequently leads to an increase in the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus into the hypothalamic–pituitary portal circulation, engendering an increase in the release of pituitary gonadotropins. Administration of a moderate gonadotropin stimulus to the ovary overcomes the ovulation disturbances and increases the cohort of follicles reaching ovulation.11,12 A marked increase in serum concentrations of luteinizing hormone (LH) in proportion to follicle-stimulating hormone (FSH) may sometimes occur,13 and this temporary change in the ratio of LH:FSH appears to bring

Anovulatory infertility is the most important indication for CC treatment. In addition, treatment is indicated for women with oligomenorrhea, or amenorrhea, who responded to progesterone (P) treatment with withdrawal bleeding. Treatment is ineffective in women with hypogonadotropic hypogonadism (WHO group I). Other controversial manifestations include luteal-phase defect, unexplained infertility, and women undergoing in vitro fertilization (IVF) when multiple follicle development is required. These include pre-existing ovarian cysts, with suspected malignancy, and liver disease.

Duration of treatment Clomiphene citrate increases secretion of FSH and LH and is administered for a period of 5 days. In women with normal cycles, administration of CC for more than 5 days resulted in an initial increase of serum FSH concentration that lasted for 5–6 days, followed by a decline in serum FSH levels, despite continuation of the drug, whereas LH levels remained high throughout the entire treatment period.16,17

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Clomiphene citrate is usually administered on day 5 of spontaneous, or induced, menstruation. This is based on the theory that on day 5 the physiologic decrease in serum FSH concentration provides the means for selection of the dominant follicle. Initiation of the drug on day 2 induces earlier ovulation, which is analogous to the physiologic events of the normal menstrual cycle. The starting dose is usually 50 mg/day, owing to the observation that 50% of pregnancies occur with the 50-mg dose.18 In order to obtain good results, CC therapy should be carefully monitored. Obviously, serial measurements of LH, FSH, estradiol, and P and ultrasound measurements provide the most detailed information on the patient’s response to treatment.

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Several reports have associated long-term (>12 months) CC therapy with a slight increase in future risk of ovarian cancer (relative risk [RR]=(1.5–2.5).21 Owing to these initial reports, the Committee on Safety of Medicines (CSM) in the UK advised doctors to adhere to the manufacturers’ recommendations of limiting treatment to a maximum of 6 months. However, this increased risk has not been confirmed by subsequent reports. Several case reports have linked CC with congenital malformations, especially neural tube defects (NTDs).22–28 Data available on 3751 births after CC treatment included 122 children born with congenital malformations (major and minor), representing an incidence of 32.5/1000 births.29 This figure is within the range found among the normal population.30

Results of treatment Summary Clomiphene citrate induces ovulation in the majority of women. The ovulation rate ranges between 70 and 92%; however, the pregnancy rate is much lower. The discrepancy between the high ovulation rates and relatively low pregnancy rates may be due to the following factors:

Clomiphene citrate is one of the most popular drugs for ovulation induction because it is easy to administer, highly effective, and considerably safe, there is no need for close monitoring, and the cost is minimal.

Aromatase inhibitors 1. 2. 3. 4. 5. 6. 7.

Antiestrogen effects on the endometrium. Antiestrogen effects on the cervical mucus. Decrease of uterine blood flow. Impaired placental protein 14 synthesis. Subclinical pregnancy loss. Effect on tubal transport. Detrimental effects on the oocytes.19

The Cochrane review20 of clinical data regarding the use of CC for unexplained subfertility in women, based on five randomized trials of CC (doses ranging from 50 to 250 mg/day for up to 10 days) compared with placebo, or no treatment, showed that the odds ratio (OR) for pregnancy per patient was 2.38 (95% confidence interval [CI] 1.22–4.62). The OR for pregnancy per cycle was 2.5 (95% CI 1.35–4.62). It was concluded from this review that CC appeared to improve pregnancy rates modestly in women with unexplained subfertility.

Side effects and safety The most common side effects are hot flushes (10%), abdominal distention, bloating or discomfort (5%), breast discomfort (2%), nausea and vomiting (2%), visual symptoms, and headache (1.5%). A rise in basal body temperature may be noted during the 5-day period of CC administration. Visual symptoms include spots (floaters), flashes, or abnormal perception. These symptoms are rare, universally disappear upon cessation of CC therapy, and have no permanent effect. The multiple pregnancy rate is approximately 5% and almost exclusively due to twins.

Aromatase, a cytochrome P450-dependent enzyme, acts as the ultimate step in the synthesis of estrogen, catalyzing the conversion of androgens to estrogens. 31 The conversion of androgens to estrogens occurs also at peripheral sites, such as in muscle, fat, and the liver.32 Recently, a group of new, highly selective aromatase inhibitors has been approved to suppress estrogen production in postmenopausal women with breast cancer. Aromatase inhibitor is a competitive inhibitor of the aromatase enzyme system, and inhibits the conversion of androgens to estrogens. It inhibits the aromatase enzyme by competitively binding to the heme of the aromatase–cytochrome P450 subunit of the enzyme, resulting in a reduction of estrogen biosynthesis in all tissues where it is present (Fig 36.2). Treatment significantly lowers serum estrone, estradiol, and estrone sulfate, and has not been shown significantly to affect adrenal corticosteroid synthesis, aldosterone synthesis, or synthesis of thyroid hormones. Maximum suppression is achieved within 48–78 hours. The first aromatase inhibitor to be developed was aminoglutethimide, but its usage was stopped owing to side effects, one of which was adrenal insufficiency.33 However, this development stimulated the formulation of numerous other aromatase inhibitors that were described as first-, second-, and thirdgeneration inhibitors according to chronologic development. They were further classified as type I (steroid analogs of androstenedione) and type II (nonsteroidal) (Table 36.1).

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17α-OH

Table 36.1 The different types and generations of aromatase inhibitors

Pregnenolone

Generation

Type I

Type II

First Second

None Formestane

Third

Exemestane

Aminoglutethimide Fadrozole Rogletimide Anastrozole Letrozole Vorozole

Androstenedione 17α-OH Testosterone

Progesterone

AROMATASE AROMATASE Estrone

Estradiol

Fig 36.2 Aromatase inhibitor. Aromatase, an enzyme found in the liver, is responsible for the conversion of androgens – androstenedione and testosterone – into estrogens – estrone and estradiol. By inhibiting aromatase the body produces less estrogen and maintains a higher testosterone state. DHEA, dehydroepiandrosterone sulfate.

noted with CC. The drug is now under extensive evaluation in anovulatory patients.

Metformin Pharmacokinetics Third-generation aromatase inhibitors are administered by mouth, and have a half-life of approximately 48 hours, which allows once-daily dosing.34,35 These drugs metabolize mainly in the liver, and are excreted through the biliary (85%) and the urinary (11%) systems.

Side effects Reported side effects are bone pain (20%), hot flashes (18%), back pain (17%), nausea (15%), and dyspnea (14%). These side effects are typically observed after long-term administration.

Drugs available Letrozole: this is chemically described as 4,4′-(1H1,2,4-triazole-1-ylmethylene) dibenzonitrile, with a molecular weight of 285.31 and empirical formula C17H11N5. Anastrozole: the molecular formula is C17H19N5 with a molecular weight of 293.4. Both drugs were approved for treatment of postmenopausal women with breast cancer. The first clinical study using an aromatase inhibitor (letrozole: AstraZenica) for ovulation induction was published by Mitwally and Casper in 2001.36 With letrozole treatment in patients with PCOS, ovulation occurred in 75% and pregnancy was achieved in 25%. Letrozole appears to prevent unfavorable effects on the endometrium that are frequently observed with antiestrogen use for ovulation induction. Since the initial observation, several studies have been published on the use of aromatase inhibitors in the treatment of infertile patients.37–39 Recently, the same group of investigators40 were able to show that the use of an aromatase inhibitor reduced the FSH dose required for controlled ovarian stimulation, without the undesirable antiestrogenic effects occasionally

The biguanide metformin (dimethylbiguanide) is an oral antihyperglycemic agent widely used in the management of noninsulin-dependent diabetes mellitus. It is an insulin sensitizer that reduces insulin resistance and insulin secretion. Over the last few years there has been increased interest in the use of metformin (at doses of 1500–2500 mg/day) to increase ovulatory frequency, particularly in women described as having polycystic ovary syndrome (PCOS). There is, however, some recent conflicting evidence regarding the usefulness of metformin in PCOS patients. In a Cochrane systematic review,41 metformin was concluded to be an effective treatment for anovulation in women with PCOS, with it being recommended to be a first-line treatment, and with some evidence of benefit on parameters of the metabolic syndrome. Ovulation rates were higher when combined with clomiphene (76% vs 46% when used alone). Finally, the authors recommended that it should be used as an adjuvant to general lifestyle improvements, and not as a replacement for increased exercise and improved diet. Subsequently, both the American Society for Reproductive Medicine (ASRM) Practice Committee (USA)42 and the National Institute for Clinical Excellence (NICE) (UK)43 have made recommendations for its use in treating anovulatory PCOS. In previously untreated women with PCOS, no superiority of the combination of CC and metformin, rather than CC alone, was demonstrated in a large, Dutch multicenter study.44 In a ‘head-to-head’ study comparing CC with metformin as first-line treatment, although ovulation and pregnancy rates were similar, significantly fewer miscarriages and, therefore, more live births, were achieved with metformin. 45 In a meta-analysis of randomized trials in PCOS patients undergoing OI or IVF/embryo transfer (ET), 46 co-administration of metformin to gonadotrophins did not significantly improve ovulation (OR = 3.27, 95% CI 0.31–34.72) or pregnancy

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(OR = 3.46, 95% CI 0.98–12.2) rates. Metformin coadministration in an IVF treatment did not improve the pregnancy rate (OR = 1.29; 95% CI 0.84–1.98) but was associated with a reduction in the risk of ovarian hyperstimulation syndrome (OHSS) (OR = 0.21, 95% CI 0.11–0.41). 46 However, the authors concluded that the review was inconclusive in terms of not being able to exclude an important clinical treatment effect because of the small number of trials and small sample sizes of the individual trials limiting the power of the meta-analysis. Neveu et al47 carried out an observational comparative study to determine which first-line medication (CC or metformin) was more effective in PCOS patients undergoing OI and to verify whether any patient characteristic was associated with a better response to therapy. The authors included ‘154 patients who had never been treated for ovulation induction to avoid confounding effects of a previous fertility treatment.’ Patients receiving metformin alone had an increased ovulation rate compared with those receiving CC alone (75.4% vs 50%). Patients on metformin had similar ovulation rates compared with those in the combination group (75.4% vs 63.4%). Pregnancy rates were equivalent in the three groups. Response to metformin was independent of body weight and dose. Finally, nonsmoking predicted better ovulatory response overall as well as lower fasting glucose for CC and lower androgens for metformin. A recent literature review48 was carried out to establish if metformin was efficacious when given to CCresistant PCOS patients (The Medline database was searched from January 1, 1980 to January 1, 2005). When the data from four prospective double-blind placebo controlled trials were pooled, the overall effect of the addition of metformin in the CC patient was p = 0.0006 with a 95% CI of OR of 1.81–8.84. In only two trials the randomization was prospective; when the data of these two trials were pooled, the overall effect of the addition of metformin in the CCresistant patient was p < 0.0001, with a 95% CI of OR of 6.24–70.27. Combining all data gave an overall positive effect of p <0.0001, with a 95% CI of OR of 3.59 – 12.96. The authors concluded that the addition of metformin in the CC-resistant patient is highly effective in achieving ovulation induction. In the largest study to date, Legro and colleagues49 randomized 626 subfertile women with PCOS who had received previous fertility therapy but were not known to be CC resistant, to have CC + placebo, extended-release metformin + placebo, or a combination of metformin + CC for up to 6 months. The dose of extended-release metformin was gradually increased until a maximum dose of 2000 mg/day. Medication was discontinued when pregnancy was confirmed, and subjects were followed until delivery. The primary endpoint of the study was live birth rate. The live birth rate was 22.5% (47 of 209 subjects) in the CC group, 7.2% (15 of 208) in the metformin group, and 26.8% (56 of 209)

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in the combination therapy group (p <0.001 for metformin vs both CC and combination therapy; p = 0.31 for CC vs combination therapy). Among pregnancies, the rate of multiple pregnancies was 6.0% in the CC group, 0% in the metformin group, and 3.1% in the combination therapy group. The rates of firsttrimester pregnancy loss did not differ significantly among the groups. However, the conception rate among subjects who ovulated was significantly lower in the metformin group (21.7%) than in either the CC group (39.5%, p = 0.002) or the combination therapy group (46.0%, p <0.001). With the exception of pregnancy complications, adverse-event rates were similar in all groups, though gastrointestinal side effects were more frequent, and vasomotor and ovulatory symptoms less frequent, in the metformin group than in the CC group. They concluded that CC was superior to metformin in achieving live birth in women with PCOS, although multiple births are a complication. In spite of the non-significant difference in live birth rates, between CC and combination therapy, the latter group had superior ovulation rates vs CC or metformin alone (60.4 vs 49.0 vs 29.0%; Fig 36.3) and a trend to an improvement in the pregnancy rate (absolute difference = 7.2%) following use of CC + metformin vs CC. There were some important reductions in body mass index (BMI) testosterone, insulin, and insulin resistance in patients treated with the combination vs CC alone. Some of the differences in results reported in Legro et al49 compared with Palomba45 may have been due to the inclusion of a large percentage of patients with a BMI >30. However in a post-hoc analysis, the largest differences in pregnancy rate and live birth rate in the CC vs CC + metformin groups were found in women with a BMI >34. To conclude, whereas the adverse features of PCOS can be ameliorated with lifestyle intervention, such as diet and exercise, some further short-term benefits related to ovulation and cardiac risk factors may be derived from medication with metformin. Further studies are warranted to examine the role of metformin in managing the long-term metabolic implications of PCOS.

Pharmacokinetics Metformin is administered orally and has an absolute bioavailability of 50–60%, and gastrointestinal absorption is apparently complete within 6 hours of ingestion. Metformin is rapidly distributed following absorption and does not bind to plasma proteins. No metabolites or conjugates of metformin have been identified. Metformin undergoes renal excretion and has a mean plasma elimination half-life after oral administration of between 4.0 and 8.7 hours. Food decreases the extent of and slightly delays the absorption of metformin.

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70 60.4 60 49 50 40

Ovulation

31.1

29

26.8

23.9 22.5

30

Pregnancy Live birth

20 8.7 7.2

10 0

Met alone

CC alone

CC+Met

Side effects In one US double-blind clinical study of metformin in patients with type 2 diabetes, the most reported adverse reactions (reported in >5% patients) following metformin use were diarrhea (53%), nausea/vomiting (25.5%), flatulence (12.1%), asthenia (9.2%), indigestion (7.1%%), abdominal discomfort (6.4%), and headache (5.7%).

Human chorionic gonadotropin: the luteinizing hormone surge surrogate Owing to inconsistency of the spontaneous LH surge, in controlled ovarian stimulation, and its inefficacy in patients being treated with GnRH agonists, human chorionic gonadotropin (hCG) has been uniformly adopted by all successful ovarian-stimulation programs to effect the final triggering of ovulation. When preovulatory follicles are present, administration of hCG is followed by granulosa cell luteinization, a switch from estradiol to P synthesis, resumption of meiosis and oocyte maturation, and subsequent follicular rupture 36–40 hours later. These processes will occur only if the follicle is of appropriate size and granulosa and theca cell receptivity is adequate, depending on LH receptor status. Human chorionic gonadotropin has been used as a surrogate LH surge because of the degree of homology between the two hormones. Both LH and hCG are glycoproteins with a molecular weight of approximately 30 kDa, and both have almost identical α subunits and a high cystine content (Fig 36.4). Most important, they have the same natural function, i.e. to induce luteinization and support lutein cells. Major differences include the sequence of the β subunit, the regulation of secretion of both hormones, and the pharmacokinetics of clearance of hCG as opposed to LH (Table 36.2).

Figure 36.3 Ovulation, pregnancy, and live birth rates (%) in PCOS patients treated with Metformin(MET) alone, CC alone, or Metformin +CC. Reproduced from Legro et al,49 with permission.

The plasma metabolic clearance rate of hCG is slower than that of LH; i.e. a rapid disappearance phase in the first 5–9 hours after intramuscular (i.m.) injection, and a slower clearance rate in the 1–1.3 days after administration (Fig 36.5). The calculated initial and terminal half-life of recombinant hCG is 5.5 + 1.3 and 3.1 + 3.0 hours, respectively, as opposed to 1.2 + 0.2 and 10.5 + 7.9 hours, respectively, for recombinant human LH, as determined after intravenous (i.v.) administration of the drugs.50 By day 10 after administration, <10% of the originally administered hCG was measurable.51 Some authors have advocated the presence of a serum factor directed against hCG preparations, which significantly prolongs the half-life of hCG administration to women who have received repeated courses of gonadotropins.52 Others have not found such a correlation.51 Ludwig et al suggested that the main differences between LH and hCG lie within the N-linked oligosaccharides and the C-terminal sequence, in which the latter, and especially the O-linked oligosaccharides in this peptide, are responsible for the longer half-life of hCG compared with LH.53 It is of interest that hCG does not inhibit the subsequent spontaneous LH surge by the intact pituitary, confirming that an ultrashort loop feedback of LH (here hCG) with its own secretion is not functional.54–56 It has been found that elevated P levels immediately after hCG administration subsequently induce pituitary LH surges in CC/hMG (human menopausal gonadotropin) cycles.54 The long serum half-life of hCG is likely to be an undesirable characteristic in clinical practice. Residual hCG may be mistaken for early detection of de novo synthesis of hCG by a newly implanted pregnancy. Additional consequences of hCG administration are the sustained luteotropic effect, development of multiple corpora lutea, and supraphysiologic levels of estradiol and P synthesis. Sustained high-level stimulation

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Glycosylation on β subunit β subunit

Glycosylation on α subunit

N-ter N-ter

O-glycosylation on CTP

C-ter-α

α subunit

Glycosylation on α subunit

Fig 36.4 Human chorionic gonadotropin (hCG) model. Computerized model of hCG with full glycosylation and cytidine triphosphate (CTP). (This model was created and provided by the scientific department of Serono Laboratories, USA.)

Table 36.2 Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) pharmacokinetics and characteristics. Pharmacokinetics of recombinant human LH(rLH), urinary human menopausal gonadotropin (uhMG), urinary hCG(uhCG), and recombinant hCG(rhCG). Results are expressed as mean ± SD. Modified from references 50 and 50a Test drug Subjects (n) Route Dose (IU) Cmax* (IU/l) t1/2(1)*(h) t1/2*(h)

rhLH

uhMG

uhCG

rhCG

12 i.v. 300 32.1 ± 5.0 0.8 ± 0.2 10.5 ± 7.9

12 i.v. 300 24.0 ± 4.2 0.7 ± 0.2 12.4 ± 12.3

12 i.v. 5000 906 ± 209 5.5 ± 1.3 31 ± 3

12 i.v. 5000 1399 ± 317 4.7 ± 0.8 28 ± 3

*Based on serum concentrations measured with immunoradiometric assay (mean ± SD). i.v., intravenous; Cmax, maximum concentration; t1/2 (1), initial half-life; t1/2, terminal half-life.

of the corpora lutea may lead to OHSS, a major complication of gonadotropin therapy.57 Administration of hCG results in an increase in LH-like activity, but does not reconstitute the midcycle physiologic FSH surge. Another disadvantage of hCG vs the physiologic LH surge is that of higher luteal-phase levels of estradiol and P induced by supraphysiologic hCG concentrations. Excessive levels of circulating estradiol have

been implicated in the relatively high rates of implantation failure and early pregnancy loss observed in ovarian-stimulation programs.58,59 Another possible disadvantage of the prolonged activity of hCG is that of small-follicle, delayed ovulation, which could be the cause of the development of multiple pregnancies. Almost universal use of GnRH agonists and pituitary desensitization protocols has made the fear of

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Gonadotropins

a

Serum β hCG level (IU/l)

300 250 200 150

Historical overview

100 50 0 b 1200 1000 800 600 400 200 0 0

50

100

150

200

250

300

Time after hCG administration (h)

Fig 36.5 Pharmacokinetics of serum β hCG in two hypogonadotropic women; (a) the first woman; (b) the second woman. Three regimens of human chorionic gonadotropin (hCG) injections were applied in each woman: 10 000 IU administered subcutaneously, or intramuscularly, and 5000 IU administered intramuscularly. (Modified from Weismann et al. 49a.)

untimely LH surges relatively obsolete; hence hCG, the timing of the LH-like stimulus, has been given greater flexibility. Tan et al60 actually showed that there was no difference in cycle outcome with random timing of hCG administration over a 3-day period. Unfortunately, invalidation of the pituitary mechanism that releases us from an inappropriate LH surge has also made us completely dependent on hCG, with all its inherent problems, for the final stage of ovulation triggering. Another issue requiring clarification is the minimal effective dose of hCG in order to trigger oocyte maturation and ovulation. In a study examining the minimal effective dose of hCG in IVF,61 dosages of 2000, 5000, and 10 000 IU of urinary hCG (uhCG) were administered to 88, 110, and 104 women, respectively. No differences in oocyte recovery were noted when comparing the groups that received 5000 and 10 000 IU. However, a significantly lower number of oocytes were aspirated in the 2000-IU group, compared with the 5000- and 10 000-IU groups. With the recent development of recombinant technology, recombinant hCG (rhCG) became available for clinical use, and appears to be as efficacious as uhCG with the benefit of improved local tolerance.51,62,63 Using r-hCG in the IVF63 program, administration of 250 µg r-hCG compared with 5000 IU uhCG revealed that r-hCG is at least as effective as 5000 IU of uhCG. Using a higher dose of rhCG, such as 500 µg, resulted in retrieval of more oocytes, but a three-fold increase of the OHSS occurred. It was also noted that local reaction to the injection was significantly better than to the urinary product of equal dose.53

In 1927, Aschheim and Zondek discovered a substance in the urine of pregnant women with the same action as the gonadotropic factor in the anterior pituitary.64 They called this substance gonadotropin or ‘prolan.’ Furthermore, they believed that there were two distinct hormones, prolan A and prolan B. They subsequently used their findings to develop the pregnancy test that carries their names. In 1930, Zondek reported that gonadotropins were also present in the urine of postmenopausal women,65 and in the same year, Cole and Hart found gonadotropins in the serum of pregnant mares.66 This hormone, pregnant mare serum gonadotropin (PMSG), was found to have a potent gonadotropic effect in animals. However, it was only in 1937 that Cartland and Nelson were able to produce a purified extract of this hormone.67 It was not until 1948, as a result of the work of Stewart, Sano, and Montgomery, that gonadotropins in the urine of pregnant women were shown to originate from the chorionic villi of the placenta, rather than the pituitary. It was subsequently designated ‘chorionic gonadotropin.’68 After years of experiment, it gradually became apparent that the pituitary factor was needed for the production of mature follicles, and that chorionic gonadotropin could induce ovulation only when mature follicles were present.69 Within years, it became apparent that the use of gonadotropin extracts from nonprimate sources was of limited clinical value owing to the development of antibodies that neutralized their therapeutic effect. In 1947, Piero Donini, a chemist at the Pharmaceutical Institute, Serono, in Rome tried to purify hMG from postmenopausal urine. This purification method was based on a method used by Katzman et al, published in 1943.70 The first urine extract of gonadotropin contained LH and FSH and was named Pergonal, inspired by the Italian words ‘per gonadi’ (for the gonads).71 The approval to sell Pergonal was first granted by the Italian authorities in 1950 (Table 36.3). Only in 1961, with Pergonal treatment, was the first pregnancy achieved in a patient with secondary amenorrhea, which resulted with the birth (in 1962 in Israel) of the first normal baby girl.72 Urinary FSH (Metrodin) and highly purified FSH became available with the development of new technologies using specific monoclonal antibodies to bind the FSH and LH molecules in the hMG material in such a way that unknown urinary proteins could be removed. Metrodin has a specific activity of 100–200 IU of FSH/mg of protein, whereas Metrodin-HP highly purified has an activity of approximately 9000 IU/mg of protein.

Human menopausal gonadotropin Human menopausal gonadotropin contains an equivalent amount of 75 IU FSH and 75 IU LH in vivo bioactivity. Cook et al73 demonstrated that hMG preparations

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Table 36.3 treatment 1927 1959 1960 1966 1970 1978 1984 1985 1990

URINE

Milestones of development in infertility

The discovery of pituitary hormone controlling ovarian function Purification and clinical use of pituitary and urine gonadotropins Clinical use of clomiphene citrate Use of clomiphene citrate and gonadotropin becomes common practice Development of radioimmunoassay for measuring hormone levels Ultrasound imaging of ovarian follicles Use of gonadotropin-releasing hormone (GnRH) agonists in infertility treatment Further purification of urinary gonadotropins Use of recombinant gonadotropins

Kaolin Crude material

hMG

Monoclonal antibodies that react with FSH

Antibody that reacts with LH

Metrodin

Recovery of FSH

Metrodin-HP

also contain up to five different FSH isohormones and up to nine LH species. These differences may cause discrepancies in patients’ responses occasionally observed when using various lots of the same preparation. Follicle-stimulating hormone, which is the major active agent, accounts for <5% of the local protein content in extracted urinary gonadotropin products.74 The specific activity of these products does not usually exceed 150 IU/mg protein. The different proteins found in various hMG preparations include tumor necrosis factor binding protein I, transferrin, urokinase, Tamm– Horsfall glycoprotein, epidermal growth factor, and immunoglobulin-related proteins.75 Although hMG preparations are effective and relatively safe, local side effects, such as pain and allergic reactions, that were possibly attributed to immune reactions related to nongonadotropin proteins, have been documented in certain cases.76 Information is scarce regarding the metabolism of gonadotropin hormones. It was shown that purified preparations of hFSH, hLH, and hCG injected (i.v.) in humans had serum half-lives (as determined by bioassays) of 180–240 minutes, 38–60 minutes, and 6–8 hours, respectively. Measuring levels of gonadotropins by in vivo bioassays serves to compare biologic effects of gonadotropin preparations in a quantitative manner in animals. In the extensively used Steelman–Pohley assay,77 21-day-old female Sprague–Dawley rats are injected subcutaneously (s.c.) for 3 days and their ovaries weighed on the fourth day. Disadvantages of this assay are that its sensitivity is too low to detect small amounts of FSH in the serum, reproducibility is poor (+20% variation), and the procedure is cumbersome. The reliance on this assay, in effect, signifies that an ampoule of hMG, which appears to have 75 IU of FSH, may actually contain between 60 and 90 IU. Circulating levels of the gonadotropins measured at any given moment represent the balance between pituitary release and metabolic clearance. After i.v. injection, the initial half-life of urinary FSH

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Protein and LH

Fig 36.6 Schematic presentation of the production of human menopausal gonadotropin (hMG) and purification of urinary follicle-stimulating hormone (uFSH) and high-purity (HP)-FSH. LH, luteinizing hormone.

was demonstrated to be approximately 2 hours,78 and the true terminal (elimination) half-life appeared to be 17 + 5 hours. After i.m. injection of urinary FSH preparations, the half-life was estimated to be approximately 35 hours.51

Purified follicle-stimulating hormone Further purification of hMG substantially decreased LH-like activity, leading to a commercial pFSH preparation. Metrodin was introduced in the mid-1980s and is a product from the same source as hMG, but the LH component has been removed by immunoaffinity chromatography (Fig 36.6). Apart from obtaining a more purified product, the rationale of developing a pFSH preparation was that ovulation induction using gonadotropins in patients with elevated endogenous LH serum levels could, on theoretical grounds, preferably be performed without exogenously administered LH. It was also suggested that FSH alone could increase folliculogenesis.79 Furthermore, it was speculated that LH in gonadotropin preparations could be responsible for the high incidence of complications in patients with elevated serum LH levels.80,81 However, other studies82,83 have indicated that the effectiveness of gonadotropin preparations and the occurrence of OHSS were not dependent on the LH:FSH ratio,51 albeit the administration of pFSH to patients with PCOS did result in decreased LH levels, compared with hMG.84 The desirable goal of having an FSH preparation of high purity led to the development of an immunopurified product (Metrodin-HP) of >95% purity.85

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Recombinant human gonadotropins (follicle-stimulating hormone, luteinizing hormone, and chorionic gonadotropin)

vial of the MCB.86 MCB and WCB are routinely tested for sterility, mycoplasma, and viral contaminanation.

Following the development of highly purified urinary FSH, considerable improvements have facilitated both separation of FSH from human LH (hLH) and its production using recombinant technology. Early technology focused on the production of biological molecules in bacterial cells (usually Escherichia coli). However, the structural complexity of human gonadotropins such as FSH and the need for post-translational modification of the molecule by protein folding and glycosylation, made functional protein production impossible in prokaryotes. Thus, a mammalian cell culture system was employed with functional molecules being produced in Chinese hamster ovary (CHO) cells. The world’s first recombinant hFSH (rhFSH; follitropin α) preparation for clinical use was produced by Serono Laboratories in 1988, and was licensed for marketing in the European Union as Gonal-F in 1995. An rhFSH (follitropin β; Puregon) product was also licensed by Organon Laboratories in 1996. The genes for the other gonadotropins have also been transfected into mammalian cell lines, and recombinant hLH (rhLH) and human chorionic gonadotropin (rhCG) are now commercially available (rhLH as Luveris, Merck Serono International, Switzerland; rhCG as Ovidrel/Ovitrelle, Merck Serono International; and rhFSH and rhLH in a 2:1 ratio, Pergoveris, Merck Serono International). However, for the purposes of this section, the focus will be on rhFSH (follitropin α). The production of hFSH by recombinant technology required isolation and cloning of genes for two subunits, the α subunit – which is also common to hLH and hCG – and a hormone-specific β subunit. Appropriate vectors were prepared and transfected into suitable immortalized mammalian cell lines. The cell line originally chosen by Serono Laboratories was well established (CHO–DUKX), and already being used to produce proteins such as recombinant human erythropoietin. These cells are normally dihydrofolate reductase (DHFR) deficient, and therefore sensitive to tetrahydrofolate analogues such as methotrexate. Cells were co-transfected with the human α and β FSH genes and then treated with methotrexate, in order to select successfully transfected cells which could express the newly introduced genes. A stable line of transformed cells was selected, which secreted high quantities of rhFSH. These cell lines were used to establish a master cell bank (MCB), which now serves as the source of working cell banks (WCBs). The MCB consists of individual vials containing identical cells, which are cryopreserved until required. Thus, a continuous supply of rhFSH with guaranteed consistency from WCB to WCB is now available by expansion of cells recovered from a single

Traditionally, quantification of hFSH, LH, and hCG for clinical use has involved the use of in vivo bioassays. For hFSH, a number of bioassays have been assessed for this purpose, but one of the most robust and specific remains the Steelman–Pohley in vivo assay, first developed in the 1950s.77 FSH activity is quantified by rat ovarian weight gain and FSH vials or ampoules are subsequently filled according to the desired bioactivity, measured in international units (IUs). However, the assay has a number of limitations: it is time consuming, cumbersome, uses large numbers of rats (which is of ethical concern) and is limited in its precision – the European Pharmacopoeia defines an activity range (80–125% of the target value) within which an FSH batch is acceptable for clinical use. Recent advances in the manufacturing process for the rhFSH follitropin α, however, enable high batchto-batch consistency in both isoform profile and glycan species distribution.87,88 The most significant advantage of this over other commercially available gonadotropins is that it permits FSH to be quantified reliably by protein content (mass in µg) rather than by biologic activity. The coefficient of variation for an in vivo bioassay is typically ± 20%, compared with less than 2% for physicochemical analytic techniques, such as size exclusion HPLC (SE-HPLC).87,88 As a result, Merck Serono International now quantify their rhFSH (Gonal-F), rhLH, and rhCG protein by SE-HPLC, a precise and robust assay that results in a significant improvement in batch-to-batch consistency.90

Quantifying and standardizing gonadotropin content

Physicochemical consistency of rhFSH: glycan mapping and isoelectric focusing Glycan mapping provides a fingerprint of the glycan species of rhFSH and an estimation of the degree of sialylation of the oligosaccharide chains. For each rhFSH batch, intact glycan species are released by hydrazinolysis and labeled with a fluorescent derivative. As each glycan molecule is labeled with a single molecule of the dye, the response coefficient is the same for all glycan species, which are separated and detected by anion exchange chromatography and fluorimetry. Results are expressed as the relative percentage of the glycan species grouped as a function of their charge, which is related to the number of sialic acids they carry. The hypothetical charge number, Z, is defined as the sum of the percent areas under the curve in the neutral, mono-, di-, tri- and tetrasialylated glycan regions, multiplied by their corresponding charge.90 The Z number was demonstrated to be a very precise estimate of the degree of sialylation, with a coefficient

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of variation of 2% or better. Evaluation of Gonal-F batch data over time has demonstrated a highly consistent glycoform distribution, which reflects the high consistency of its molecular profile.87,88,91 The second physicochemical technique, isoelectric focusing, is performed in a gel matrix across a pH range of 3.5–7.0. After scanning the gel, the PI values and band intensities of the sample isoforms are compared with the reference standard. The distribution of the main bands from Gonal-F has remained similar to the reference standard over time, indicating a high consistency of isoform distribution.87

Follitropin α filled by mass Between-batch analysis of the ratio of Gonal-F bioactivity, measured in IU using the Steelman–Pohley assay, and protein content, measured in µg by SEHPLC, has demonstrated a stable, normal distribution of specific activity with no bioreactor run effect.87 Similarly, drug substance production data over time also confirmed the well-controlled behavior and consistency of the Gonal-F manufacturing process.87,88 The highly consistent physicochemical and biologic properties of the product now permit FSH quantification by SE-HPLC, and vials or ampoules can be filled by mass (FbM) rather than by specific bioactivity. This product is referred to as Gonal-F FbM (Merck Serono International, Switzerland). Once the physicochemical consistency of Gonal-F FbM had been demonstrated, the clinical relevance of the improved manufacturing process was assessed. A total of 131 women were enrolled into a multicenter, double-blind, randomized, parallel group study comparing the efficacy and safety of four batches each of Gonal-F FbM and Gonal-F filled and released by IU (FbIU) in stimulating multiple follicular development prior to IVF.92 Adequate levels of ovarian stimulation were achieved with both preparations, resulting in a large number of embryos. The clinical pregnancy rate per treated cycle was 30.3% with the FbM preparation compared with 26.2% with FbIU. Both preparations showed similar levels of adverse events. However, it is the consistency of clinical response between batches that is of particular importance to physicians. The study demonstrated that the improved manufacturing process for the FbM over the FbIU preparation was associated with an improvement in the consistency of ovarian response (p<0.039), including significantly improved between-batch consistency in the clinical pregnancy rate (p<0.001). Compared with Gonal-F FbIU, the FbM preparation reduced the between-batch variability in clinical outcome. Similar results were also demonstrated in larger studies in ART and OI of Gonal-F FbM vs FbIU.93-97 In a retrospective study by Balasch et al,93 the clinical results during the introduction of Gonal-F FbM were compared to standard Gonal-F FbIU. The study included the last 125 patients treated with Gonal-F FbIU and

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the first 125 patients receiving Gonal-F FbM for ART ovarian stimulation. The patient demographics, oocyte yield, the number of metaphase II oocytes, and the fertilization rates were similar in both groups of patients. However, embryo quality as assessed on day 2 and implantation rates were significantly higher (18.6 vs 28.6% p = 0.008) in the rhFSHFbM group. Accordingly, in spite of the mean number of embryos transferred being significantly lower in the rhFSH FbM group, there was a trend for higher clinical pregnancy rates (44 vs 35.2%) in this group of patients. In a large UK multicenter observational study carried out using Gonal-F FbM in 1427 ART patients,94 the safety and efficacy of Gonal-F FbM was confirmed in routine clinical practice. The patients’ mean age was 34.3 years old and an average of 10.3 oocytes were retrieved. Only 2.7% of the patients who started FSH therapy did not receive hCG. The incidence of severe OHSS was 0.4% and the clinical pregnancy rate per cycle was 29.2%. In the OI study,95 following use of Gonal-F FbM vs FbIU, fewer patients required an adjustment in the FSH dose (37% vs 60%) and there were fewer cancelled cycles (13% vs 21%) during treatment using a chronic low-dose protocol. Hence, the quality of gonadotropin preparation may play an important role in the consistency of the clinical response, including a reduction in the cycle cancellation.97

Molecular modeling and the development of ‘designer’ gonadotropins One approach for expanding the range of recombinant gonadotropins available for the treatment of subfertility is through protein engineering. Using today’s medications, daily injections of FSH are necessary for effective ovarian follicle stimulation. One scenario for the future is the development of new FSH molecules engineered to possess an extended half-life and duration of therapeutic action. Such molecules will enable the physician to provide single injections to drive follicle growth for up to a week in a controlled and predictable fashion. One such long-acting protein, designated FSH– C-terminal peptide (FSH–CTP) was developed by Organon and described by Bouloux and colleagues in 2001.98 FSH–CTP consists of the α subunit of rhFSH together with a hybrid β subunit made up of the β subunit of hFSH and the C-terminal part of the β subunit of hCG. FSH–CTP has a longer half-life than standard rhFSH. A study in healthy female volunteers showed that a single dose of FSH–CTP induced multiple follicular growth accompanied by a dose-dependent rise in serum inhibin-B.99 The first live birth resulting from a stimulation cycle with FSH–CTP was reported in 2003100 and further studies have been carried out in subfertile patients undergoing ART and OI.101,102 Another such protein has been identified at the Serono Reproductive Biology Institute (SRBI) through the use of the three-dimensional structure of FSH combined with powerful bioinformatics computer

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AS900672

β

β

α

α

FSH = Four N-glycans

AS900672 = Five N-glycans

modeling approaches to predict how new molecular structures might ‘resist’ physiologic mechanisms responsible for its inactivation and clearance from the body. This protein, which is a hyperglycosylated FSH (AS900672) (Fig 36.7), displays an extended half-life, such that 2 days following its delivery to rats via subcutaneous injection, circulating levels remain some sixfold higher than those seen after an identical administration of rhFSH Gonal-F. This hyperglycosylated FSH is currently under further clinical investigation in ART patients.

Safety profile of gonadotropins Accumulation of data on 1160 babies born after induction of ovulation with gonadotropins29 revealed that major and minor malformations were found in 63 infants, representing an overall incidence of 54.3/1000 (major malformations 21.6/1000; minor malformations 32.7/1000). This rate of malformation is not significantly different from that of the general population.

Gonadotropin-releasing hormone Introduction Control of gonadotropin secretion is exerted by hypothalamic release of GnRH, initially known as luteinizing hormone-releasing hormone (LHRH), but the lack of evidence for a specific FSH-releasing hormone (FSHRH) prompted a change in terminology. Gonadotropinreleasing hormone is produced and released from a group of loosely connected neurons located in the medial basal hypothalamus, primarily within the arcuate nucleus, and in the preoptic area of the ventral hypothalamus. It is synthesized in the cell body, transported along the axons to the synapse, and released in a pulsatile fashion into the complex capillary net of the portal system of the pituitary gland.103

Fig 36.7 Increased glycosylation by site-directed amino acid substitution results in a long-acting FSH (Hyperglycosylated FSH enriched: AS900672).

Gonadotropin-releasing hormone was first isolated, characterized, and synthesized independently in 1971 by Andrew Schally and Roger Guillemin, who were subsequently awarded the Nobel Prize for their achievement.104,105 Gonadotropin-releasing hormone is a decapeptide that, similar to several other brain peptides, is synthesized as part of a much larger precursor peptide, the GnRH-associated peptide (GAP), that has a 56 amino acid sequence. The structure of GnRH is common to all mammals, including humans, and its action is similar in both males and females. Gonadotropinreleasing hormone is a single-chain peptide comprising 10 amino acids with crucial functions at positions 1, 2, 3, 6, and 10. Position 6 is involved in enzymatic cleavage, positions 2 and 3 in gonadotropin release, and positions 1, 6, and 10 are important for the threedimensional structure (Fig 36.8). In humans, the critical spectrum of pulsatile release frequencies ranges from the shortest interpulse frequency of approximately 71 minutes in the late follicular phase to an interval of 216 minutes in the late luteal phase.106,107

Gonadotropin-releasing hormone agonists: biosynthesis and mechanism of action Native GnRH has a short plasma half-life and is rapidly inactivated by enzymatic cleavage. The initial concept was to create substances that prolong the stimulation of gonadotropin secretion. Analogs with longer half-life and higher receptor activities were created by a structural change at the position of enzymatic breakdown of GnRH. The first major step in increasing the potency of GnRH was the substitution of glycine number 10 at the C-terminus. While 90% of the biologic activity is lost with splitting of the 10th glycine, it is predominantly restored with the attachment of NH2-ethylamide to the

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Structure of GnRH 1

2

3

4

5

6

7

8

9

10

pyro (Glu)-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2

Activation of the GnRH receptor

Regulation of GnRH receptor affinity

Regulation of biologic activity

Structure of GnRH agonists pyro (Glu)-His-Trp-Ser-Tyr-

-Leu-Arg-Pro-Gly-NH2

GnRH antagonists Ser Ser

Table 36.4

Tyr

Leu

Arg

Pro

-NH2

Fig 36.8 Gonadotropin-releasing hormone (GnRH) analog structure. A schematic illustration of native GnRH, GnRH agonist, and GnRH antagonist. Position 6 is involved in enzymatic cleavage, positions 2 and 3 in gonadotropin release, and positions 1, 6, and 10 are important for the three-dimensional structure.

The structure of gonadotropin-releasing hormone (GnRH) and GnRH agonistic analogs 6th position

Compound Amino acid (no) Native GnRH

1 Glu

2 His

3 Trp

4 Ser

5 Tyr

6 Gly

10th position 7 Leu

8 Arg

9 Pro

10 GlyNH2

Nonapeptides Leuprolide Buserelin Goserelin Histrelin

Leu Ser(OtBu) Ser(OtBu) DHis(Bzl)

NHEt NHEt AzaGlyNH2 AzaGlyNH2

Decapeptides Nafarelin Triptorelin

2Nal Trp

GlyNH2 GlyNH2

proline at position 9.108 The second major modification was the replacement of the glycine at position 6 by D-amino acids, which decreases enzymatic degradation (Fig 36.8). The combination of these two modifications was found to have synergistic biologic activity. Agonistic analogs with D-amino acids at position 6, and NH2-ethylamide substituting the Gly10-amide, are not only better protected against enzymatic degradation but also exhibit a higher receptor binding affinity. The affinity could be further increased by introduction of larger, hydrophobic, and more lipophilic amino acids at position number 6 (Table 36.4). The increased lipophilicity of the agonist is associated with a prolonged half-life, which may be attributed to reduced renal excretion through increased plasma protein binding, or fat tissue storage of nonionized fat-soluble compounds.108 Thus, in all analogs, position 6 is substituted with a D-amino acid or a D-amino acid with different radicals.

Insertion of D-amino acid blocks degradation and thus renders more stability and higher receptor affinity109 (Table 36.4). The agonists leuprolide (D-Leu6,Pr9NHEt) and buserelin (D-Ser(OtBu)6, Pr9-NHEt) contain an ethylamide, and goserelin (D-Ser(OtBu)6,Pro9AzaGlyNH2) and histrelin (Nt-Bzl-D-His6,Pro9AzaGlyNH2) contain azaglycine at position 10 and are, therefore, nonapeptides. Nafarelin (D-Nal(2)6) and triptorelin (D-Trp6) contain the original Gly10-amide, and are, therefore, decapeptides. More than 1000 GnRH analogs have been synthesized and tested, but only a few have been introduced into clinical practice. Differences between analogs are mainly related to methods of administration and potency. The available data usually describe the relative potency of a certain GnRH agonist compared with native GnRH (Table 36.5). Direct comparison between the clinically available GnRH agonists under identical conditions has never been undertaken.

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Table 36.5 Trade names, plasmatic half life, relative potency, route of administration, and recommended dose for the clinically available gonadotropin-releasing hormone analogs (GnRH-a) Generic name

Trade name

Half-life

Relative potency

Native GnRH

1

Nonapeptides Leuprolide

Lupron

90 min

Buserelin

Superfact, Supercur

80 min

50–80 20–30 20–40

Histrelin Goserelin

Supprelin Zoladex

< 60 min 4.5 h

100 50–100

Decapeptides Nafarelin Triptorelin

Synarel Decapeptyl

3–4 h 3–4.2 h

200 36–144

Therefore, translation of data from these models to humans should be performed with caution. All GnRH agonistic analogs are small polypeptide molecules that need to be administered parenterally, as they would otherwise be susceptible to gastrointestinal proteolysis. The oral and rectal administration of analogs is associated with very low biopotency (0.0–1% vs parenteral administration). Intranasal spray is extremely effective, but the bioavailability is only 3–5%, and the relatively fast elimination kinetics require frequent dosing (2–6 times/day) to obtain continuous stimulation and down-regulation.110 For long-term treatment a depot formulation is available. The drug is formulated as controlledrelease depot preparations with the active substance dissolved, or encapsulated, in biodegradable material. Intramuscular injections provide maintained therapeutic levels for 28–35 days. Thus, monthly injections are sufficient for maintaining down-regulation. Although the exact cellular basis for desensitization of the gonadotroph has not been fully delineated, the extensive use of GnRH agonistic analogs in research facilitated an explosive augmentation of information and knowledge. Acute administration of GnRH agonistic analogs increases gonadotropin secretion (the flare-up effect) and usually requires 7–14 days to achieve a state of pituitary suppression. Prolonged administration of GnRH agonistic analogs leads to down-regulation of GnRH receptors. This phenomenon was first shown in 1978, when Knobil and co-workers published their classic paper demonstrating downregulation of gonadotropin secretion by sustained stimulation of the pituitary with GnRH.111 The agonist-bound receptor is internalized via receptor-mediated endocytosis,112 with kinetics determined by the potency of the analog. The internalized complex subsequently undergoes dissociation, followed by degradation of the ligand and partial recycling of the receptors.113

Administration route

Recommended dose

i.v. s.c. s.c. i.m. depot s.c. Intranasal s.c. s.c. implant

500–1000 µg/day 3.75–7.5 mg/month 200–500 µg/day 300–400 × 3–4/day 100 µg/day 3.6 mg/month

Intranasal s.c. i.m. depot

200–400 × 2/day 100–500 µg/day 3.75 mg/month

Side effects Side effects of GnRH agonist therapy are related to the fall in sex hormone serum concentration. As GnRH agonist interacts with GnRH receptors, which are mainly present in the pituitary, no systemic effects are common. The main symptoms of low serum concentrations of estrogen are flushes, decreased libido, impotence, vaginal dryness, reduced breast size, and emotional instability. One of the matters of concern is the effect of estrogen depletion on bone mineral density (BMD), as estrogen is of major importance in preventing the development of osteoporosis. A summary of data from different trials114 showed that GnRH analog therapy caused significant but reversible bone loss. The mechanism appears to be similar to the development of postmenopausal osteoporosis; i.e. high bone turnover with elevated alkaline phosphatase and osteocalcin levels.

Teratogenic effects There does not appear to be an increased risk of birth defects, or pregnancy wastage in human pregnancies exposed to daily low-dose GnRH agonist therapy in the first weeks of gestation. Although placental transfer of GnRH agonists in pregnant rhesus monkeys was demonstrated, no deleterious effects were observed.115 From their toxicology studies in animals, no toxic effects were reported by the drug manufacturers.116 Although several authors claimed a normal outcome of pregnancy following inadvertent administration of a GnRH agonist during early pregnancy,117–119 Ron-El et al120 reported the birth of a newborn with a small soft cleft palate. Lahat et al reported a high incidence of attention deficit hyperactivity disorder in long-term follow-up of children inadvertently exposed to GnRH agonists in early pregnancy.121 Therefore, as this complication is purely iatrogenic, it should best be avoided.

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Table 36.6 Comparing mechanisms of action of gonadotropin-releasing hormone (GnRH) agonists and antagonists GnRH antagonist Receptor blockage without receptor activation Competitive inhibition Immediate and dose-dependent suppression Rapid reversibility

GnRH agonist

Receptor downregulation Pituitary desensitization Initial flare-up Slow reversibility

Gonadotropin-releasing hormone antagonist Mechanism of action Antagonist analogs of GnRH have a direct inhibitory, reversible suppressive effect on gonadotropin secretion. Antagonistic molecules compete for and occupy pituitary GnRH receptors, thus competitively blocking the access of endogenous GnRH and precluding substantial receptor occupation and stimulation. Suppression attained by GnRH antagonists is immediate (no flare-up effect), and, as receptor loss does not occur, a constant supply of antagonists to the gonadotroph is required to ensure that all GnRH receptors are continuously occupied. Consequently, compared with agonistic analogs, a higher dose range of antagonists is required for effective pituitary suppression (Table 36.6).

Synthesis of GnRH antagonists Over the past three decades, thousands of GnRH analogs, both agonists and antagonists, have been Table 36.7

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synthesized. The first generation of antagonistic analogs were hydrophilic, and contained replacements for His at position 2 and for Trp at position 3. Inhibitory activity increased after incorporation of a D-amino acid at position 6. However, histamine release also increased, resulting in anaphylactic reactions which prevented their clinical use. In thirdgeneration antagonistic analogs, the undesirable risk of anaphylaxis and edema was eliminated by replacing the D-Arg at position 6 by neutral D-ureidoalkyl amino acids, to produce compounds such as cetrorelix, iturelix, azaline B, ganirelix, abarelix, and antarelix122–128 (Table 36.7).

Safety and tolerability studies The introduction of GnRH antagonists in clinical use was delayed owing to the property of the first generation of antagonists to induce systemic histamine release and a subsequent general edematogenic state. Studies in rat mast cells confirmed that incorporation of D-Cit at position 6 of antagonists results in reduced histamine release.129,130 This characteristic of cetrorelix was first assessed in in vitro assays that demonstrated effective plasma concentrations to be significantly lower (<103) than the median effective dose (ED50) for systemic histamine secretion, and, therefore, could confidently be regarded as insignificant. Owing to large disparities in such assays, cetrorelix safety was further tested in in vivo settings. Cetrorelix injected at doses of 1.5 mg/kg s.c., and 1 and 4 mg/kg i.v., into rats caused no systemic adverse effects, such as edema, respiratory dysfunction, or cardiovascular compromise. In these animal studies no teratogenic effects, or detrimental influence on implantation rates or on embryonic development, were noted when administered in the periconceptional period. Several thousand human

Structure formulation of native gonadotropin-releasing hormone (GnRH) and GnRH antagonists

Name

Amino acid sequence

GnRH

pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2

First generation 4F Ant

NAc∆1, 1Pro-D4FPhe-DTrp-Ser-Tyr-DTrp-Leu-Arg-Pro-GlyNH2

Second generation NalArg Detirelix

NACD2Nal-D4lFPhe-pTrp-Ser-Tyr-DArg-Leu-Arg-Pro-GlyNH2 NACD2Nal-D4CIPhe-pTrp-Ser-Tyr-DHarg(Et2)-Leu-Arg-Pro-DAlaNH2

Third generation NalGlu Antide Org30850 Ramorelix Cetrorelix Ganirelix A-75998 Azaline B Antarelix

NACD2Nal-D4C7Phe-D3Pal-Ser-Arg-DGlut(AA)-Leu-Arg-Pro-DAlaNH2 NACD2Nal-D4CIPhe-D3Pal-Ser-Lys(Nic)-DDLys(Nic)-Leu-Lys(Isp)Pro-DAlaNH2 NACD4CIPhe-D4CIPhe-DBal-Ser-Tyr-DLys-Leu-Arg-Pro-DAlaNH2 NACD2Nal-D4CIPhe-DTrp-Ser-Tyr-DSet(Rha)-Leu-Arg-Pro-AzaglyNH2 NACD2Nal-D4CIPhe-D3Pal-Ser-Tyr-DCit-Leu-Arg-Pro-DAlaNH2 NACD2Nal-D4CIPhe-D3Pal-Ser-Tyr-DHarg(Et2)-Leu-Harg(Et2)-Pro-DAlaNH2 NACD2Nal-D4CIPhe-D3Pal-Ser-NMeTyr-DLys(Nic)-Leu-Lys(Isp)-Pro-DAlaNH2 NACD2Nal-D4CIPhe-D3Pal-Ser-Aph(atz)-DAph(atz)-Leu-Lys(Isp)-Pro-DAlaNH2 NACD2Nal-D4CIPhe-D3Pal-Ser-Tyr-DHcit-Leu-Lys(Isp)-Pro-DAlaNH2

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patients have been treated with third-generation GnRH antagonists (i.e. ganirelix, cetrorelix, or abarelix) without evidence of systemic, or major local skin reactions, and no cessation of therapy was warranted due to side effects.129,131–135 The common side effects observed were injection-site reactions and possibly nausea, headache, fatigue, and malaise. No drug interactions were demonstrated in vitro with medications metabolized through the P450 cytochrome pathway. Since the discovery of extrapituitary human GnRH receptors, the safety of GnRH agonists and antagonists with respect to various structures has become a cardinal question. Effects of GnRH antagonists on the ovary, oocytes, granulosa cells, endometrium, and the embryo in relation to fertility and implantation rates are being investigated. Direct effects of GnRH antagonists on human ovarian steroi do genesis in vitro have not been demonstrated. In preliminary in vitro and animal studies, recent data revealed that some adverse effects on oocyte maturation and on preimplantation development of embryos may be inflicted by GnRH antagonists through inhibition of GnRH receptors in these structures.136–138 The hypothesis that GnRH may play a role in the preimplantation development of embryos was examined by Raga et al.139 This group of investigators found that preimplantation embryonic development was significantly enhanced by incubation with increasing concentrations of GnRH agonists, and was significantly decreased by GnRH antagonists compared with that of the control group. Moreover, GnRH antagonist (5 and 10 µmol/l) was able to completely block embryo development. The deleterious effect of GnRH antagonists on embryo development was reversed by increasing concentrations of the agonist, as determined by the number of embryos reaching the blastocyst stage. Based on the lower implantation rates of the higher-dosage groups of ganirelix in the large dosefinding study,140 the possibility of direct effects of antagonists on human embryos is of concern, and remains unresolved.

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37 The role of FSH and LH in ovulation induction: current concepts Juan Balasch

Introduction and overview The ovary has two essential physiologic responsibilities: the periodic release of oocytes and the production of the steroid hormones, estradiol and progesterone. Both activities are integrated into the continuous repetitive process of follicle growth and maturation, ovulation, and corpus luteum formation and regression, which constitute the so-called ovarian cycle. The ovarian cycle is under pituitary gonadotropic control: follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are synthesized and secreted by the pituitary, and together play a central part in regulating the menstrual cycle and ovulation. Therefore, a basic knowledge of gonadotropic control of ovarian function is an essential requirement for a proper understanding of ovulation-induction techniques using exogenously administered gonadotropins. Thus, this chapter begins with a review of the role of FSH and LH in the control of follicular growth and function. This is followed by a section addressing the development of pituitary gonadotropins, from urinary products to recombinant medications, and stressing the advantages of using the biotech drugs. The three gonadotropins involved in ovulation induction (FSH, LH, and human chorionic gonadotropin [hCG]) are now commercially available and produced in vitro by recombinant DNA technology (rhFSH, rhLH, rhCG). These highly specific monohormonal products have permitted important advances in our understanding of gonadotropin action at the cellular level, and also provide us with the perspective of preparing consistent-formulation regimens for ovulation induction or tailoring therapy with FSH and LH, individually or combined, according to the individual patient’s needs.1–3 On the above evidence, the third section in this chapter is devoted to contemporary strategies for ovulation induction in the anovulatory patient. The object of ovulation induction is to restore the ovulatory state and restore fertility potential but producing ideally only one ovulatory follicle. Both the most appropriate gonadotropin to use and pros and cons of different regimens of gonadotropin administration are discussed separately for World Health Organization

(WHO) group II and WHO group I anovulation. The chapter concludes by providing the reader with current therapeutic modalities for inducing multiple follicular development (the so-called controlled ovarian hyperstimulation) in the already ovulating patient undergoing treatment with assisted reproductive technologies (ART). These different goals require different approaches to how the ovaries are stimulated.

Gonadotropic control of follicular growth and function Although the physiologic effects of FSH and LH are intimately connected, and both gonadotropins are necessary for normal gonadal function and gamete maturation, it has recently become possible to define better the specific spectrum of both FSH and LH actions. Although it is a continuum, the life cycle of a preovulatory follicle can be broken down into three successive phases: 1. 2. 3.

Initiation, which occurs from birth to senescence independent of gonadotropic support. FSH-dependent progression, requiring tonic stimulation by FSH. LH-responsive maturation, when FSH-induced genes fall under LH control, leading to final follicular maturation and ovulation4.

This subject has recently been reviewed elsewhere,2–8 and is summarized here.

Follicle-stimulating hormone At birth, the human ovaries contain ~2 000 000 follicles arrested at the primordial stage of development. Throughout infancy, childhood, and adolescence, continuing throughout the reproductive years, this endowment is progressively depleted as individual follicles exit the primordial pool and folliculogenesis begins. However, ~99.9% of follicles will never complete their development; instead, they default to atresia, owing to inadequate stimulation by FSH. This

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means that only ~400 follicles sequentially mature and ovulate during an average woman’s reproductive lifetime. Theoretically, every primordial follicle has the potential to mature, secrete estrogen, and ovulate. However, until puberty, blood concentrations of FSH and LH remain too low to stimulate full preovulatory follicular development. The follicles start to grow more rapidly in the luteal phase of the cycle preceding ovulation, but pre-antral stages of follicular growth occur independently of gonadotropic stimulation. However, antrum formation requires tonic stimulation by FSH, beginning when follicles are ~0.25 mm in diameter (the socalled gonadotropin-regulated growth phase) (Fig 37.1). The selective rise in serum FSH beyond a critical ‘threshold’ level that occurs during the luteal– follicular transition is a potent stimulus for follicle recruitment (Fig 37.2), and several early antral follicles begin to enlarge in this phase of the cycle because of the mitogenic action of FSH on granulosa cells. Only one of these follicles, however, is eventually ‘selected’ to ovulate, while the others become atretic. Selection of the dominant follicle can be explained by development-related changes in responsiveness to FSH and LH, which occur in granulosa and theca cells modulated by ovarian para(auto)crine mechanisms. The follicle whose granulosa cells are most responsive to FSH (lowest FSH ‘threshold’) becomes first in the cohort to secrete estrogen, which feeds back through the hypothalamo–pituitary axis and begins to suppress pituitary FSH secretion. Blood FSH, therefore, declines to a concentration insufficient to sustain the development of other follicles that have higher FSH thresholds. These latter become nonovulatory and undergo atresia, while the dominant follicle continues to mature and secrete estrogen. It is possible that the maturing follicle reduces its dependence on FSH by acquiring LH receptors, as discussed below.3,4,8

Luteinizing hormone Traditionally, the roles of LH in folliculogenesis have been considered to be limited to stimulating thecacell androgen production, triggering ovulation, and supporting the corpus luteum. However, it is now accepted that in the late stages of follicle development, granulosa cells become receptive to LH stimulation and LH becomes capable of exerting its actions on both theca cells and granulosa cells.2,4,8 At the midfollicular phase, the dominant follicle reaches ≥10 mm in diameter, thus being recognizable as the largest healthy follicle in either ovary, and increasingly synthesizes estradiol. LH is capable of stimulating androgen-substrate production from theca cells, to be transformed into estrogen by FSH-stimulated aromatase activity in granulosa cells (the so-called twocell, two-gonadotropin model). In addition, a major development-related response of granulosa cells to

LH FSH

Dominance > 20 mm

Selection ∼10 mm Recruitment <5 mm

Estrogen

0

4

8 Day of cycle

10

16

Fig 37.1 Gonadotropin-dependent stages in preovulatory follicular development. Recruitment: at the beginning of each menstrual cycle, plasma follicle-stimulating hormone (FSH) concentrations increase sufficiently to stimulate the proliferation and functional maturation of granulosa cells in multiple immature follicles, including induction of luteinizing hormone (LH) receptors, aromatase activity, and inhibin biosynthesis. Tonic stimulation by LH maintains thecal androgen synthesis in these follicles. Selection: the next follicle that will ovulate emerges as the follicle that is most responsive to FSH (that is, has the lowest FSH ‘threshold’). Dominance: by the midfollicular phase, the preovulatory follicle becomes recognizable as the largest healthy follicle in either ovary, containing granulosa cells that express LH receptors coupled to aromatase and inhibin synthesis. Since this follicle is uniquely responsive to both FSH and LH, it continues to grow and secrete estrogen despite decreasing plasma concentrations of FSH. Development-dependent paracrine signals (including inhibin) maintain the dominance of this follicle, amplifying its responsiveness to FSH and LH. From Hillier,3 with permission.

FSH is their increased expression of LH receptors, functionally coupled to steroid synthesis. LH receptors are constitutively present on theca cells, and appear on granulosa cells that have been adequately stimulated by FSH. This development enables mature granulosa cells in the preovulatory follicle (and subsequently in

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The role of FSH and LH in ovulation Dominant follicle

RECRUITMENT−SELECTION

Follicles

Gonadotropins

atresia Ovarian factors M ~7 DAY OF MENSTRUAL CYCLE

14

Fig 37.2 Gonadotropin–ovarian interaction. Diagram of gonadotropin-mediated follicular recruitment, selection, and maturation of the dominant follicle by the rise in follicle stimulating hormone (FSH) secretion at the beginning of the menstrual cycle. From Yen,9 with permission.

the corpus luteum) to respond directly to LH. Recent findings indicate that the process of preovulatory follicular development may be regulated by a single intracellular message (cyclic adenosine monophosphate, cAMP) which, in turn, is controlled in succession by two different messengers, FSH and LH.8 In fact, most of FSH’s physiologic actions on granulosa cells, including stimulation of the aromatase system, can be exerted by LH once its receptors are expressed.2,4,8 As would be predicted by the common intracellular cAMP pathway, granulosa cells from FSH-stimulated follicles respond similarly to both FSH and LH, and, moreover, at nonsaturating levels of FSH and LH, the responses are additive.8 The overall significance of these findings is that while granulosa cells from early antral follicles are responsive only to FSH, granulosa cells from FSH-stimulated follicles are responsive to either FSH or LH.4,8 In fact, LH seems to be able to sustain preovulatory follicular endocrine activity previously induced by FSH,2,10,11 but definite in vivo evidence in the human is still lacking. During the human menstrual cycle, LH released by the pituitary gland is also a major paracrine regulator.3,4 In fact, multiple growth/differentiation factors and sex steroids produced in reponse to ovarian stimulation with FSH and LH mediate short-loop feedback signaling between granulosa cells and theca during preovulatory follicular development. Ovarian paracrines include theca-derived androgens and granulosa-derived insulin-like growth factors (IGFs) and inhibins, which orchestrate preovulatory follicular estrogen secretion. Thus, there is experimental evidence that granulosa cells in immature follicles express androgen receptors, through which theca-derived androgens act to potentiate follicular responsiveness to FSH. Conversely,

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experimental studies have revealed that in the presence of minimal stimulation by LH, FSH is able to activate paracrine signaling (granulosa on theca signaling mediated by IGFs and inhibins), which sustains thecal androgen synthesis and thereby explains why treatment with FSH alone is capable of stimulating ovarian estrogen synthesis in many clinical situations.3,4 In spontaneous ovulatory cycles, the most responsive follicle to FSH at the beginning of the cycle is the first to produce estrogen and express granulosa-cell LH receptor. Paracrine signaling activated by FSH and LH sustains growth and estrogen secretion by the preovulatory follicle until ovulation. In addition, both experimental and clinical evidence clearly indicates that while follicular growth may not require LH, LH plays a primary part in complete maturation of the follicle and oocyte competence.10,12–14 However, it is important to note that while LH, like FSH, is capable of dose-dependently stimulating steroid synthesis, with respect to granulosa cell proliferation, LH, unlike FSH, is inhibitory at high doses. This may provide a basis for interpreting some ‘antifolliculogenic’ effects of inappropriately high concentrations of LH, which are discussed below. Finally, when the midcycle LH surge occurs, LH follicle interactions disrupt granulosa cell contacts in the cumulus oophorus and induce oocyte maturation (meiosis), cause follicular rupture, and induce granulosa cell luteinization.7 Until now, no pharmaceutically pure human LH preparation has been commercially available; thus, hCG has been used for years in infertility treatment protocols as a surrogate for LH in order to stimulate ovulation. hCG acts on the ovary through a LH/CG receptor, exerting a luteinizing effect that is more prolonged than that of LH because of its longer half-life.15 This may have important implications with respect to the risk of developing ovarian hyperstimulation syndrome (OHSS), as discussed below.

The window for LH: the ‘threshold’ dose and ‘ceiling’ value concepts There is basic, experimental and clinical evidence unequivocally indicating that ovarian follicles have development-related requirements for stimulation by LH: that is, there is a ‘threshold’ for LH requirements during folliculogenesis.2,4,16,17 The amount of LH activity actually necessary for normal follicle and oocyte development, however, is not known, but is likely to be very low, since less than 1% of follicular LH receptors need to be occupied in order to elicit a maximal steroidogenic response, and, accordingly, resting levels of LH (1–10 IU/l) should be sufficient to provide maximal stimulation to theca cells.18 Defining this threshold in clinical practice became possible when both rhFSH and rhLH were available as two separate preparations. Thus, two multicenter studies conducted in patients with hypogonadotropic hypogonadism (the ideal

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models for investigating gonadotropin actions on ovarian steroidogenesis and follicular development) confirmed the pivotal role of LH in normal follicular function, and established that a daily dose of 75 IU rhLH is sufficient for promoting optimal follicular development in most patients, as measured by estradiol secretion and the ability to luteinize when exposed to hCG.19,20 Remarkably, the dose-finding European multicenter study19 showed that those patients who received the highest dose (225 IU/day) of rhLH developed a smaller number of growing follicles than those who received 75 IU rhLH/day, which may reflect an LH ‘ceiling’ effect as discussed next. Although LH is essential for estrogen synthesis and maintenance of follicular dominance, there is clinical evidence that excessive stimulation of the ovaries by LH adversely affects normal preovulatory development. Depending on the stage of development, follicles exposed to inappropriately high concentrations of LH enter atresia or become prematurely luteinized, and oocyte development may be compromised.2,18,21,22 Thus, developing follicles appear to have finite requirements for stimulation by LH, beyond which normal development ceases. Whereas each follicle has a threshold beyond which it must be stimulated by FSH to initiate preovulatory development, it may also have a ‘ceiling’ within which it should be stimulated by LH, unless further normal development is terminated (Table 37.1).2,23 The LH ‘ceiling’ hypothesis is further supported by two well-known clinical conditions which may be associated with reproductive failure: ovulation induction with clomiphene citrate in the anovulatory patient, and the use of the short protocol with gonadotropin-releasing hormone (GnRH) agonists in in vitro fertilization (IVF).16 Clomiphene is used in WHO group II anovulatory patients (mainly polycystic ovary syndrome [PCOS]). The main mode of action of clomiphene is to boost FSH in the early to midfollicular phase but, unfortunately, it also raises LH levels at this apparently critical stage and, mainly for those patients who already have a high baseline level, the additional discharge of LH may prejudice their chances to conceive. Both the lack of conception in the face of an apparent ovulatory pattern and an increased risk of miscarriage have been reported with clomiphene citrate. Inappropriate LH action interfering with follicular and oocyte maturation would explain these adverse reproductive effects.24 Similarly, exposure of the developing follicle to inappropriately high levels of LH with the flare-up protocol in assisted reproduction may adversely affect the reproductive process in the form of lower pregnancy rates and increased early pregnancy losses.25 Finally, it is noteworthy that a study in profoundly down-regulated young oocyte donors showed that the inclusion of exogenous LH activity (in the form of 1 ampule/day HMG from stimulation day 5) in the ovarian stimulation protocol with rhFSH can have beneficial or detrimental effects on oocyte yield and quality, depending on the level of endogenous LH, thus supporting the concept of a ‘window’ for LH requirement in ovarian stimulation.26

Table 37.1

The LH ‘ceiling’ hypothesis23

• Ovarian follicles have development-related requirements for stimulation by LH • LH, beyond a certain ‘ceiling’ level, suppresses granulosa proliferation, and initiates atresia (immature follicles) or premature luteinization (preovulatory follicles) • Mature follicles are more resistant (higher ‘ceiling’) to LH than immature ones • During ovulation induction, LH dose should not exceed the ‘ceiling’ of the most mature follicles

In summary, current concepts of gonadotropic control of ovarian function and clinical evidence have established that both a ‘threshold’ and a ‘ceiling’ for LH levels (framing the so-called LH ‘window’) exist during the follicular phase of menstrual and induced cycles. Therefore, levels of LH should be neither too high nor too low during ovulation induction.27 During the second half of the follicular phase, as plasma FSH concentrations decline, the LH-dependent phase of preovulatory follicular development proceeds normally only if LH is present at concentrations over the threshold level and beneath the ceiling value. When the ceiling is exceeded at the midcycle surge of LH, further division of granulosa cells ceases as luteinization proceeds (Fig 37.3).

Clinical implications According to the above evidence, follicular responsiveness to FSH and LH is developmentally regulated. FSH plays a crucial part in recruitment, selection, and dominance, while LH contributes to dominance, final maturation, and ovulation. On the basis of physiology, now that pharmaceutically ‘pure’ rhFSH and rhLH are available, it is possible to develop improved clinical strategies for stimulating ovarian function. Those who stand to benefit are women receiving treatment for ovulatory dysfunction and those with normal ovarian function undergoing ART. The therapeutic aim in each group, however, is quite different. In the former, it is desirable to stimulate mono-ovulation with a view to conception occurring in vivo. In the latter, the aim is to stimulate multiple follicular development. The challenge is to tailor therapy with FSH and LH, alone or in combination, according to the outcome desired, based on the principles summarized here and the available gonadotropin preparations. This is discussed below.

Developments in gonadotropin preparations: from urinary products to recombinant gonadotropins Pharmaceutical preparations containing biologically active gonadotropins for ovulation induction have been in use for about 75 years. As recently

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The role of FSH and LH in ovulation Suppression of granulosa proliferation Follicular atresia (nondominant follicles) Premature luteinization (preovulatory follicle) Oocyte development compromised LH ceiling

The LH window

Normal follicular growth and development Paracrine signaling activated by FSH and LH Adequate granulosa proliferation and functional maturation Normal androgen and estrogen biosynthesis Full follicular and oocyte maturation LH threshold Follicular growth by FSH action (granulosa cell proliferation) Induction of granulosa cell aromatase activity by FSH No paracrine signaling between granulosa and theca No androgen (and estrogen) synthesis No full oocyte maturation

Fig 37.3 Diagram illustrating the ‘luteinizing hormone (LH) window’ concept. FSH, follicle stimulating hormone. Modified from Balasch and Fábregues,16 with permission.

follitropin β (Puregon; NV Organon, Oss, the Netherlands). Both follitropins are structurally identical to native FSH, and each comprises the α and β subunits which compose this gonadotropin; the nomenclature for these recombinant products does not refer to those subunits, but is merely a means of distinguishing chronologically one from another.30 Like rhFSH, recombinant human LH (rhLH) (Luveris; Merck-Serono International) and recombinant hCG (rhCG) (Ovitrelle and Ovidrel; Serono International) are produced under the most stringent manufacturing conditions, and have been assessed successfully for clinical use.5,6,31–33 The three gonadotropins now produced in vitro by recombinant DNA technology share several advantages over urinary products:28,30,32,34,35 1.

reviewed,28 major advances in technology have brought the field of gonadotropin therapy a very long way since the era of animal-, human pituitary-, and urinary-derived hormones. For years, human menopausal gonadotropin (hMG) has been the only urinary gonadotropin available for clinical use. The FSH and LH content of hMG is theoretically equal (75 IU of FSH and 75 IU of LH), albeit with different FSH/LH ratios. In addition, hMG has a low specific activity and is of <4% purity, as only ~3–4% of the protein content is gonadotropin. Over the past 20 years, urinary FSH-only preparations became new therapeutic options for ovulation induction. In the mid-1980s, urinary ‘purified’ FSH (pFSH) (with <1% LH contamination but still having 95% protein impurity) was developed, which was followed by the availability of highly purified FSH (FSH-HP) in 1993. FSH-HP contained <0.1% LH contamination, and was the first highly pure biologic extract (~4% impurity), and, as a result of this, could be injected subcutaneously (s.c.), unlike the earlier preparations which had to be administered intramuscularly (i.m.). Biochemical analysis of a new formulation of hMG described as highly purified indicated that the hMG preparation contains a mixture of FSH, LH (0.85 IU/vial), and human chorionic gonadotropin (hCG; 11.3 IU/vial), together with other urinary proteins. Specific FSH activity was about 2000 IU/mg for the new hMG, compared with 8000 IU/mg of FSH-HP. The purity of the new hMG was estimated from highperformance liquid chromatography at 50–60% in terms of area percentage, compared with values of 99% for FSH-HP.29 Recombinant human FSH (rhFSH) which is completely devoid of both LH activity and nonspecific urinary proteins, represents the final transition to a true drug, where the starting material and complete manufacture are under rigorous control.30 Two rhFSH preparations have been registered as follitropin α, which was marketed first in 1995 (Gonal-F; MerckSerono International, Geneva, Switzerland), and

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A fully controlled production process from bulk to finished product, a fact to be considered taking into account a recent report showing that there is transmission of prion infectivity from scrapieinfected mice with lymphocytic nephritis, and thus urine may provide a vector for prion transmission.36 Especially in the current era of ‘prion scare,’ this may be an important argument in favor of biotech substitutes.37 Full traceability from the starting material (cell line) to the final product. ‘Traceability’ means that for every manufactured batch of gonadotropin it is possible to identify the source. However as far as urine collection goes, being able to record exactly which donor contributed to the pool of material used to produce the batch is impossible. This is noteworthy considering that there is a continuing and growing difficulty in maintaining control on the sourcing of large volumes of urine needed to supply commercial demands28 and a recent consensus statement from an international panel of experts recommended that the urine should be sourced from donors not at risk from human transmissible spongiform encephalopathies.38 Along this line of action, national regulatory/health authorities from different countries encouraged the replacement of urinary-derived gonadotropins of human origin with recombinant products, advised that human plasma or urine used in the production of medicines should not be sourced from any country with one or more indigenous cases of human transmissible spongiform encephalopathies, outlined new labeling requirements for products derived from human urine (the labels are to state that risk of trasmission of infective agents of known or unknown nature cannot be definitively excluded when administered products are derived from human urine), and required full traceability for urinary derived products. As an example, in 2002 an endogenous case of variant Creutzfeldt–Jakob disease was confirmed in Sicily (Italy), and as urine was sourced from this

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country for production of urinary FSH-HP, the product was withdrawn from the UK market as a precautionary measure.39 High purity and specific activity, as reported above. Unlimited supply with batch-to-batch consistency. This is important considering the current increasing demand for exogenous gonadotropins, which requires a big availability of raw material and the fact that an ampule of urinary FSH labeled to contain 75 IU may range in true activity from 50 to 120 IU FSH.40 In addition, consistency in the biopotency of the preparation becomes important in order not to overstep the thin red line between mono- and multifollicular development, especially where small incremental dose rises are concerned,41 as discussed below. Complete absence of contamination by the other gonadotropins (i.e. they are absolutely monohormonal products).

This leads to a circumvention of adverse immune reactions owing to contaminant urinary proteins, the possibility of s.c. self-administration with ready-to-use pen devices (either preloaded [follitropin α] or loaded by cartridge [follitropin β] for multiple injections), ensuring patients an accurate and correct dose while facilitating treatment individualization, and prevention of variability in ovarian response to gonadotropin administration observed cycle-to-cycle in the same patient. Interestingly, a new filled-by-mass manufacturing process for follitropin α resulted in an even more consistent ovarian response, less need for dose adjustments, and fewer cancelled cycles.42–46 At present, rhFSH, rhLH, and rhCG are commercially available and they offer risk reduction for patients as well as the assurance of superior quality control over the final product.28 In the clinical setting, these recombinant gonadotropins have proved to be, at least equal, or even more efficacious and/or efficient than their urinary counterparts, and thus they should be considered as the gold standard of today for ovarian stimulation.28,39 In fact, it has been claimed that unit for unit, rhFSH is more potent than urinary FSH.41 Remarkably, the first biotech drugs substitutes, which were introduced in the 1980s, namely recombinant insulin and recombinant growth hormone, have never really needed to show superiority compared with the previously used products they were replacing and the debate was not whether recombinant insulin was better than animal insulin, but whether it was not worse. Similarly, there has been a drive to use synthetic recombinant clotting factors in preference to plasma-derived products.37

Ovulation induction in the anovulatory patient Ovulatory disturbances are present in about 15–25% of couples presenting for an infertility evaluation.47–49

Most infertile anovulatory patients fall into the WHO group II (normogonadotropic anovulation) category, and the great majority of these women are diagnosed as having PCOS.48–50 These women are well estrogenized and have normal FSH levels, but LH may be elevated. In contrast, WHO group I anovulation or hypogonadotropic hypogonadism (HH) is a much less frequent condition, characterized by reduced hypothalamic or pituitary activity and resulting in abnormally low serum concentrations of FSH and LH and negligible estrogen activity. It can be caused by a number of abnormalities of endogenous hypothalamic GnRH secretion, all of which are incompatible with normal folliculogenesis and subsequent ovulation.49,51 Both groups of patients have different gonadotropin requirements for ovulation induction and are discussed separately. The most important principle in ovulation induction is to provide as close as possible physiologic restoration of cyclic ovarian function. In particular, the aim should be to achieve the ovulation of a single follicle. Multiple follicular development is a complication which is characteristic of ovulation induction with exogenous gonadotropins, particularly in women having PCOS who are very sensitive to gonadotropin stimulation.52 Excessive follicular development (>35–40 follicles), usually associated with very high estradiol levels (>4000–5000 pg/ml), may lead to two important iatrogenic complications: OHSS and multifetal pregnancy. OHSS is a potentially lethal condition, the pathophysiologic hallmark of which is marked hemodynamic derangement caused by peripheral arterial vasodilatation and vascular leakage of fluid from the intravascular space into the peritoneum, causing ascites and hemoconcentration.53,54 Multifetal pregnancies are associated with considerable maternal–fetal morbidity and mortality, and, according to some studies, as many as 75% of iatrogenic multifetal pregnancies are due to ovulation induction in anovulatory women, whereas only the remaining 25% are the product of ART.55–59

Induction of ovulation in PCOS patients (WHO group II anovulation) Which gonadotropin to use? Elevated serum LH and disturbed intraovarian regulation of FSH action are endocrine features in PCOS,60,61 and early studies both in vitro62 and in vivo63 provided evidence that the self-perpetuating state of biochemical imbalance so characteristic of PCOS could be interrupted in a physiologic way when FSH is administered in a chronic low dose. Thus, although hMG and FSH preparations have both been used successfully for ovulation induction in PCOS,64 it is accepted that when endogenous LH is already elevated (for example in PCOS), FSH alone is conceptually better,2,5,61 Elevated LH concentrations may directly or indirectly hasten late follicular phase meiotic maturation,

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and abnormal oocyte maturation may be responsible for the reduced fertility and increased miscarriage rates frequently encountered in women with PCOS.7,65,66 This notwithstanding, it has been questioned whether this can be applied to ovulation induction with gonadotropins on the basis that the administration of hMG to patients with PCOS who are not receiving GnRH agonist does not result in significant increases in serum LH concentrations.67–69 It is postulated that, during ovulation induction, gonadotropin-stimulated estrogens and inhibins feed back on the hypothalamic– pituitary axis and reduce endogenous gonadotropin secretion, and thus daily LH serum levels remain low.7 However, in those previous studies investigating LH levels during ovulation induction with hMG in PCOS patients, serum LH concentrations were judged by daily single blood samples. Owing to the pulsatile mode of LH secretion, no single blood sample can be used reliably to evaluate gonadotropin pathophysiology.70 This is because the 95% confidence limits of a single blood sample taken to measure LH range from 50 to 150% of the measured value.71 In addition, endogenous LH and exogenously administered LH (either urinary or recombinant) have a short terminal half-life of around 10–11 hours.72 Thus, it is not surprising that several studies have reported normal serum LH but abnormal urinary LH, and emphasized that earlymorning urinary measurements are more informative than those in serum because they reflect nocturnal LH secretion.73,74 Also, that fact may explain normal LH levels during exogenous LH administration, because blood sampling is performed after the hormone has been cleared from the serum. On the above evidence, we carried out a pharmacokinetic and endocrine comparison of rhFSH and hMG in PCOS patients, including LH measurements, in 8-hour urine samples reflecting overnight renal urine secretion.75 We found that a peak in LH serum levels was observed 4 hours after a single i.m. injection of 225 IU hMG, LH returning to basal values 10–11 hours later. Remarkably, in such patients receiving ovulation induction according to a low-dose step-up protocol with either rhFSH or hMG, we found that LH levels in urine were significantly higher in the hMG group.75 Finally, two Cochrane reviews on clinical trials investigating gonadotropin therapy for ovulation induction in women with clomiphene-resistant PCOS concluded that no significant benefit could be demonstrated from urinary FSH vs hMG in terms of pregnancy rate, but a significant reduction in OHSS associated with FSH was observed.76,77 According to experimental data, this could be explained by the reciprocal paracrine signaling between LH-stimulated theca cells and FSH-stimulated granulosa cells, which could bring about follicular hypersensitivity to FSH.78 As discussed above, hypersecretion of LH is one of the endocrine phenomena usually associated with PCOS. Thus, in principle, administration of exogenous LH for ovulation induction in these patients is

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Table 37.2 Results of conventional step-up protocol (starting dose two ampules human menopausal gonadotropin [hMG]/day) for gonadotropin ovulation induction in World Health Organization (WHO) group II infertile patients No of patients No of cycles Ovulatory cycles (%) Conceptions (per ovulatory cycle) (%) Multiple pregnancy rate (%) Abortion rate (%) Hyperstimulation rate (%)

1047 >2500 62–98 10–20 15–36 24–42 1.1–14

Summary of six series. Adapted from Fauser et al.60

not warranted. Recently, however, a new strategy for clinical research was devised, which, on the basis of the ‘LH ceiling’ hypothesis discussed above, explores the effect of exogenous administration of high doses of LH during ovarian stimulation in patients with PCOS having hypersensitive ovaries.17,79 The ultimate therapeutic goal in this line of research was to try and minimize the preovulatory follicles to reduce multiple pregnancy rates. A prospective, randomized, double-blind, multicenter, dose-finding study was carried out to evaluate the effects of four different doses of rhLH (6.8, 13.6, 30, or 60 µg daily for a maximum of 7 days) administered in the late follicular phase in WHO group II anovulatory women over-responding to FSH treatment. It was concluded that doses of up to 30 µg rhLH/day appear to increase the proportion of patients developing a single dominant follicle, thus providing further support to the LH ceiling concept discussed above.80 Therefore, this study suggests the clinical efficacy of high-dose rhLH for inducing atresia of secondary follicles and promoting mono-ovulation in WHO group II anovulatory women.

Which regimen of gonadotropin administration? Gonadotropin induction of ovulation has been traditionally performed since the early 1970s by using hMG in the individualized conventional step-up dose regimen. This is characterized by initial daily doses of two ampules of hMG (~150 IU of bioactive FSH), which is increased by ≥50% every 3–5 days until an ovarian response occurs. This treatment modality is effective, but the complication rate is relatively high (Table 37.2). On the other hand, the use of hMG containing fixed proportions of FSH and LH to stimulate ovarian function ignores the fact that follicular responsiveness to FSH and LH varies characteristically with preovula10,11 tory development, as discussed above. Thus, the need to re-evaluate the use of gonadotropins became imperative once ‘LH-free’ forms of urinary FSH became available, and led to the implementation of low-dose treatment programs, which have been used in step-up, step-down, and sequential regimens.

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FSH therapy Threshold level FSH Plasma level

Rescued follicle

Atretic follicles

Fig 37.4 The threshold theory. When the follicle-stimulating hormone (FSH) level is above threshold, a follicle will be ‘rescued’ (continue to growth).

Gonadotropins have a narrow therapeutic range: the difference between the dose that achieves adequate follicular growth and the dose that causes hyperstimulation is small, particularly in women with estrogenicity and anovulation (i.e. PCOS patients).80a Thus, the three low-dose regimens proposed for treatment in WHO group II anovulation are focused to fulfill the two essential requirements for successful ovulation induction in PCOS patients: to allow FSH to rise slowly to just above the FSH threshold level (which is increased in PCOS patients – as evidenced by normal endogenous FSH concentrations – but has great inter-individual variability), while avoiding an ‘explosive’ ovarian response because of the exquisite sensitivity of polycystic ovaries to exogenous gonadotropins (Fig 37.4). The chronic low-dose step-up regimen for gonadotropin induction of ovulation has been the preferred method of ovarian stimulation in PCOS patients since 1990.52,60,64 This regimen is based on the threshold concept suggested by Brown et al.81,82 and amplified by Zeleznik83 which argues that the development of multiple follicles results from the failure to reproduce the precise dosage requirements that are normally maintained by feedback regulation. These authors established that initiation of follicular growth requires only a 10–30% increment in the dose of exogenous FSH, and thus advocated small, stepwise increments of FSH at 5-day intervals. In practice, however, the results of this approach were complicated by an overstimulation rate of 3% and a 26% rate of multiple pregnancy.82 The failure to achieve a high proportion of uniovulatory cycles has been related to both too high a starting dose and too short a time before increasing the dose.84 At present, this step-up approach is characterized by a low initial daily FSH dose, usually 75 IU, and the dose is increased gradually by small amounts (37.5

IU/day) until a dominant follicle emerges on ultrasound monitoring. According to the clinical features of the patient and history of multiple follicles developed within the first treatment week on 75 IU/day or OHSS in previous treatment cycles, the starting dose may be lower (one-half to two-thirds ampule per day). A feature of this regimen is that the first increase in the daily dose is performed only after 14 days of therapy if there is no evidence of an ovarian response on ultrasound (Fig 37.5). Early large series of PCOS patients treated with this protocol have shown that this treatment modality is characterized by low complication rates while maintaining fair pregnancy rates (Table 37.3).64,85,86 Also, two comparative prospective studies of the conventional regimen, with the chronic low-dose step-up protocol using urinary FSH88 or rhFSH89 for ovulation induction in PCOS patients, showed that the low-dose approach eliminated complications of OHSS and multiple pregnancies without jeopardizing the incidence of pregnancy. A review of results of published series of low (75 IU) starting-dose FSH therapy for women with PCOS, including 1391 cycles completed in 717 patients, indicates that mono-ovulatory cycles are observed consistently in approximately 70% of cases, pregnancy rates of 20% per cycle and 40% per woman are achieved, the incidence of OHSS is very low (0.14%), and the multiple pregnancy rate is only 6% (consisting of twins in 88% of cases).90 Finally, the safety and effectiveness of chronic low-dose FSH administration for ovulation induction is further supported by a recent review of studies using this approach in intrauterine insemination (IUI) cycles and including 681 pregnancies among 6670 IUI cycles initiated and reporting a satisfactory pregnancy rate per initiated cycle (10%), with a mean rate of high-order multiple pregnancy of 0.3% (being 0% in most of the studies included).91 The step-down protocol applies decremental doses of gonadotropins once ovarian response is established, but the starting dose is higher than in the stepup approach (Fig 37.6). The aim is to approximate physiologic circumstances mimicking the natural intercycle FSH elevation and the subsequent decreasing dependence of the dominant follicle with respect to FSH.60 According to this ‘threshold/ window’ concept, the duration, rather than the magnitude, of FSH increase affects follicle development.92 Monitoring of follicular growth is, however, more stringent than with the step-up approach. In addition, the long halflife of currently available FSH preparations makes it difficult to judge the correct reduction of dose in order to maintain follicle growth without risk of hyperstimulation.52 Notwithstanding the above, results from a pioneering team suggest the step-down protocol is an effective approach for FSH administration in PCOS patients.87,93 However, difficult reproducibility of this approach in daily clinical practice may be a problem as suggested by a randomized multicenter study comparing the step-up vs step-down protocol in PCOS and concluding that the step-up

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Cycle day 3: 75 IU FSH/day

7 days

– Follicle ≤ 10 mm – Maintain dose

– Follicle > 10 mm

7 days

– Maintain dose until follicle ≥ 18 mm – Follicle ≤ 10 mm – Follicle >10 mm – Increase dose by 37.5 IU/day

hCG injection

7 days

– Follicle ≤ 10 mm – Follicle > 10 mm – Increase dose by 37.5 IU/day weekly to a maximum of 225 IU/day

– Follicle > 10 mm – Follicle ≤ 10 mm – Cancel cycle – Start new cycle increasing starting dose by 37.5 IU/day

Fig 37.5 The chronic low-dose step-up protocol for ovulation induction with follicle-stimulating hormone (FSH) in polycystic ovarian syndrome (PCOS) patients. hCG, human chorionic gonadotropin.

Table 37.3 Results of low-dose regimens for gonadotropin ovulation induction in World Health Organization (WHO) group II infertile women in four large series Parameter Low-dose regimen No of patients No of cycles Ovulatory cycles (%) Cycle fecundity (per started cycle) (%) Multiple pregnancy rate (%) Abortion rate (%) Severe OHSS

Balen et al (1994)85

White et al (1996)64

Balasch et al (1996)86

van Santbrink et al (1995)87

Step-up 103 603 68 14

Step-up 225 934 72 11

Step-up 234 534 78 17

Step-down 82 234 91 16

18 16 0.5

OHSS, ovarian hyperstimulation syndrome.

6 28 0

15 11 0

12 19 0

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Ultrasonography (every 2–3 days)

– Follicle > 9 mm

– Decrease 37.5 IU/day every 3 days

– Follicle ≤ 9 mm

– Increase 37.5 IU/day – Maintain 10 days

– Follicle > 9 mm – Follicle ≤ 9 mm – Maintain 75 IU/day until hCG injection – Cancel cycle

protocol is more efficient in obtaining a monofollicular development and ovulation than the step-down protocol. In addition, it was found that although the duration of stimulation is longer, the rate of ovarian hyperstimulation is much lower using the step-up protocol.94 An alternative method for ovulation induction with FSH in PCOS patients is the so-called sequential protocol, which combines an initial step-up gonadotropin administration followed by a stepdown regimen after follicular selection (leading follicle ≥14 mm). In an early comparative study with a low-dose step-up regimen where the incremental dose increase was performed after 7 days of treatment, both approaches were shown to be safe and effective.95 However, a recent large prospective multicenter study comparing the standard chronic lowdose step-up protocol vs the sequential approach, showed that the chronic low-dose step-up regimen for rhFSH administration is efficacious and safe for promoting monofollicular ovulation in women with WHO group II anovulation. In addition, this study confirmed that maintaining the same starting rhFSH dose (75 IU/day) for 14 days before increasing the dose in the step-up regimen is critical to adequately controlling the risk of over-response. During this interval, most women (>90%) will respond to a dose of 75 IU.96 Remarkably, rhFSH has proved to be effective and safe for ovulation induction in PCOS patients with a history of severe OHSS.97 In summary, according to the current evidence, the strict adherence to the principle of the classic chronic low-dose step-up regimen, which is to employ a 75-IU FSH starting dose for 14 days and then use small incremental dose rises (37.5 IU) when necessary, at intervals of not less than 7 days, until follicular development is initiated, should be the standard approach

Fig 37.6 The step-down protocol for ovulation induction with follicle-stimulating hormone (FSH) in polystic ovarian syndrome (PCOS) patients according to van Santbrink et al.87 hCG, human chorionic gonadotropin.

to be used for ovulation induction in patients with WHO group II anovulation. Although rhFSH has been a major advance with respect to recombinant gonadotropins for the induction of ovulation in anovulatory infertility associated with PCOS, rhCG has also been successfully used in such a condition to trigger ovulation (when used instead of urinary hCG). The dose of 250 µg of rhCG provides the optimal dose for final follicular maturation in treatment cycles for timed intercourse or IUI, and this recombinant gonadotropin is better tolerated, as adverse reactions at the injection site are more likely to occur after treatment with the urinary hCG.98,99 hCG may also be used to support the luteal phase after ovulation induction. Although this is a controversial topic in PCOS patients,64,86,88 we are in favor of using repetitive hCG supplementation during the luteal phase (whenever no risk of OHSS exists), because it has been suggested that this may decrease pregnancy loss.100 In a large multicenter study,86 we found a 10% rate of spontaneous miscarriage, which contrasts sharply with spontaneous abortion rates >25–30% reported by others.64,88

Ovulation induction in hypogonadotropic hypogonadism (WHO group I anovulation) In the HH woman having intact pituitary function, pulsatile GnRH therapy can be used to restore physiologically the periodic release of FSH and LH, resulting in ovulation and pregnancy rates of 75% and 18%, respectively.101 The alternative therapeutic option is gonadotropin treatment, and no definite consensus exists with respect to which of the two regimens is the more optimal, considering costs, drug availability, chances of ovulation and conception, risks and complications, patient’s comfort, and physician’s preferences.102–104

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Which gonadotropin to use? The treatment of profoundly hypogonadotropic women with urinary FSH or rhFSH alone induces multiple follicle development but is associated with ovarian endocrine abnormalities and low oocyte fertilization rates.13,14,105–108 These findings, which are in agreement with the above-discussed current concepts on gonadotropic control of folliculogenesis, indicate that, in spite of apparently normal follicular development induced by FSH, some exogenous LH is required to optimize ovulation induction in terms of both drug requirements and clinical results. rhLH thus appears to be an ideal adjunct therapy to rhFSH in HH women. Until recently, hMG was the only source of exogenous LH for this group of anovulatory women. Over the past 10–15 years, however, a number of case reports and studies have suggested that rhLH is effective and safe when administered in association with rhFSH in WHO group I anovulatory patients.13,19,20,109–111 The use of rhLH as a separate therapeutic agent allows the clinician to tailor the dose in order to stay below the ‘LH ceiling’ discussed above.2,23 Once the efficacy and safety of the combination of rhFSH and rhLH for ovulation induction in HH women were proved, the next step was to determine the minimal effective dose of rhLH for supporting rhFSHinduced follicular development in these LH- and FSH-deficient anovulatory patients. This was done in a multicenter dose-finding study, where patients were randomized to receive rhLH (0, 25, 75, or 225 IU/day) in addition to a fixed dose of rhFSH (150 IU/day).19 The study concluded that rhLH was found to: 1.

2.

3.

Promote dose-related increases in estradiol and androstenedione secretion by rhFSH-induced follicles. Increase ovarian sensitivity to FSH, as demonstrated by the proportion of patients who developed follicles after the administration of a fixed dose of rhFSH. Enhance the ability of these follicles to luteinize when exposed to hCG.

In the study,19 it was shown that a daily dose of 75 IU rhLH was effective in most women in promoting optimal follicular development, but a minority of patients may require up to 225 IU/day. Therefore, this pioneering study confirmed that there is individual variation in the dose of rhLH required to promote optimal follicular development. Furthermore, it was found that increasing exposure to LH during the follicular phase reduces the number of growing follicles, which might reflect an LH ceiling effect as discussed above.19 Another multicenter study20 confirmed that combined rhFSH and rhLH treatment induces follicular growth, ovulation, and pregnancy in a good proportion of hypogonadotropic anovulatory patients and is well tolerated. The doses of 150 IU rhFSH and 75 IU

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rhLH daily were found to be the most appropriate, but in some patients doses >75 IU rhLH/day were necessary. Interestingly, this study clearly suggested that hypogonadotropic patients having very low levels of endogenous LH (<1 IU/l, i.e. below the threshold for normal estradiol biosynthesis and full follicular maturation) would need higher doses of gonadotropins, compared with women having basal LH levels of at least 1 IU/l, to reach the criteria necessary for hCG administration.20 In fact, there was individual variation in the dose of both LH and FSH necessary to induce ovulation depending on basal LH level,20 thus emphasizing the importance of administering FSH and LH separately, at least in some women. Therefore, both studies19,20 confirmed that there is individual variation in the dose of LH (and also FSH) required to promote optimal (mono)follicular development. The use of hMG containing fixed proportions of FSH and LH for ovulation induction in HH women has been linked to a high prevalence of multiple folliculogenesis, which is considered a major drawback to its use.101,103 Further refinement of the dosing schedule of both FSH and LH to minimize the likelihood of multiple ovulation occurring in these patients is now possible, with the availability of monotherapeutic recombinant gonadotropic agents.1,112 Thus, enhancing the LH environment would provide a means of inducing atresia in secondary follicles and promoting growth of a minimal number of preovulatory follicles (‘LH ceiling concept’). In fact, a pilot study on the subject involved patients with HH who were treated with increasing doses (every 7 days) of rhFSH (starting dose of 112.5 IU/day), according to patients’ ovarian response, along with a fixed dose of 225 IU/day of rhLH. When at least one follicle reached a diameter of 10–13 mm, the patients were randomized to three different groups: the first group continued treatment with both drugs; the second group continued rhLH and received a placebo substitute for rhFSH; and the third group continued rhFSH and received a placebo substitute for rhLH. When one follicle reached 18 mm in mean diameter, ovulation was triggered by the administration of 10 000 IU of hCG. The results of this study clearly demonstrated that the number of follicles >11 mm in diameter on the day of hCG injection was significantly lower in the rhLH/placebo group in comparison with the rhFSH/placebo group.17,79 This study performed in HH patients, who are the best and only true models for investigating the physiology of gonadotropin actions on the ovary, emphasizes the delicate balance and need for both FSH and LH in normal follicular development. Thus, it is possible that a dual advantage of high-dose rhLH may exist in the form of promoting the terminal maturation of a single preovulatory follicle, and simultaneously arresting the development of multiple less mature follicles that would otherwise occur in response to treatment with FSH.27

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3 2.5−3

2−3 2−2.5 1.5−2

Ampules/day (1 amp. = 75 IU FSH + 75 IU LH)

1

2

7

7

7

7

Days

FSH therapy

Threshold level FSH

3.

Fig 37.7 The combined (step-down and step-up) protocol for gonadotropin therapy in World Health Organization (WHO) group I anovulatory patients. FSH, follicle-stimulating hormone; LH, luteinizing hormone.

basis according to the ovarian response and the principles of the step-up regimen (Fig 37.7). Now that rhLH is commercially available, it is possible to stimulate with 2 × 75 IU rhFSH coupled with 75 IU rhLH, or according to the individual needs of patients as discussed above. Because tonic ovarian stimulation by pituitary gonadotropin is absent in these patients, luteal phase support, preferentially with hCG, is indicated.

Plasma level

Superovulation or stimulation of multiple follicular development

Fig 37.8 Stimulation of multiple follicular development for assisted reproductive technologies (ART). Maintenance of a superthreshold follicle-stimulating hormone (FSH) level during the time of multiple follicular recruitment.

Which regimen of gonadotropin administration? A chronic step-up regimen is the usual gonadotropin treatment approach.85,103,106 However, because these patients usually have long-standing HH associated with extremely low concentrations of FSH/LH and estradiol serum levels, the following facts should be considered when inducing ovulation: 1.

2.

Pretreatment with a sequential estrogen–progestin combination for one or two cycles ‘primes’ the endometrium and cervical glands, and may result in a better response to gonadotropins. Both the initial dose (traditionally 2 or 3 75-IU ampules of hMG) and dose increments are usually higher than in low-dose protocols used in PCOS patients. In addition, the first dose adjustment is performed after 7 rather than 14 days of therapy. This author prefers a combined step-down and stepup approach, where patients receive two to three ampules daily of hMG (according to the patient’s body mass index [BMI]) on stimulation days 1 and 2, and one ampule on days 3–7. From day 8 onward, hMG is administered on an individual

While the goal of induction of ovulation in anovulatory infertile women for conception in vivo is to approach the normal menstrual cycle as closely as possible, the aim of multiple follicular development (MFD) for ART is completely different: here the objective is to interfere with the selection of a single dominant follicle to obtain multiple oocytes for IVF. In fact, exogenous gonadotropins are used to ensure the maintenance of a superthreshold level during the time of follicle recruitment, thus overriding ovarian mechanisms of follicle selection (Fig 37.8). In addition, most ART patients are normally ovulating women. Therefore, as previously stressed,60 the use of the term ‘induction of ovulation’ for ART is confusing and should be abandoned.

Which gonadotropin to use? Although both urinary FSH (either in the form of hMG or pFSH/FSH-HP) and rhFSH alone can be successfully used for ovarian stimulation in nondownregulated cycles,113,114 at present, most patients undergoing IVF or intracytoplasmic sperm injection (ICSI) also receive concomitant GnRH analogs to prevent spontaneous LH surges and improve follicular response. The low endogenous LH levels achieved with GnRH analogs in some cases may amplify the differences, if any, in treatment outcome seen with the use of hMG and FSH preparations. The recent availability of GnRH antagonists, which can cause more profound LH suppression than GnRH agonists, adds further interest to the subject.

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Assisted reproduction treatment in general population Treatment with GnRH agonists does not usually result in total elimination of LH, and it is accepted that <1% of LH receptors need to be occupied to elicit a maximal steroidogenic response.18 However, there seems to be a range of LH concentrations obtained in patients treated with GnRH agonists, and these can be maintained for a considerable duration; with FSHonly preparations containing negligible LH activity, it is possible that there may be a subgroup of patients with low LH concentrations in whom ovarian responses are influenced.78,115 This can become especially relevant considering the following: 1. 2.

3.

Such women cannot be identified in advance by measuring LH levels after down-regulation.116 Oocyte maturity and fertilization rate in ART are influenced by the particular hormonal stimulation that preceded oocyte retrieval.117 The availability of both rhFSH preparations, which are fully devoid of LH activity, and potent GnRH antagonists, suppressors of pituitary gonadotropin secretion.

Considerable debate exists as to whether the LH activity contained in hMG preparations could affect the outcome of ART in GnRH agonist-downregulated women. Some authors have postulated a negative impact of using ‘LH-free’ gonadotropins for ovarian stimulation in ART. On the opposite side, the idea persists that elevated concentrations of LH (endogenous or superimposed through the use of hMG) during follicular development and in the periovulatory phase are unnecessary, and may not be desirable because of their potential detrimental effects on oocyte health and subsequent fertilization and implantation rates.18,118 Thus, while the relative importance of FSH and LH in the human process of follicular growth and maturation is still being investigated, considerable debate exists in the literature as to whether the LH component in hMG preparations could make a difference with regard to the outcome of ART treatment in GnRH agonist-downregulated women. Several facts support the concept that LH administration is not needed in the vast majority of patients undergoing ART in cycles stimulated with rhFSH in down-regulated women (long protocol of GnRH agonist) or in association with GnRH antagonist:

•

•

•

• •

•

The switch in stimulation regimens using downregulation with GnRH agonist to a more widespread use of FSH-only preparations, without LH supplementation, has been associated with an increased rate of overall program success.119–122 According to both case-control and cohort studies by us, LH serum measurements in the mid-follicular phase and even throughout the follicular phase during ovarian stimulation with rhFSH cannot predict ovarian response and ART outcome in

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down-regulated women.123,124 Even in conditions of profound LH suppression, such as cycles treated with a depot GnRH and a fixed low gonadotropin dose (both of which are neither standard practices nor absolutely first choice in ART), we found that supplemental LH may be required in terms of treatment duration and gonadotropin consumption but, in spite of this, oocyte and embryo yield and quality were significantly higher with the use of rhFSH compared with hMG.125 rhLH supplementation to rhFSH does not improve ovarian stimulation and ART outcome in pituitary-suppressed women receiving the long protocol of GnRH agonist; even more, it may have a negative impact on oocyte maturation and/or implantation rates mainly in patients younger than 35 years old.126,127 In the early clinical trials comparing GnRH agonist and GnRH antagonist for ART, pregnancy rates in the GnRH antagonist groups were similar irrespective of using rhFSH or hMG for ovarian stimulation.128 On the other hand, recent studies have shown that LH concentrations after GnRH antagonist administration do not influence pregnancy rates in IVF–embryo transfer, and even more, profound LH suppression after GnRH antagonist administration is associated with a significantly higher ongoing pregnancy rate after IVF.129,130 Finally, recent clinical trials have demonstrated that rhLH supplementation to rhFSH during GnRH antagonist administration in ART cycles does not improve IVF outcome.131,132 A very recent systematic review of the literature133 concluded that the available evidence suggests that, among women with normal ovulation or WHO II oligo-anovulation, low endogenous LH levels during ovarian stimulation for IVF using GnRH analogs (agonist or antagonist) are not associated with a decreased probability of ongoing pregnancy beyond 12 weeks. On the contrary, this review concluded that there is evidence to suggest that the opposite may be true.133 In this respect, it is noteworthy that the inclusion of exogenous LH activity (in the form of hMG) to the ovarian stimulation protocol for down-regulated young oocyte donors can have beneficial or detrimental effects on oocyte yield and quality depending on the level of endogenous LH.26 An even more recent meta-analysis investigating the efficacy of rhLH supplementation to rhFSH for ovarian stimulation in GnRH antagonist protocol for IVF/ICSI cycles failed to show any statistically significant difference in implantation and pregnancy rates.134

Therefore, according to the above evidence, it seems clear that there is no need to administer exogenous LH to a general ART population if daily doses of an appropriate GnRH agonist (in terms of the substance, formulation,

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and dosage) and the appropriate approach of rhFSH administration are used. Notwithstanding this, a need for some LH supplementation may be evidenced in some women, depending on the extent to which the endogenous serum LH is suppressed by concomitant GnRH agonist therapy, the direct effect of the latter on the ovary, and the protocol of gonadotropin administration used. Thus, recent randomized studies tested whether LH supplementation during controlled ovarian hyperstimulation, as opposed to increasing the daily rhFSH dose, can improve the outcome in down-regulated normo-ovulatory normogonadotropic patients who show an initially hyporesponsiveness to rhFSH in the form of a steady response characterized by a normal follicular recruitment to age- and BMIappropriate rhFSH dosages on treatment days 5–7, but show a plateau on follicular growth (no increase in the estradiol level and in the follicular size) on days 8–10 of stimulation in spite of continuing the same rhFSH dosage.135–138 These women have to be distinguished from the typical poor responder in whom the detection of a few antral follicles during the early stages of stimulation is followed by later cancellation of the cycle due to insufficient follicular growth. From these studies135– 138 it was concluded that LH activity supplementation (in the form of hMG or rhLH) is more effective than increasing the dose of rhFSH in terms of ovarian outcome in patients showing a hyporesponsiveness to monotherapy with rhFSH in the midfollicular phase of ART cycles. In addition, those studies demonstrated that the use of rhLH is more effective than hMG in order to rescue the ART cycles, and the daily dose of 150 IU rhLH seems to give better results than 75 IU in this regard.136,138 However, some of those studies employed a too-low daily starting dose (150 IU) of rhFSH, mainly considering that a depot GnRH agonist preparation (having a more profound suppressive effect on the pituitary and ovaries than daily doses) was used.136,137 In fact, the agonist seems to be the major effect modifier,139 and it has been shown that the currently used dosages of GnRH agonists in ART are too high, resulting in unphysiologic low LH levels.140 Thus, using daily doses of an appropriate GnRH agonist (leuprolide or triptorelin having lower potency than buserelin) and a step-down regimen of rhFSH administration as described below, we found that the proportion of LH-suppressed women is lower than previously reported,123 and we need to add some LH during ovarian stimulation in no more than 1–2% of patients in our ART general program. These are patients usually having a low estradiol response and/or an apparent discrepancy between estradiol serum levels and developing follicles.16 In conclusion, FSH-only products alone are useful tools for the vast majority of patients undergoing MFD under pituitary suppression for ART, provided that an appropriate GnRH agonist (substance, formulation, and dosage) is selected.

Low responders and patients of advanced reproductive age. The introduction of urinary purified FSH preparations first, and then rhFSH, was heralded as a stride forward for controlled ovarian hyperstimulation regimens. Thus, prospective studies in poor responders demonstrated significant benefits with rhFSH.141,142 Other investigators, however, have found that the association of LH activity in the form of hMG143 or rhLH144,145 to rhFSH during ovarian stimulation for ART improves success rates in previous poor responders or in patients with reduced ovarian reserve. Not infrequently, however, the use of hMG in previously poor responders to FSH-only preparations is associated with an increase in estradiol levels, but oocyte recovery and overall IVF results are still poor.16,146 Age-related infertility is due to oocyte abnormalities and decreased ovarian reserve.147 Two recent reports148,149 suggested that rhLH supplementation from the mid to late follicular phase in women undergoing assisted reproduction with GnRH agonist down-regulation and stimulation with rhFSH may increase implantation rates in patients ≥35 years old but not in younger women. One of these studies,149 however, was based on a low number of patients aged ≥35 years old and, as stressed by the authors, their results require additional studies for confirmation. In the second study,148 the clinical pregnancy rate was similar in women aged ≥35 years old who received both rhFSH and rhLH and those stimulated with rhFSH alone, but the difference in clinical pregnancy rates was significantly higher in favor of the rhLHsupplemented patients when only the subgroup of women undergoing their first ART cycle were considered. This subgroup of patients, however, also had significantly more embryos transferred in the rhFSH + rhLH group. Predicted clinical pregnancy rates from a regression logistic model adjusted for the number of embryos transferred indicated no significant difference between rhFSH + rhLH and rhFSH treatment groups, although the regression model also demonstrated that the higher number of embryos replaced in the LH-supplemented group did not explain the higher pregnancy rate.148 In our even more recent prospective randomized clinical trial, comprising a total of 120 consecutive normogonadotropic infertile women aged ≥35 years old undergoing their first cycle of ART, we concluded that rhLH supplementation does not increase ovarian response and implantation rates in patients of older reproductive age stimulated with rhFSH under pituitary suppression.150 Interestingly, a ‘ceiling effect’ was evidenced in our study where rhLH-supplemented patients showed impaired follicular development and oocyte yield compared with patients receiving rhFSH alone.150 Considering some methodological differences between the three studies148–150 mainly regarding the sample size, patients’ selection criteria, the type of GnRH agonist used, the stimulation day when LH supplementation was started, and even the rhFSH/rhLH

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ratios used, further studies on the subject are warranted before a definite indication for routine rhLH supplementation in women of reproductive age undergoing ART is established. Thus, it has been reported that supplementation with rhLH 75 IU/day in ART patients over 38 years old stimulated with rhFSH under pituitary down-regulation may improve early follicular recruitment and the number of metaphase II oocytes obtained.151 However, no beneficial effect in the rhLH-treated group was observed in that study in terms of embryo yield and pregnancy rates.151 In conclusion, in spite of transient enthusiasm for specific stimulation protocols, no compelling advantage for one stimulation protocol over another has been established in poor responders and patients of advanced reproductive age. Currently, treatment of infertility when the cause is limited to decreased ovarian reserve is empiric, except for oocyte donation.147,152 However, at present, the possibility that androgen treatment directly,153,154 or indirectly through the use of an aromatase inhibitor (which induces a temporary accumulation of intraovarian androgens)155 or rhLH (considering that androgens are a direct secretory product of LH action on thecal cells),156 may amplify the FSH effects on the ovary, improving follicular recruitment, is a matter of great interest and research.

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observed that tapering of the FSH dose later in the course of ovarian stimulation for IVF reduces the risk of OHSS, despite a higher starting dose of stimulation. This would be explained by a reduction in circulating FSH levels during the days before hCG injection.165 The use of a step-down regimen of rhFSH administration may also be important with respect to ovarian paracrine signaling. If, as reported above, FSH activates a paracrine mechanism that up-regulates LHresponsive androgen synthesis, and hence estradiol synthesis, it is tempting to postulate that higher doses of FSH used at a critical period of ovarian stimulation during the early follicular phase can overcome toolow ‘residual’ LH concentrations existing in some women once pituitary–ovarian suppression has been achieved. In vitro studies showing dose-dependent stimulation by FSH of paracrine regulators (inhibin and IGFs) production by granulosa cells from immature human ovarian follicles support that contention.3,4 This is important, taking into account that: 1.

2.

The LH isohormone profile may alter following GnRH agonist administration, resulting in differences in biopotency not reflected in immunoassays. Measurements of serum LH either before or during ovarian stimulation are not useful to predict ovarian response. Circulating LH measurements do not accurately reflect LH administration.19,20,135

Which regimen of gonadotropin administration?

3.

The daily dose of gonadotropin administered in ART cycles may be fixed, or progressively increased or tapered according to the given patient’s response. We prefer a tapering (step-down) regimen after pituitary suppression, wherein the highest dose of FSH is given on stimulation days 1 and 2 (300–450 IU) and is then reduced to 150 IU/day once follicular recruitment has been achieved. This regimen has proved to be clinically efficacious157,158 and is further supported by the following. First, it has been shown that for successful induction of multiple folliculogenesis in normally ovulating women there is a critical period during the early follicular phase of the cycle when FSH values should remain above the physiologic level to stimulate follicle recruitment maximally in the primary cohort.159,160 Secondly, follicles recruited by exogenous FSH require an FSH threshold concentration that is higher than that in the natural cycle.159 Thirdly, marked inter-individual variation exists in FSH thresholds as well as in FSH metabolic clearance and ovarian sensitivity to FSH.161–163 Remarkably, in clinical studies, such a threshold level was reached with a single injection of six ampules of FSH (75 IU per ampule) on cycle day 2, and further growth of the follicles was obtained with extra FSH from cycle day 4 onward at the daily dose of two ampules.159 Fourthly, studies in primates have shown that the step-down regimen leads to greater synchronization of follicular maturation when compared with conventional stepup stimulation.164 Finally, it has been empirically

The above notwithstanding, defining the optimal starting dose of FSH for each patient is certainly one of the most important issues in the management of ART cycles. For a ‘standard’ IVF patient, suggested optimal starting doses range from 100 to 350 IU of FSH per day, according to whether minimal or large numbers of oocytes are considered a success. An ‘appropriate’ response has been arbitrarily defined as retrieval of 5– 14 oocytes, but some European countries are now legally restricted in the number of oocytes that may be used in patients’ treatment, and the ability to predict an appropriate response for each patient in this situation without wastage, both of oocytes and in cost of drugs, has become a crucial issue.166,167 Interestingly, in the largest data series so far analyzed to determine predictive factors of ovarian response, basal FSH, BMI, age, and number of follicles <11 mm at screening were the most important variables in ART patients less than 35 years of age who were treated with rhFSH monotherapy. Using these four predictive factors, a follitropin α starting dose calculator was developed that can be used to select the FSH starting dose required for an optimal response. The relevance of this dose calculator is being evaluated in a prospective clinical trial where follitropin α filled by mass is used. As consistency in the biopotency of the preparation becomes important with respect to precise dose scheduling, the use of follitropin α filled by mass in that trial may

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enhance the modeling observed in the previous retrospective study.167 Irrespective of the regimen of FSH administration used, once MFD has been accomplished, hCG is used to induce final oocyte maturation in ART cycles. A recent Cochrane review concluded there is no evidence of a difference in clinical outcomes between urinary and recombinant gonadotropins used for induction of final follicular maturation in IVF/ICSI cycles but rhCG showed better local tolerance.99 Owing to marked differences in the manufacturing process, a potential additional advantage of rhCG over urinary hCG is in order to avoid the so-called emptyfollicle syndrome, a not frequent but frustrating condition causing expense and inconvenience that is considered to be a drug-related problem.168,169 Apart from the potential usefulness of rhLH in inducing MFD in the ART general population receiving GnRH analogs (agonists or antagonists), low responders, and patients of advanced reproductive age, an important additional contribution of rhLH to ovulation induction in ART patients is related to the risk of developing OHSS. As manifestations of OHSS occur within a predictable time frame in the presence of hCG, this seems to indicate that the administration of hCG at culmination of the MFD cycle is the key event in the induction of OHSS. Clinical resolution of OHSS seems to parallel the decrease of residual exogenous hCG serum levels after induction of oocyte maturation in MFD cycles. Furthermore, pregnancy and its associated increase in endogenous hCG may prolong or worsen the course of an episode of OHSS or initiate a ‘late form’ of OHSS. Finally, OHSS rarely occurs when hCG is withheld.170 hCG is structurally related to the pituitary LH, and the actions of LH and hCG are mediated by the same receptor. Most important, both hormones have the same natural function: to cause luteinization and support lutein cells. The most relevant structural feature of hCG is its elevated content of sialic acid residues, which are responsible for its longer serum half-life and enhanced biologic activity. The potency of hCG appears to be approximately 6–7-fold that of LH, although systematic information on this subject is limited.171 The longer serum half-life of hCG, with its prolonged effect on the follicle population,15 may be an undesirable characteristic in clinical practice. Thus, two studies have shown that rhLH is as effective (in terms of oocyte recovery) as, but safer (in terms of propagating OHSS) than, hCG when used in ART to induce final follicular maturation and luteinization.172,173 A single dose of rhLH, between 15 000 and 30 000 IU, gave the highest efficacy/safety ratio in IVF patients.172 It was comparable with 5000 IU urinary hCG in terms of efficacy, but resulted in a statistically significant reduction in moderate OHSS and midluteal serum progesterone levels. However, further studies are warranted to establish the protocol and dose for the optimal efficacy (number and competence of oocytes

retrieved)/safety (incidence of OHSS) ratio when rhLH is given to induce final follicular maturation and luteinization in ART patients.

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103. Martin KA, Hall JE. Pulsatile GnRH in hypogonadotropic hypogonadism. In: Filicori M, Flamigni C, eds. Ovulation Induction. Update ’98. New York: Parthenon Publishing, 1998: 47–54. 104. Messinis IE. Ovulation induction: a mini review. Hum Reprod 2005; 20: 2688–97. 105. Couzinet B, Lestrat N, Brailly S, et al. Stimulation of ovarian follicular maturation with pure folliclestimulating hormone in women with gonadotropin deficiency. J Clin Endocrinol Metab 1988; 66: 552–6. 106. Shoham Z, Balen A, Patel A, Jacobs HS. Results of ovulation induction using human menopausal gonadotropin or purified follicle-stimulating hormone in hypogonadotropic hypogonadism patients. Fertil Steril 1991; 56: 1048–53. 107. Schoot DC, Coelingh-Bennik HJ, Mannaerts BM, et al. Human recombinant follicle-stimulating hormone induces growth of preovulatory follicles without concomitant increase in androgen and estrogen biosynthesis in a woman with isolated gonadotropin deficiency. J Clin Endocrinol Metab 1992; 74: 1471–3. 108. Schoot DC, Harlin J, Shoham Z, et al. Recombinant human follicle-stimulating hormone and ovarian response in gonadotropin-deficient women. Hum Reprod 1994; 9: 1237–42. 109. Hull M, Corrigan E, Piazzi A, Loumaye E. Recombinant human luteinizing hormone: an effective new gonadotropin preparation. Lancet 1994; 344: 334–5. 110. Kousta E, White DM, Piazzi A, et al. Successful induction of ovulation and completed pregnancy using recombinant luteinizing hormone and follicle stimulating hormone in a woman with Kallmann’s syndrome. Hum Reprod 1996; 11: 70–1. 111. Agrawal R, West C, Conway GS, et al. Pregnancy after treatment with three recombinant gonadotropins. Lancet 1997; 349: 29–30. 112. Hillier SG. Ovarian stimulation with recombinant gonadotropin: LH as an adjunct to FSH. In: Jacobs HS, ed. The New Frontier in Ovulation Induction. Carnforth, UK: Parthenon Publishing, 1993: 39–47. 113. Jansen CA, Van Os MC. Puregon without analogs: an oxymoron. Gynecol Endocrinol 1996; 10(Suppl 1): 34. 114. Strowitzki T, Kentenich H, Kiesel L, et al. Ovarian stimulation in women undergoing in vitro fertilization and embryo transfer using recombinant human follicle stimulating hormone (Gonal-F) in non-downregulated cycles. Hum Reprod 1995; 10: 3097–101. 115. Fleming R, Chung CC, Yates RW, Coutts JR. Purified urinary follicle stimulating hormone induces different hormone profiles compared with menotrophins, dependent upon the route of administration and endogenous luteinizing hormone activity. Hum Reprod 1996; 11: 1854–8. 116. Loumaye E, Engrand P, Howles CM, O’Dea L. Assessment of the role of serum luteinizing hormone and estradiol response to follicle-stimulating hormone on in vitro fertilization outcome. Fertil Steril 1997; 67: 889–99. 117. Pieters MH, Dumoulin JC, Engelhart CM, et al. Immaturity and aneuploidy in human oocytes after different stimulation protocols. Fertil Steril 1991; 56: 306–10.

118. The Ganirelix Dose-Finding Study Group. A double-blind, randomised, dose-finding study to assess the efficacy of the GnRH antagonist Ganirelix (Org 37462) to prevent premature LH surges in women undergoing controlled ovarian hyperstimulation with recombinant FSH (Puregon). Hum Reprod 1998; 13: 3023–31. 119. FIVNAT. Dossier FIVNAT-99. Bilan de l’année 98, Paris, 1999. 120. FIVNAT. Dossier FIVNAT-2000. Bilan de l’année 99, Paris, 2000. 121. Wikland M. Progress of ART; the role of the clinician. Presented at the 11th World Congress on In Vitro Fertilization and Human Reproductive Genetics, Sydney, 1999. 122. Cramer DW, Liberman RF, Powers D, et al. Recent trends in assisted reproductive techniques and associated outcomes. Obstet Gynecol 2000; 95: 61–6. 123. Balasch J, Vidal E, Peñarrubia J, et al. Suppression of LH during ovarian stimulation: analysing threshold values and effects on ovarian response and the outcome of assisted reproduction in downregulated women stimulated with recombinant FSH. Hum Reprod 2001; 16: 1636–43. 124. Peñarrubia J, Fábregues F, Creus M, et al. LH serum levels during ovarian stimulation as predictors of ovarian response and assisted reproduction outcome in down-regulated women stimulated with recombinant FSH. Hum Reprod 2003; 18: 2689–97. 125. Balasch J, Peñarrubia J, Fábregues F, et al. Ovarian responses to recombinant FSH or HMG in normogonadotrophic women following pituitary desensitization by a depot GnRH agonist for assisted reproduction. Reprod Biomed Online 2003; 7: 35–42. 126. Balasch J, Creus M, Fábregues F, et al. The effect of exogenous luteinizing hormone (LH) on oocyte viability: evidence from a comparative study using recombinant human follicle-stimulating hormone (FSH) alone or in combination with recombinant LH for ovarian stimulation in pituitary-suppressed women undergoing assisted reproduction. J Assist Reprod Genet 2001; 18: 250–6. 127. Marrs R, Meldrum D, Muasher S, et al. Randomized trial to compare the effect of recombinant human FSH (follitropin alfa) with or without recombinant human LH in women undergoing assisted reproduction treatment. Reprod Biomed Online 2004; 8: 175–82. 128. Huirne JAF, Lambalk CB. Gonadotropin-releasinghormone-receptor antagonists. Lancet 2001; 358: 1793–803. 129. Kolibianakis EM, Zikopoulos K, Schiettecatte J, et al. Profound LH suppression after GnRH antagonist administration is associated with a significantly higher ongoing pregnancy rate in IVF. Hum Reprod 2004; 19: 2490–6. 130. Merviel P, Antoine JM, Mathieu E, et al. Luteinizing hormone concentrations after gonadotropinreleasing hormone antagonist administration do not influence pregnancy rates in in vitro fertilizationembryo transfer. Fertil Steril 2004; 82: 119–25. 131. Cédrin-Durnerin I, Grange-Dujardin D, Laffy A, et al. Recombinant human LH supplementation during GnRH antagonist administration in IVF/ICSI

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cycles: a prospective randomized study. Hum Reprod 2004; 19: 1979–84. Griesinger G, Schultze-Mosgau A, Dafopoulos K, et al Recombinant luteinizing hormone supplementation to recombinant follicle-stimulating hormone induced ovarian hyperstimulation in the GnRH-antagonist multiple-dose protocol. Hum Reprod 2005; 20: 1200–6. Kolibianakis EM, Collins J, Tarlatzis B, et al. Are endogenous LH levels during ovarian stimulation for IVF using GnRH analogues associated with the probability of ongoing pregnancy? A systematic review. Hum Reprod Update 2006; 12: 3–12. Baruffi RLR, Mauri AL, Petersen CG, et al. Recombinant LH supplementation to recombinant FSH during induced ovarian stimulation in the GnRH-antagonist protocol: a meta-analysis. Reprod Biomed Online 2007; 14: 14–25. De Placido G, Mollo A, Alviggi C, et al. Rescue of IVF cycles by hMG in pituitary down-regulated normogonadotrophic young women characterized by a poor initial response to recombinant FSH. Hum Reprod 2001; 16: 1875–9. De Placido G, Alviggi C, Mollo A, et al. Effects of recombinant LH (rLH) supplementation during controlled ovarian hyperstimulation (COH) in normogonadotrophic women with an initial inadequate response to recombinant FSH (rFSH) after pituitary downregulation. Clin Endocrinol 2004; 60: 637–43. De Placido G, Alviggi C, Perino A, et al. Recombinant human LH supplementation versus recombinant human FSH (rFSH) step-up protocol during controlled ovarian stimulation in normogonadotrophic women with initial inadequate ovarian response to rFSH. A multicentre, prospective, randomized controlled trial. Hum Reprod 2005; 20: 390–6. Ferraretti AP, Gianaroli L, Magli MC, et al. Exogenous luteinizing hormone in controlled ovarian hyperstimulation for assisted reproduction techniques. Fertil Steril 2004; 82: 1521–6. Westergaard LG, Erb K, Laursen SB, et al. Human menopausal gonadotropin versus recombinant folliclestimulating hormone in normogonadotropic women down-regulated with a gonadotropin-releasing hormone agonist who were undergoing in vitro fertilization and intracytoplasmic sperm injection: a prospective study. Fertil Steril 2001; 76: 543–9. Janssens RM, Lambalk CB, Vermeiden JP, et al. Dose-finding study of triptorelin acetate for prevention of a premature LH surge in IVF: a prospective, randomized, double-blind, placebo-controlled study. Hum Reprod 2000; 15: 2333–40. Raga F, Bonilla-Musoles F, Casañ EM, Bonilla F. Recombinant follicle stimulating hormone stimulation in poor responders with normal basal concentrations of follicle stimulating hormone and oestradiol: improved reproductive outcome. Hum Reprod 1999; 14: 1431–4. De Placido G, Alviggi C, Mollo A, et al. Recombinant follicle stimulating hormone is effective in poor responders to highly purified follicle stimulating hormone. Hum Reprod 2000; 15: 17–20.

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143. Meo F, Rainieri DM, Khadum I, Serhal P. Ovarian response and in vitro fertilization outcome in patients with reduced ovarian reserve who were stimulated with recombinant follicle-stimulating hormone or human menopausal gonadotropin. Fertil Steril 2002; 77: 630–2. 144. Laml T, Obruca A, Fischl F, Huber JC. Recombinant luteinizing hormone in ovarian hyperstimulation after stimulation failure in normogonadotropic women. Gynecol Endocrinol 1999; 13: 98–103. 145. Lisi F, Rinaldi L, Fishel S, et al. Use of recombinant FSH and recombinant LH in multiple follicular stimulation for IVF: a preliminary study. Reprod Biomed Online 2001; 3: 190–4. 146. Phelps JY, Figueira-Armada L, Levine AS, et al. Exogenous luteinizing hormone (LH) increases estradiol response patterns in poor responders with low serum LH concentrations. J Assist Reprod Genet 1999; 16: 363–8. 147. Practice Committee of the American Society for Reproductive Medicine. Aging and infertility in women: a committee opinion. Fertil Steril 2006; 86(Suppl 4): S248–52. 148. Marrs R, Meldrum D, Muasher S, et al. Randomized trial to compare the effect of recombinant human FSH (follitropin alfa) with or without recombinant human LH in women undergoing assisted reproduction treatment. Reprod Biomed Online 2004; 8: 175–82. 149. Humaidan P, Bungum M, Bungum L, Yding Andersen C. Effects of recombinant LH supplementation in women undergoing assisted reproduction with GnRH agonist down-regulation and stimulation with recombinant FSH: an opening study. Reprod Biomed Online 2004; 8: 635–43. 150. Fábregues F, Creus M, Peñarrubia J, et al. Effects of recombinant human luteinizing hormone supplementation on ovarian stimulation and the implantation rate in down-regulated women of advanced reproductive age. Fertil Steril 2006; 85: 925–31. 151. Gómez-Palomares JL, Acevedo-Martin B, Andrés L, et al. LH improves early follicular recruitment in women over 38 years old. Reprod Biomed Online 2005; 11: 409–14. 152. Mahuette NG, Arici A. Poor responders: does the protocol make a difference? Curr Opin Obstet Gynecol 2002; 14: 275–81. 153. Balasch J, Fábregues F, Peñarrubia J, et al. Pretreatment with transdermal testosterone may improve ovarian response to gonadotrophins in poor-responder IVF patients with normal basal concentrations of FSH. Hum Reprod 2006; 21: 1884–93. 154. Massin N, Cédrin-Durnerin, Coussieu C, et al. Effects of transdermal testosterone application on the ovarian response to FSH in poor responders undergoing assisted reproduction technique – a prospective, randomized, double-blind study. Hum Reprod 2006; 21: 1204–11. 155. García-Velasco JA, Moreno L, Pacheco A, et al. The aromatase inhibitor letrozole increases the concentration of intraovarian androgens and improves in vitro fertilization outcome in low responder patients: a pilot study. Fertil Steril 2005; 84: 82–7.

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156. Fleming R; Luveris Pre-treatment Group. Pretreatment with rhLH: respective effects on antral follicular count and ovarian response to rhFSH. Hum Reprod 2006; 21(Suppl 1): 54–5. 157. Balasch J, Fábregues F, Creus M, et al. Pure and highly purified follicle-stimulating hormone alone or in combination with human menopausal gonadotrophin for ovarian stimulation after pituitary suppression in in vitro fertilization. Hum Reprod 1996; 11: 2400–4. 158. Davis OK, Rosenwaks Z. In vitro fertilization. In: Adashi EY, Rock JA, Rosenwaks Z, eds. Reproductive Endocrinology, Surgery, and Technology. Philadelphia: Lippincott-Raven, 1996; 2: 2319–34. 159. Lolis DE, Tsolas O, Messinis IE. The follicle-stimulating hormone threshold level for follicle maturation in superovulated cycles. Fertil Steril 1995; 63: 1272–7. 160. Messinis IE, Templeton AA. The importance of follicle-stimulating hormone increase for folliculogenesis. Hum Reprod 1990; 5: 153–6. 161. Porchet HC, le Cotonnec JY, Loumaye E. Clinical pharmacology studies of recombinant human follicle-stimulating hormone. III. Pharmacokinetic– pharmacodynamic modeling after repeated subcutaneous administration. Fertil Steril 1994; 61: 687–95. 162. Ben-Rafael Z, Levy T, Schoemaker J. Pharmacokinetics of follicle stimulating hormone: clinical significance. Fertil Steril 1995; 63: 689–700. 163. van Santbrink EJ, Hop WC, van Dessel TJ, et al. Decremental follicle-stimulating hormone and dominant follicle development during the normal menstrual cycle. Fertil Steril 1995; 64: 37–43. 164. Abbasi R, Kenigsberg D, Danforth D, et al. Cumulative ovulation rate in human menopausal/ human chorionic gonadotropin-treated monkeys: “step-up” versus “step-down” dose regimens. Fertil Steril 1987; 47: 1019–24. 165. Meldrum DR. Vascular endothelial growth factor, polycystic ovary syndrome, and ovarian hyperstimulation syndrome. Fertil Steril 2002; 78: 1170–1.

166. Popovic-Todorovis B, Loft A, Linhard A, et al. A prospective study of predictive factors of ovarian response in ‘standard’ IVF/ICSI patients treated with recombinant FSH. A suggestion for a recombinant FSH dosage normogram. Hum Reprod 2003; 18: 781–7. 167. Howles CM, Saunders H, Alam V, Engrand P; FSH Treatment Guidelines Clinical Panel. Predictive factors and a corresponding treatment algorithm for controlled ovarian stimulation in patients treated with recombinant human follicle stimulating hormone (follitropin alfa) during assisted reproduction technology (ART) procedures. An analysis of 1378 patients. Curr Med Res Opin 2006; 22: 907–18. 168. Ndukwe G, Thornton S, Fishel S, et al. "Curing" empty follicle syndrome. Hum Reprod 1997; 12: 21–3. 169. Peñarrubia J, Balasch J, Fábregues F, et al. Recurrent empty follicle syndrome successfully treated with recombinant human chorionic gonadotrophin. Hum Reprod 1999; 14: 1703–6. 170. Whelan JG III, Vlahos NF. The ovarian hyperstimulation syndrome. Fertil Steril 2000; 73: 883–96. 171. Stokman PG, de Leeuw R, van den Wijngaard HA, et al. Human chorionic gonadotropin in commercial human menopausal gonadotropin preparations. Fertil Steril 1993; 60: 175–8. 172. The European Recombinant LH Study Group. Recombinant human luteinizing hormone is as effective as, but safer than, urinary human chorionic gonadotropin in inducing final follicular maturation and ovulation in in vitro fertilization procedures: results of a multicenter double-blind study. J Clin Endocrinol Metab 2001; 86: 2607–18. 173. Manau D, Fábregues F, Arroyo V, et al. Hemodynamic changes induced by urinary human chorionic gonadotropin and recombinant luteinizing hormone used for inducing final follicular maturation and luteinization. Fertil Steril 2002; 78: 1261–7.

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38 Endocrine characteristics of ART cycles Jean-Noël Hugues, Isabelle Cédrin-Durnerin

Introduction The hormonal control of ovarian function by gonadotropins plays a key role in the physiologic process of follicular growth and differentiation. Over the last decade, the respective contributions of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) to follicular development have been better defined mainly through clinical data obtained from assisted reproduction technology (ART) cycles performed with gonadotropinreleasing hormone (GnRH) agonist protocols. More recently, the introduction of GnRH antagonists to prevent the LH surge has provided a new model for assessing the respective roles of FSH and LH. In every situation, measurements of plasma FSH and LH levels were used to evaluate the endocrine environment of the follicle. While it is clear that hormonal assays from blood sampling cannot adequately reflect the biologic activity of gonadotropins, this approach has allowed an assessment of the required supply of exogenous FSH and LH in ART cycles. As regards the endocrine characteristics of stimulated cycles, another aspect to be considered is the evaluation of steroid output, which directly reflects the biological effect of gonadotropins on the ovary. Steroids are involved in the implantation process but may also play a paracrine or even an autocrine role on the cumulus–oocyte unit. Estradiol and progesterone measurements are currently done to determine the proper daily dose of exogenous gonadotropins, whereas the determination of androgen production is only performed in a few clinical studies. In this chapter, we will consider how the therapeutic agents currently used in ART cycles (GnRH analogs, exogenous gonadotropins) specifically modify the endocrine environment and to what extent hormonal evaluations are useful to aid the control of ovarian overstimulation and to predict the cycle outcome.

Gonadotropin profiles during ovarian stimulation for ART cycles According to the two cell–two gonadotropin model,1 both FSH and LH are required for promoting follicular

growth and differentiation. We will be considering their respective contributions in stimulation regimens separately.

Follicle-stimulating hormone It is well documented that FSH plays a crucial role in the recruitment, selection, and dominance processes during the whole follicular phase.2 On the one hand, FSH has a trophic effect on granulosa cells and is involved in the recruitment of the cohort at the early follicular phase. On the other hand, FSH stimulates transcription of several genes within the granulosa cells, leading to the synthesis of proteins such as aromatase, inhibin, and the LH receptor, whose expression clearly reflects follicle differentiation. From outstanding clinical studies performed by JB Brown in the late 1960s3 it has become clear that a certain amount of FSH secretion, defined as the ‘FSH threshold,’ is required to induce follicular growth. Moreover, as the FSH threshold is not identical for the follicles of the same cohort, the FSH supply for inducing multifollicular development should overcome the threshold of the least FSH-sensitive follicles. This concept of FSH threshold led to the postulate that increasing FSH supply in the early stage of the cycle is a key factor for the follicular recruitment process (Fig 38.1). Another aspect of the involvement of FSH in folliculogenesis is the concept of the ‘FSH window’ described by Baird.4 It means that follicular growth is maintained as long as the FSH level is above the follicle’s threshold. In a natural cycle, the decrease in FSH secretion related to a feedback effect of ovarian factors at the pituitary level largely contributes to the dominance of the selected follicle over the others. By contrast, maintaining the FSH levels above the threshold of the dominant follicle opens the window until the final stages of follicular development: a crucial component for controlled ovarian stimulation (COS). These two concepts justify the assumption that FSH is the main therapeutic agent to control folliculogenesis in all situations except that of severe hypogonadotropic hypogonadism. Indeed, in this latter case, an LH supply

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Fig 38.1 The FSH threshold and window concepts. This figure illustrates that follicular growth starts at the early follicular phase when plasma FSH concentration is above a theshold value. Differences in FSH threshold between each follicle of the same cohort account for the asynchrony of follicular development. Follicular growth will continue as long as the FSH window is opened, i.e. the plasma FSH value is above the threshold. Conversely, the reduction in plasma FSH induced by increased E2 secretion results in a progressive arrest of follicular growth. The follicle with the lowest threshold is only preserved because it becomes more sensitive to FSH and possesses LH receptor, which allows LH to contribute to ovarian steroidogenesis.

is also required to ensure adequate steroid production according to the two cell–two gonadotropin model.5 Both gonadotropin preparations and GnRH analogs are commonly used to achieve multifollicular development but the effects of each agent on FSH accumulation are quite different. As far as gonadotropin administration is concerned, it has been stated that, owing to the long elimination half-life (30–35 hours) of the FSH molecule,6 a plateau of plasma FSH is obtained after 5 consecutive days of injection.7 Conversely, FSH accumulation, which seems to be a determinant factor for the size of final cohort of mature follicles,8 is observed for a few days following the cessation of FSH administration.9 Furthermore, determination of plasma FSH levels following intramuscular or subcutaneous administration of FSH has shown that there is a modest and transient (4–8 hours) rise in plasma FSH values which cannot adequately reflect the actual bioactivity of the molecule. In another clinical study,10 Schoemaker’s group evaluated the role of plasma FSH measurements in order to determine the adequate threshold FSH dose. In this very sophisticated model, the dose of FSH administered in a pulsatile intravenous manner was adjusted daily according to the simultaneous evaluation of plasma FSH levels. In this way, the authors were able to control the minimal supply of FSH required to select the most sensitive follicle of the cohort, which is highly relevant for inducing mono-ovulation. However, the correlation between plasma FSH values and the FSH threshold dose was poor because of a large overlap of the plasma FSH values observed between patients who presented with follicular recruitment and those who did not (Fig 38.2) Consequently, it appears that determination of

plasma FSH levels is not a suitable way to assess the adequacy of the exogenous FSH supply. The effects of GnRH agonists (GnRH-a) on FSH secretion depend largely on the way these pharmaceutical agents are used. The initial flare-up effect of the agonist at the pituitary level is associated with a significant increase in plasma FSH levels which participates in the follicular recruitment in the so-called short protocol. Several studies11–13 have shown that the amplitude of the FSH response to GnRH-a is lower than that of LH. Furthermore, the dose dependence effect of the agonist on the gonadotroph response is far less evident for FSH than for LH, attesting to differences in the hypophyseal control of gonadotropin secretion. As a lower dose of GnRH-a than that usually recommended may induce a larger increase in the FSH reponse,14,15 there is a need to further evaluate the most appropriate dose of GnRH-a in this short-term protocol.16 Consistent with its lower dependence regarding GnRH control, the desensitizing effect of long-term GnRH-a administration upon FSH secretion is much less marked than for LH. Immunometric evaluation of plasma FSH has shown that the suppressive effect of the agonist is modest and may be dependent on the molecule used, buserelin being the most suppressive agent.17 Conversely, it has been also reported that FSH bioactivity may not actually decrease during GnRH-a administration.18,19 Thus, it is unlikely that determination of plasma FSH levels is relevant during the course of GnRH-a administration. Finally, the latest data concerning plasma FSH variations following administration of a GnRH antagonist provided similar conclusions. Indeed, the gonadotroph suppression was less marked for FSH than for LH, attesting once again to the relative GnRH dependence

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of FSH hypophyseal regulation.20 Nevertheless, in clinical practice, the use of GnRH antagonists in patients treated for ART is associated with a higher dosage of gonadotropins compared to gonadotropins alone in order to compensate for the suppressive effect of the antagonist on hypophyseal secretion. To sum up these data on FSH variations during treatments for ART, it does appear that determination of plasma FSH is not contributive enough to tailor a gonadotropin regimen in a proper manner. Thus, it seems more appropriate to restrict this evaluation to clinical research studies.

Luteinizing hormone The role of LH on folliculogenesis varies according to the stage of follicular development. On the one hand, LH acts directly on theca cells where LH receptors are constitutively present and ensure a tonic production of androgens during the whole follicular phase. According to the two cell–two gonadotropin theory, androgens play a key role as substrates for aromatase activity and contribute to the production of estradiol by granulosa cells. On the other hand, LH directly participates in the control of granulosa cell function through specific receptors which are gradually present as soon as cell differentiation is FSH induced. It has been shown by in vitro studies21 that, while LH induces a dose-dependent protein synthesis (aromatase activity), its effect on cell proliferation is negative, at least at high concentrations. This latter effect of LH may account for the final arrest of follicular growth at a stage of the cycle where follicular maturation is optimal. The pivotal role of LH on steroidogenesis has been well documented by studies performed in patients with hypogonadotropic hypogonadism. Indeed, in those

Fig 38.2 The FSH plasma threshold. FSH plasma ‘stable’ concentrations according to follicular growth in anovulatory patients treated with hFSH (open symbols) or hMG (closed symbols). This figure shows the overlap of plasma FSH values between patients with (right panel) or without (left panel) follicular growth.10

patients deprived of hypophyseal gonadotroph production, substitution with recombinant FSH results in follicular growth but does not allow any concomitant steroid output. In contrast, addition of recombinant LH induces a dose-dependent increase in estradiol production, a condition required to ensure endometrial preparation for embryo implantation.5 This observation emphasizes that a minimal amount of LH, defined as the ‘LH threshold,’ is required for pregnancy. However, as discussed later on, determination of plasma LH concentrations by immunometric assays may not be helpful enough for an accurate assessment of the LH threshold. Alternatively, is there any evidence for an adverse effect of high endogenous LH environment? If we look at previous reports regarding the influence of the endogenous LH on the outcome of both natural and treated cycles,22,23 it is presumed that high endogenous LH levels are often associated with an increased incidence of infertility or miscarriages. Another study24 performed in patients involved in an egg donation program suggests that this deleterious effect of high endogenous LH was related to a negative influence on the endometrium rather than on the oocyte/conceptus itself. More recently, the concept of the ‘LH ceiling’ has been proposed by Hillier25 on the basis of his own experiments showing an inhibitory effect of high LH doses on cell growth. Thus, LH, beyond a certain ‘ceiling’ level, suppresses granulosa proliferation and initiates atresia of less mature follicles. Preliminary unpublished clinical data from hypogonadotropic hypogonadal patients tend to support this concept: indeed, substitution with recombinant LH alone in the late follicular phase induces a reduction in the size of the follicular cohort and in the number of large follicles. Altogether these data clearly show that the role of LH on steroidogenesis is crucial, while its contribution to folliculogenesis and ovogenesis is still a matter of debate.

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Let us consider now the plasma LH variations when using drugs for ART cycles. Urinary human menopausal gonadotropin (hMG) preparations have been commonly used with success over the last 30 years for ovulation induction. In regimens performed with gonadotropins alone, it has been demonstrated that LH is rapidly cleared from the circulation owing to its relatively short half-life.26 The pharmacokinetics of LH have been studied in detail using recombinant human luteinizing hormone (rhLH) and the terminal half-life was found to be approximately 12 hours, half that of FSH. Thus, in contrast to FSH, there is slight evidence of plasma LH accumulation following a single injection of hMG (Fig 38.3). However, in urine, LH concentrations are significantly elevated in polycystic ovary (PCO) women treated in a chronic low-dose step-up regimen with hMG compared to rhFSH. Furthermore, determination of plasma LH from a morning blood sample following an evening injection of gonadotropins is not very informative for evaluating the actual consequences of the LH content of hMG preparations. Thus, during gonadotropin therapy, plasma LH measurements are usually restricted to the detection of the endogenous LH surge, specially required for women undergoing intrauterine inseminations. From 1982 until recently, GnRH agonists have been routinely adopted as adjunct therapy in controlled ovarian hyperstimulation. Taking advantage of the initial flare-up effect of GnRH-a injection, an ultrashort or a short-term administration of the analogs has been shown to promote follicular recruitment at the early follicular phase of the cycle. Indeed, within the 24 hours following the first GnRH-a administration, both endogenous FSH and LH are released from the hypophysis and, as mentioned earlier, the flare-up effect is more marked for LH than for FSH.11–13 Consequently, estradiol secretion is stimulated and, as discussed below, the magnitude of E2 variation proved to be the best predictor of the ovarian sensitivity to gonado-tropins. Thus, determination of plasma LH does not appear relevant during the flare-up period. In contrast, measurements of plasma LH are routinely performed at the time of hypophyseal desensitization to make sure that gonadotropin secretion is adequately downregulated after long-term administration of the GnRH-a. It is well documented that both the rapidity to achieve desensitization and the degree of LH suppression are critically dependent on numerous factors in these long-term agonist protocols: the type of molecule, the time of its first administration in the cycle, the dose and duration of GnRH-a administration and molecule formulation.29 During this period and as long as GnRH-a administration is maintained, the hypophysis is refractory to GnRH action, as attested by the disappearance of LH pulsatile secretion and a lack of response to exogenous GnRH or estradiol benzoate administration.30–32 It is also well documented that both intensity and duration of LH suppression are dose dependent.33,34 However, some unanswered questions remain regarding the state of hypophyseal desensitization. One of

them is to elucidate the reasons why there is an evident need for a higher amount of exogenous gonadotropins to obtain an adequate ovarian reponse. It is commonly stated that the more profound the hypophyseal desensitization, the worse the ovarian response to stimulation will be. This has led to the proposal of using a lower dose of GnRH-a specially for patients with a history of low response to gonadotropins. However, the effectiveness of this dose reduction is still a matter of debate. In fact, the main issue to be addressed is the actual assessment of the LH suppression. Indeed, regular immunometric assays of LH cannot properly reflect the residual hormonal LH bioactivity. After a 2-week GnRH administration, LH bioactivity seems to be completely suppressed but LH concentrations remain measurable by immunometric assays in relation to persistent secretion of presumed nonbiologically active hormones (α subunits and/or molecules with modified glycosylation).35 It has also been shown that a daily GnRH-α administration leads to a partial release of measurable α,34 and that stopping the daily agonist administration induces a sharp decrease in both plasma dimeric LH and α subunit concentrations36,37 (Fig 38.4). Thus, it must be stressed that the residual measurable LH secretion depends on the GnRH-a formulation and on the duration of administration. Moreover, it is still unclear whether bioactive residual LH secretion is totally suppressed at the time of hypophyseal desensitization in every long-term protocol. Indeed, with reference to the two cell–two gonado-tropin theory, we would predict that administration of purified or recombinant FSH during the stimulation period would not be effective in stimulating estradiol production, and it is clear that it is not the case. Moreover, the largest studies recently published definitely showed that FSH administration alone is sufficient to yield an adequate number of good-quality oocytes and embryos and to obtain a high implantation rate.38,39 Some authors have argued that, for some patients or in some situations of high LH suppression induced by some agonist formulations, the residual LH secretion may not be sufficient to ensure an appropriate estradiol secretion. Westergaard et al and Fleming et al40,41 tried to identify such subgroups of patients by evaluating the outcome of ART cycles according to the plasma level of residual LH at the time of desensitization or during the midfollicular phase. Selecting a subgroup of patients whose residual plasma levels were lower than 0.5 IU/l, they found a trend for a reduced plasma E2 concentration at the time of hCG administration and for a lower yield of oocytes and number of embryos. However, the rate of blastocyst development was unaffected. Thus, these data confirm the inability of plasma LH measurements to detect those patients who would need some addition of LH to support the ovarian stimulation. In another approach, Loumaye et al42 analyzed the E2/oocyte ratio, based on the previous observation in hypogonadotropic hypogonadal women, that the amplitude of E2 secretion per follicle is related directly to the dose of recombinant LH administered. In this model, it was shown that only a small population

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(less than 6% of patients) might benefit from exogenous LH administration and that measuring plasma LH levels after downregulation is of no practical benefit to identify this subgroup of patients. Collectively these data suggest that the LH threshold under which folliculogenesis may be impaired cannot be properly assessed by standard immunometric determination of plasma LH concentrations. Finally, the recent introduction of GnRH antagonists in the field of ART therapy provides another model for evaluating the need for LH in ART cycles. Acting as a competitor to endogenous GnRH at the receptor level,

the GnRH antagonists induce a rapid and reversible reduction in LH secretion without any interference with the hypophyseal machinery. In that respect, the hormonal situation induced by the antagonist is easier to assess than that induced by agonist: a parallel decrease in plasma dimeric and α subunit LH concentrations is elicited by GnRH antagonist administration43,44 and a rapid recovery of the pituitary–gonadal axis is predictable after discontinuation of treatment. A dose-finding study recently published45 showed that plasma LH concentrations decrease in a dose-dependent manner following the administration of Org

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) or 14 days ( ) in short-term protocol. This figure illustrates that stopping GnRH-a administration at day 7 leads to a sharp and parallel decrease in both dimeric LH and α subunit plasma concentrations. Thus, in contrast to GnRH antagonist, a daily agonist administration sustains a partial release of measurable α subunit and LH from the hypophysis.36

•

37462 (ganirelix), and no endogenous LH surge was observed whatever the dose used. This study also pointed out that the remaining endogenous LH concentrations during GnRH antagonist treatment may become critical when pituitary suppression is too profound. Thus, it is likely that assessment of residual LH concentrations may be helpful, particularly in patients treated with a single-dose GnRH antagonist protocol. To sum up these data on LH secretion during GnRH analog therapy (Fig 38.5), we may consider that the limits of plasma LH required for achievement of folliculogenesis are those defined by the LH ceiling and threshold values. Within this interval, LH support seems to be adequate to provide androgen synthesis, to ensure estradiol secretion, and to participate in the control of follicular growth. This figure also emphasizes that

assessment of LH requirements needs to take into account the joint effects of both gonadotropins and GnRH analogs on plasma LH secretion.

Steroid profiles during ovarian stimulation for ART cycles In contrast with gonadotropin, evaluation of steroid production is routinely performed during ART cycles. Plasma estradiol (E2) measurement is a good indicator of granulosa cell differentiation and is helpful to evaluate follicular maturity before triggering ovulation. Plasma progesterone (P) determination has been performed to seek for any premature luteinization, which is uncommon since GnRH analogs are regularly prescribed in

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ART cycles. Finally, plasma androgen levels are rarely determined except for clinical research.

Estradiol Estradiol plays a crucial endocrine role in the reproductive system, being involved in cervical mucus production, in endometrial proliferation for embryo implantation, and in the induction of the midcycle LH surge. In contrast, the autocrine role of estradiol in follicle development, first described in rats, is unlikely in humans, where E2 is not required for follicular growth. Thus, E2 synthesis is merely asssociated with dominant follicle development and plasma E2 concentration is a useful index to assess follicular maturity. During ART cycles, plasma E2 measurements are routinely used to calibrate the gonadotropin doses in conjunction with data obtained by ultrasound. Indeed, it is admitted that E2 synthesis is directly related to follicular size and that the contribution of mature follicles to E2 output may be estimated at about 200 pg/ml. Another aspect to be considered during ART cycles is the pattern of E2 secretion. In the early 1980s, at a time where GnRH analogs were not available for preventing any endogenous LH surge, particular attention was focused on the pattern of plasma E2 levels. The Norfolk group described several E2 patterns and correlated the outcome of the cycle with each pattern46 (Fig 38.6). Similarly, in protocols using GnRH-a, it was suggested that an increase in plasma E2 concentrations for 6 consecutive days would be optimal for the success of the cycle.47 Owing to the extreme diversity of protocols used in ART cycles, no description of a common optimal E2 pattern is available. Nevertheless, some considerations seem to be valid whatever the protocol used. A plateau of plasma E2

Fig 38.5 The LH threshold and ceiling concepts. This figure illustrates the interval defined by the respective values of LH threshold and LH ceiling. Within this interval, LH support is presumably sufficient to ensure steroidogenesis without negatively affecting follicular growth. Both gonadotropins and GnRH analogs jointly modify circulating LH levels in such a way that the residual plasma LH concentrations are most often included within these limits.

values for more than 3 days is commonly associated with a poor outcome of the ART cycle. Conversely, measurements of plasma E2 are helpful to detect the risk of excessive ovarian response and to decide coasting of gonadotropin administration, canceling the cycle or the embryo transfer. For these reasons, it seems that plasma E2 determination must be included, to some extent, in the monitoring of ART cycle treatment, and it is also clear that ultrasound may be useful to simplify the patient follow-up. As regards E2 determinations, we would also like to mention that plasma E2 variations were used as a sensitive index of ovarian responsiveness to gonadotropins and, to some extent, as a predictor of the outcome of the ART cycle. The administration of a fixed dose of exogenous FSH (300 IU) at day 3 of the cycle showed that the relative increment of plasma estradiol concentrations is a better predictor of the ovarian response than the day 3 FSH value.48 Other authors49 suggested that an early determination of plasma E2, after only few days of gonadotropin administration, may be useful to predict the subsequent ovarian response. All these data underline that a single determination of plama E2 may be a helpful predictor of a poor or high ovarian response. A similar approach, based on the evaluation of E2 response to the endogenous gonadotropin flare-up induced by GnRH-a, was proposed by Padilla et al.12 This test (Lupron screening test) aims at evaluating the increase in plasma E2 after a subcutaneous administration of leuprolide acetate (1 mg) on days 2–4 of the menstrual cycle. The authors found a good correlation between the E2 response and the ovarian response to COS and described four patterns of E2 variations with different prognoses for the cycle. In contrast to Padilla et al, Winslow et al,50 using the same agonist, correlated the relative increment of plasma estradiol from day 2 to day 3 (∆E2) with the

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ovarian response to stimulation. In a similar study using triptorelin as GnRH-a, we also demonstrated that the E2 cut-off value is reduced by a pretreatment with progestogen in programmed cycles, but the relationship between the early events of the follicular phase and the subsequent pregnancy rate still exists.51 In clinical practice, determination of the E2 response to the flare-up effect of the agonist is relevant for an early detection of potential poor responders and for tailoring gonadotropin administration accordingly. To sum up these data, the predictive E2 values for each test are presented in Table 38.1. With the long-term GnRH-a protocol, determination of plasma E2 is also recommended to assess if the hypophyseal desensitization is effective at the ovarian level. Indeed, as previously mentioned, plasma LH immunometric evaluation cannot adequately reflect

the state of pituitary desensitization. It is commonly stated that plasma E2 must be lower than 50 pg/ml to make sure that ovarian activity is actually suppressed, which usually occurs after 2 weeks of GnRH-a administration. Starting GnRH-a administration in the midluteal phase52,53 or using a long-acting formulation of the agonist54,55 may allow more rapid desensitization than when using short-acting formulations at the early follicular phase. However, it is still unclear whether there is any clinical advantage in achieving a prompt and profound des-ensitization. It was even suggested that a prompt desensitization would induce an ovarian state refractory to exogenous gonadotropins.54 In every situation, it is recommended to start ovarian stimulation with FSH only when ovarian activity is suppressed, whatever the duration of GnRH-a administration needed to achieve it.

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Table 38.1 Stimulation tests predictive of the ovarian response to gonadotropins. Different stimulation tests proposed to evaluate ovarian sensitivity to gonadotropins: hFSH (300 IU i.m.) (EFORT test) or leuprolide acetate (1 mg s.c.) or triptorelin (0.1 mg s.c. after a pretreatment by norethisterone) (GnRH-a tests) are administered at day 2 of the cycle. Plasma E2 levels are determined at day 2 before stimulation and 24 hours later at day 3. ∆E2 represents differences between plasma E2 values. Figures indicate the E2 cut-off values predictive of an adequate ovarian response. Tests Exogenous FSH (EFORT) Lupron screening test Triptorelin screening test

∆E2 (pg/ml) > 30 > 20

>5

Finally, the recent availability of GnRH antagonist in ART cycles may lead to a reassessment of the usefulness of plasma E2 determination. Indeed, the first studies performed with GnRH antagonist clearly indicate that the pattern of plasma E2 is not similar to that obtained with GnRH-a. Whether this difference may account for the lower pregnancy rate observed with GnRH antagonist protocols is still a matter of controversial debate. These new protocols may give the opportunity to revisit the interest of plasma E2 determination during the stimulation phase of ART cycles.

Progesterone Before the introduction of GnRH analogs in ART cycles, detection of premature endogenous LH surges was a constant concern because LH surges usually occurred when follicular development was still uncompleted and had some deleterious effects on oocyte quality and on the implantation rate. At that time, determination of plasma progesterone (P) was considered a complementary tool to detect partial luteinization of granulosa cells attributed to some small or short LH surges that could not have been detected even by daily blood sampling. It is clear that the current use of GnRH agonist and the recent marketing authorization of GnRH antagonists, two agents effective to prevent LH surges, have led to strictly limiting the determination of plasma P in some periods of ART cycles. It is usual to control plasma P values at the time of hypophyseal desensitization. It seems worthwhile to make sure that the corpus luteum is not still active and has not been inadvertently rescued by a prolonged GnRH-a flare-up or by a spontaneous pregnancy. Moreover, at that time, if cyst formation is observed on ultrasound, an increase in plasma P concentrations would indicate the functional nature of the cyst and would lead to perform ovarian puncture

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before FSH administration. It is recommended that ovarian stimulation not be started in a hormonal environment that may be deleterious for the oocyte or the endometrium. In this respect, an increase in plasma P that would overcome follicular phase values at the time of hypophyseal desensitization is considered deleterious for the subsequent ART cycle, and requires the administration of GnRH-a to be extended as well as the postponement of ovarian stimulation. With a similar concern, special attention has been paid to other situations where an increase in plasma P has been correlated with a risk of poor outcome for the ART cycle. The first refers to the endocrine consequences of the flare-up effect induced by GnRH-a in short-term protocols. As previously mentioned, the initial agonist administration induces a sharp increase in gonadotropin and steroid production and release. Some reports57–59 showed that increased plasma P levels during the early follicular phase may adversely affect follicular development, oocyte quality, and eventually the success rate of the cycle. However, these conclusions have been challenged by other studies. For Sims et al,60 it is only beyond a threshold of plasma P values that impairment of follicular development may be observed. Furthermore, a prospective randomized study showed that the outcomes of the ART cycles were actually similar in two groups of patients pretreated or not by a progestogen which completely prevented any plasma P increase in the flare-up period.51 Thus, there is no evidence that any increase in plasma P is detrimental at the very early follicular phase of the cycle. It is no longer necessary to perform this determination during the flare-up of short-term GnRH-a protocols. Another circumstance where plasma P determination must be considered is in the late phase of ovarian stimulation. Indeed, despite an effective suppression of endogenous gonadotropins by GnRH-a, a small increment in plasma P has been reported in up to 20% of stimulated cycles. Thus, the issue of a potential adverse effect of P increase on the cycle outcome must be addressed but is still a matter of debate: some authors61–63 reported a negative effect on the pregnancy rate through inadequate endometrial preparation, while others64–67 could not find any significant relationship. Furthermore, whether or not a P increase is detrimental, there is no consensus on the critical P threshold plasma value. Finally, the mechanisms that account for the P plasma rise despite suppressed endogenous gonado-tropins are not clearly demonstrated. Exposure to large doses of exogenous FSH seems to be associated with a higher incidence of high P plasma values,66 but it is still unclear whether the P increase is related to some disruption of the ovarian steroidogenic pathway induced by high FSH doses, or the early expression of an occult ovarian failure, as suggested recently.69 Also, the specific contribution of the adrenal gland must be considered because dexamethasone administration enables a

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partial reduction in plasma P levels.70 Nevertheless, it is likely that the impact of exogenous gonadotropins on the ovary predominantly account for this process.71 Additional studies are required to draw conclusions on this issue, and in clinical practice, plasma P cut-off values as a means of decision making should be questioned.

Androgens Determination of plasma androgens, namely testosterone and ∆4 androstenedione, is not currently performed during monitoring of ART cycles. While androgen production is mainly dependent on LH secretion, there is no evidence that assessment of androgen secretion helps in measuring low LH bioactivity. This is partly related to the fact that both ovary and adrenal gland contribute to androgen production in women with normal reproductive function. Conversely, excessive androgen production may easily be detected by plasma androgen measurements. With the exception of partial enzymatic adrenal defects, these are mainly related to ovarian hyperandrogenism with or without LH hypersecretion. During the last decade it became more evident that asessment of ovarian morphology by transvaginal probes allows a more accurate evaluation of polycystic ovary syndrome (PCOS) than plasma androgen measurements. However, while androgen determination does not appear to be a contributive factor for the assessment of COS, some reports72 show that androgens actually exert a stimulatory effect on granulosa cell proliferation in human beings and may be involved in follicular recruitment. Thus, the specific contribution of androgens in the process of folliculogenesis deserves further evaluation, and it is likely that plasma measurement may participate in better defining the potential role of androgens. One study mentioned that during ART cycles the flare-up effect of GnRH-a in short-term protocols is associated with increased androgen production. However, there is no significant evidence that oocyte quality may consequently be reduced.73 To sum up, it does seem that determination of plasma androgens need not be routinely included in the monitoring of ART cycles, but may be worthwhile in clinical research.

Inhibins A and B Inhibins A and B are secreted from granulosa cells following FSH stimulation and regulate FSH secretion by negative feedback.74,75 These two heterodimers, composed of an α subunit and one of the two β subunits forming inhibin A (αβA) and inhibin B (αβB), also exhibit unique patterns of expression and secretion during the menstrual cycle. In vitro studies demonstrated that inhibin βB-subunit messenger ribonucleic acid (mRNA) is expressed predominantly in small antral follicles and βA mRNA is expressed in the preovulatory

follicle of the human, whereas α-subunit mRNA expression is similar at both follicle stages.76 Therefore, small antral follicles have the potential to secrete inhibin B, whereas preovulatory follicles may secrete inhibin A. Many of the early observations concerning the physiology of inhibin were based on the relatively nonspecific Monash RIA, which detected both of the dimeric inhibins A and B as well as the inhibin α subunit. The recent availability of specific two-site assays for dimeric inhibin A and B measured by enzyme-linked immunosorbent (ELISA)77 has afforded the opportunity to reconsider the usefulness of their measurement in clinical practice. Serum inhibin B value in the early follicular phase of the menstrual cycle has been shown to be a valuable tool to evaluate the size of the follicular cohort.78 Moreover, the FSH dependence of inhibin A and B secretion has been demonstrated in regularly menstruating women with normal ovaries as well as in women with PCOS.79,80 Consequently, several studies have been performed to assess whether inhibin A and B measurements may be predictive of the ovarian response to gonadotropins, of the risk of hyperstimulation as well as the pregnancy rate in controlled ovarian hyperstimulation. The results of the first study81 indicated that inhibin A and pro αC are well correlated with estradiol values and the number of follicles (>10 mm) during FSH stimulation, and may be useful markers for monitoring the effects of gonadotropin stimulation. However, a subsequent study82 has shown that neither inhibin A nor inhibin B measured at the time of human chorionic gonadotropin (hCG) administration provided additional information in predicting successful outcome over age and number of oocytes. In clinical perspective, it is evident that the most useful markers are those that can be assessed in the early stages of ovarian stimulation. As a consequence, more recent analyses were focused on the predictive value of inhibin B measurement as a marker of follicular recruitment. Indeed, it did appear that inhibin B measurement between days 4 and 6 of FSH stimulation provided an early indicator of the number of recruited follicles destined to form mature oocytes.83,84 Moreover, another study85 recently showed that a similar relationship between inhibin B measured after 2 days of FSH stimulation and oocyte number may be applied in both normal and low responders. Therefore, inhibin B measurement in the early stage of ovarian stimulation may provide useful information to clinicians in making decisions regarding cancellation of the cycle or modulation of the gonadotropin dose. This predictive value of inhibin B is likely to be true for patients treated with protocols that include hMG and GnRH antagonist.86

Anti-Müllerian hormone Anti-Müllerian hormone (AMH), a member of the transforming growth factor beta (TGF-β) family, is produced by granulosa cells.87 The highest level of AMH expression is present in granulosa cells of secondary, preantral,

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and small antral follicles up to 6 mm in diameter,88 whereas, in follicles growing into dominance, this expression is progressively reduced.89,90 Consequently, AMH is barely detectable at birth, reaches the highest values after puberty, then decreases progressively with age and becomes undetectable at menopause.91,92 Serum AMH levels have been shown to strongly correlate with the number of antral follicles93,94 and has the major advantage over other markers of ovarian reserve (FSH, estradiol, and inhibin B) of being cycle independent. This allows blood sampling at any period of the cycle.95,96 The main interest of measuring AMH prior to ART cycles is actually related to its ability to predict the ovarian responsiveness to FSH. It was shown that as the number of antral follicles in the ovaries is proportionally related to the size of primordial follicle stock from which they were recruited, 97 the antral follicle count (AFC) may be a useful predictor of poor response in the in vitro fertilization (IVF) program.98 More recently, several studies have reported that AMH could also be one of the best predictor of the ovarian response to hyperstimulation99,100 and, in only one study, of the chance of becoming pregnant after IVF.101 A systematic review has recently assessed the true accuracy of AMH as a prognostic factor for the outcome of IVF/intracytoplasmic sperm injection (ICSI) treatment compared with AFC.102 Thirteen studies reporting on the capacity of AMH to predict ovarian response and/or nonpregnancy for IVF cycles, and considered suitable for data extraction and metaanalysis, were identified.94,103–114 Receiver operating characteristic (ROC) curves showed a high accuracy for AMH and AFC for the prediction of poor ovarian response but limited accuracy for nonpregnancy prediction. Furthermore, these data did not suggest a clearly better predictive ability for AMH than for AFC because the difference was not statistically significant (p = 0.73). However, AMH determination has some advantages over AFC. Indeed, it does need to be carried out on a specific day of the cycle, because AMH levels fluctuate only marginally and prediction by samples of any cycle day will be equally accurate.95,110,115 Currently, the availability of the AMH assay may present some problems, but surely it will soon become part of one of the large automated platforms, with inherent validity checks and limited assay variation. In contrast, AFC necessitates skilled ultrasound operators who carefully identify, measure, and count ovarian follicles. Although observer bias may be limited technically,116,117 a new source of bias may arise from the fact that the ultrasound operator is aware of the cut-off for test judgment and may become influenced by the consequences of the test for the treatment of the couple. Such test inflation has recently been suggested from a study in older IVF patients who

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were allowed or refused IVF treatment on the basis of this test.118 Also, AFC has to be carried out in the early follicular phase of the cycle, although variation of counts across the cycle may be very modest.119 Finally, the performance for nonpregnancy prediction is clearly poor for both AMH and AFC. This observation makes sense because AMH, like AFC, is strongly thought to represent only the size of the cohort of FSH-sensitive follicles continuously present in the ovaries while the relation between quantity and oocyte or embryo quality is much less clear. Another issue to be addressed regards the interest of measuring plasma AMH levels during ovarian stimulation. From experimental data mainly obtained in rodents, the potential functions of AMH are inhibition of the initial recruitment of primordial follicles,120 inhibition of aromatase activity in granulosa cells,121,122 and decrease of FSH-stimulated follicle growth in the mouse, both in vitro and in vivo.123 These data suggest that AMH is a negative regulator of follicle growth and reduces follicle sensitivity to FSH. Conversely, it has been suggested that AMH expression might be regulated by FSH itself. Indeed, in human follicular fluid, FSH exerts a negative influence on AMH secretion in the small follicles.124 Furthermore, in vitro FSH treatment significantly reduced AMH expression in cultured granulosa cells retrieved from patients with PCOS.125 Additionally, in PCOS patients, during administration of low doses of recombinant hFSH according to a chronic low-dose step-up protocol to mimic the typical pattern of the FSH secretion of the early follicular phase of normal nonstimulated cycles, a significant AMH decrease was gradually observed up to the day of follicular dominance.126 Even if the precise mechanism is still not clearly understood, it is likely that the decrease in serum AMH observed prior to the establishment of dominance during FSH treatment in PCOS patients results from the shift of small antral follicles to larger ones expressing less AMH. Similarly, during controlled ovarian hyperstimulation for IVF in normo-ovulatory women, three studies have reported a marked serum AMH decline up to more than 50% in close relationship with the diminution of the small antral follicular number and the establishment of multifollicular dominance.89,127,128 Therefore, in contrast to the physiological follicular phase where no significant change in plasma AMH levels is usually reported,127,129 a decrease in AMH level would be only observed in situations of small antral follicle excess such as PCOS or during controlled ovarian hyperstimulation in normal women undergoing IVF. Presumably, this decrease in plasma AMH levels might also result from the evolution of the unselected follicles to atresia.127 Accordingly, a contemporary decrease in the number of small follicles was observed at ultrasound in the IVF study.89

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Overall, these data indicate that measurement of plasma AMH levels is not actually useful during ovarian stimulation, except in studies designed to investigate the regulation of follicular development and arrest.

6.

Conclusions It appears from this review that the endocrine characteristics of ART cycles depend largely on the drugs used to achieve COS. It is clear that FSH therapy is mandatory in every stimulation, but assessment of FSH plasma values is not sufficiently predictive of the adequacy of FSH supply to be routinely determined. As far as plasma LH determinations are concerned, immunometric LH assays cannot properly reflect the bioactivity of the circulating residual LH following GnRH analog administration. Furthermore, there is no evidence that plasma LH measurements could help detect patients who might need the addition of some LH during ART cycles. Consequently, plasma LH determinations may be restricted to control of hypophyseal desensitization. Conversely, while there is a trend to minimize the cost of ART cycle monitoring and, in that respect, to pay special attention to ultrasound data during ovarian stimulation, it seems to us that a concomitant evaluation of estradiol secretion must be recommended to assess the secretory pattern of this hormone. Indeed, whether or not the plasma estradiol profile is relevant to the outcome of the cycle is still a matter of debate, especially with the recent use of GnRH antagonists. Thus, more information is needed before a definitive conclusion can be drawn. Similarly to LH, plasma progesterone determination may be restricted to the time of hypophyseal desensitization. Finally, while plasma androgens are not currently evaluated during ovarian stimulation, it may be questioned whether measuring their levels are worthwhile to indirectly assess the residual LH bioactivity in patients treated with GnRH analogs.

7.

8.

9.

10.

11.

12.

13.

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References 1. Short R. Steroids in the follicular fluid and the corpus luteum of the mare: a “two-cell type” theory of ovarian steroid synthesis. J Endocrinol 1962; 24: 59–63. 2. Messinis IE, Templeton AA. The importance of follicle-stimulating hormone increase for folliculogenesis. Hum Reprod 1990; 5: 153–6. 3. Brown J. Pituitary control of ovarian function – concepts derived from gonadotrophin therapy. Aust NZ J Obstet Gynaecol 1978; 18: 47–54. 4. Baird DT. A model for follicular selection and ovulation: lessons from superovulation. J Steroid Biochem 1987; 27: 15–23. 5. The European Recombinant Human LH Study Group. Recombinant human luteinizing hormone (LH) to support recombinant human follicle-stimulating hormone (FSH)-induced follicular development in LH

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39 The use of GnRH agonists Judith AF Huirne, Roel Schats

Introduction Gonadotropin-releasing hormone (GnRH) is the primary hypothalamic regulator of reproductive function. With the help of a very small amount (250 mg) of GnRH derived from 160 000 porcine hypothalami, a group of scientists at Andrew Schally’s peptide laboratory in New Orleans was able to unravel the chemical structure of this compound in 1971.1,2 Roger Guillemin was able to characterize and also synthesize independently this neuroendocrine hormone. They both received the Nobel prize for their achievement. GnRH is a decapeptide that, like several other brain peptides, is synthesized as a part of a much larger precursor peptide, the GnRH-associated peptide (GAP). This peptide is made up of a sequence of 56 amino acids. The availability of the synthetic hormone for dynamic endocrine testing and receptor studies created new insights into the physiological role of GnRH in the hypothalamic– pituitary–gonadal axis.3 GnRH is produced and released from a group of loosely connected neurons located in the medial basal hypothalamus, primarily within the arcuate nucleus, and in the preoptic area of the ventral hypothalamus. It is synthesized in the cell body, transported along the axons to the synapse, and released in a pulsatile fashion into the complex capillary net of the portal system of the pituitary gland.4 GnRH binds selectively to highly specific receptors of the anterior pituitary gonadotropic cells and activates intracellular signaling pathways via the coupled G proteins (Gαs), leading to the generation of several second messengers, among which are diacylglycerol and inositol-4,5-triphosphate. The former leads to activation of protein kinase C and the latter to the production of cyclic AMP and release of calcium ions from intracellular pools.5–7 Both events result in secretion and synthesis of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). A pulsatile mode of GnRH release from the hypothalamus to the pituitary is required to ensure gonadotropin secretion.8–10 In humans the pulsatile release frequencies range from the shortest interpulse frequency of about 71 minutes

in the late follicular phase to an interval of 216 minutes in the late luteal phase.11–13 High frequent (>3 pulses/hour) and continuous exposure of the pituitary to GnRH failed to produce normal LH and FSH release patterns,14–16 owing to pituitary desensitization. This mechanism is still not clear, except that postreceptor signaling is involved, true receptor loss (downregulation) having only an initial role.17 The pulsatile release by the GnRH neurons is hypothesized to be based on an ultrashort loop feedback by GnRH itself; this autocrine process could serve as a timing mechanism to control the frequency of pulsatile neurosecretion. Several mechanisms, based on calcium and cyclic AMP signaling, have been proposed to account for the pulse secretion. Another role of intracellular signaling in pulsatile generation has been suggested by the marked inhibition of Gi protein activation by LH, human chorionic gonadotropin (hCG), muscarine, E2, and GnRH levels.7,18,19 After the discovery of the chemical structure of native GnRH type I, which proved to be the classic reproductive neuroendocrine factor, many were synthetically produced. Most were able to elicit a huge FSH and LH release from the pituitary and were therefore called GnRH agonists. However, under continuous administration of a GnRH agonist, the normal synthesis and subsequent release of LH, and to a lesser extent FSH, became blocked (Fig 39.1). Other analogs caused an immediate fall in gonadotropin secretion from the pituitary by competitive receptor binding, and were designated GnRH antagonists. In contrast to the agonistic compounds, the introduction of the GnRH antagonists into clinical practice has been hampered for a long time by problems concerning solubility and direct allergy-like side effects due to histamine release.20,21 Recently, these problems have been resolved, leading to the third-generation GnRH antagonists: two are on the market and many others are under investigation.22 Nowadays, GnRH agonists have gained a wide field of clinical applications.23 Suppression of the pituitary ovarian (or testicular) axis for a limited or even an extended period is the main goal to be achieved in these treatments.

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26 24 22 20 18 16 14 12 10 8 6 4 2 0 Day

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LH

LHRH 0.5 mg/min RIA MLCA IRMA

1

8 6 4

37.0

bbt °C

0.8 0.6 0.4

5

7

9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65

3

5

7

9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65

3

5

7

9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65

3 FSH

2 0 Day

800

1

E2

600 400 200

0 Day

1

Fig 39.1 Hormone levels for FSH, LH, and estradiol (E2) in a patient with continuous intravenous infusion of 0.5 mg/min LHRH. LH was measured with three and FSH with two different assays (RIA, radioimmunoassays; MLCA, Magic Lite Chemoluminescence Assay; ISMA, Immunoradiometric assay). bbt, basal body temperature. (Private collection Professor J Schoemaker.)

Structural modifications The elucidation of the structure, function, and metabolic pathways of native GnRH has prompted an intensive effort by research laboratories and the pharmaceutical industry to synthesize potent and longer-acting agonists and antagonists.24 Over the past three decades, thousands of analogs of GnRH have been synthesized. Only seven of the agonistic analogs of GnRH have been approved and become clinically used drugs. The first major step in increasing the potency of GnRH was made with substitutions of glycine number 10 at the C terminus. Although 90% of the biologic activity is lost by splicing of glycine number 10, most of it is restored with the attachment of NH2-ethylamide to the proline at position 9, leading to nonapeptides.25 The second major modification was the replacement of the glycine at position 6 by D-amino acids, which slows down enzymatic degradation. The combination of these two modifications was found to

have synergistic biologic activity and proved to exhibit a higher receptor-binding affinity. The affinity can be increased further by the introduction of larger, hydrophobic and more lipophilic D-amino acids at position number 6. The increased lipophilicity of the agonist is associated with a prolonged half-life, which may be attributed to reduced renal excretion through increased plasma protein binding, or fat tissue storage of nonionized fat-soluble compounds.25 For details about the structure see Table 39.1.

Clinical applications The original goal for the development of agonistic analogs of GnRH was that they would eventually be used for the treatment of anovulation. However, soon after the elucidation of the structure of GnRH, the ‘paradoxical’ ability of agonistic analogs to inhibit reproductive function in experimental animals was

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Table 39.1

531

Amino acid sequence and substitution of the GnRH agonists

Compound Amino acid no Native GnRH

Position 6 1 Glu

2 His

3 Trp

4 Ser

5 Tyr

6 Gly

Position 10 7 Leu

8 Arg

9 Pro

10 GlyNH2

Nonapeptides Leuprolide (Lupron, Lucrin) Buserelin (Suprefact) Goserelin (Zoladex) Histrelin (Supprelin) Deslorelin (Ovuplant)

Leu Ser(O’Bu) Ser(O’Bu) D-His(Bzl) D-Tr

N-Et-NH2 N-Et-NH2 AzaGlyNH2 AzaGlyNH2 N-Et-NH2

Decapeptides Nafarelin (Synarel) Triptorelin (Decapeptyl)

2Nal Trp

GlyNH2 GlyNH2

demonstrated.26 The most important clinical applications of the potent GnRH agonists were derived from their capacity to cause rapid desensitization of the pituitary gland as a result of prolonged nonpulsatile administration, leading to a decrease in serum gonadotropin levels and subsequently inhibition of ovarian steroidogenesis and follicular growth. The potential for reversibly inducing a state of hypogonadotropic hypogonadism, which was also termed ‘medical gonadectomy’ or ‘medical hypophysectomy,’ allowed for the relatively rapid and extensive introduction of GnRH agonists into clinical practice. For a variety of indications, complete abolition of gonadotropin secretion with subsequent suppression of gonadal steroids to the levels of castrated subjects was considered beneficial. This therapeutic approach has already had its efficacy and merits proved in the treatment of metastatic prostatic cancer, breast cancer, central precocious puberty, external endometriosis, uterine fibroids, hirsutism, and other conditions.27,28 Since the first report on the use of the combination of the GnRH agonist buserelin and gonadotropins for ovarian stimulation for in vitro fertilization (IVF) in 1984,29 numerous studies have demonstrated the efficacy of this concept. Subsequently the use of GnRH agonists has gained widespread popularity, and the vast majority of assisted reproductive technology (ART) programs use this approach for controlled ovarian hyperstimulation (COS)–IVF. The major advantage initially offered by the agonists was the efficient abolition of the spontaneous LH surge.30 The incidence of premature LH surges and subsequent luteinization in cycles with exogenous gonadotropin stimulation, without the use of a GnRH agonist, was observed by several investigators to range between 20 and 50%, leading to an increased cancellation rate.31 Moreover, a deleterious effect on both fertilization and pregnancy rates was noted.30,32 A meta-analysis of randomized controlled trials has shown that the use of GnRH agonists has not only reduced cancellation rates but has also increased the number of oocytes and

embryos, allowing better selection33 so that, on average, the outcome in terms of pregnancy rates was improved.34 A number of controversial issues remain concerning the use of GnRH agonists in assisted reproduction. The problems can be divided into the following four categories: 1. 2. 3. 4.

Which route of administration is the best? Which agonist(s) should be used in ART? What is the optimal dose? What is the optimal scheme?

Which route of administration is the best? Administration routes of GnRH agonists are intramuscular or subcutaneous depot injection, intranasal or subcutaneous administration. Although there is an advantage for the patient in the usually single injection of the depot preparations, the duration of action is prolonged. The effect can last until the first weeks of pregnancy.35 Broekmans et al showed that rapid induction of a hypogonadotropic and hypogonadal state is possible in regularly cycling women by administration of a single depot of triptorelin. However, suppression of pituitary and ovarian function appears to be continued until the eighth week after the injection.35 This is far longer than is actually needed. Devreker et al found obvious negative effects of depot preparations: longer stimulation phase, consequently more ampules needed, but more importantly lower implantation and delivery rates (32.8% vs 21.1%; 48.9% vs 29.1%, respectively). Their conclusion was that as a long-acting GnRH agonist might interfere with the luteal phase and embryo development, short-acting GnRH agonists should be preferred in ART.36 Based on a recent meta-analysis comparing depot vs daily administration, it can be concluded that no evident differences could be observed in terms of pregnancy rates. However, the use of depot GnRH

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analogs is associated with increased gonadotropin requirements and longer stimulation periods and should therefore not be advocated in terms of costeffectiveness.37 Moreover, on a theoretical basis it seems to be more elegant to avoid any possible direct effect on the embryo, although several authors claim a normal outcome of pregnancy following inadvertent administration of a GnRH agonist during early pregnancy.38–43 Lahat et al reported a high incidence of attention deficit hyperactivity disorder (ADHD) in long-term follow-up of children inadvertently exposed to GnRH agonists early in pregnancy.44 Thus, although depot preparations seem attractive because of their ease of administration for the patient, they cannot be advocated for routine use in IVF. One exception to this statement might be the prolonged use of GnRH analogs before IVF–ET (embryo transfer) in patients with endometriosis, which seems to be associated with higher ongoing pregnancy rates.45 With the intranasal route the absorption of the GnRH agonist fluctuates inter- and intra-individually, giving an unpredictable desensitization level, but most times this is sufficient to prevent premature LH surges. For research or study purposes the daily subcutaneous injections deserve preference, because of their more stable effect. The clinician has to make up the balance between comfort for the patient and a more stable effect in selecting the intranasal vs the subcutaneous route of administration.

Which agonist(s) should be used in ART? In Table 39.1 seven GnRH agonists are mentioned. In fact, only four are commonly used in IVF programs. An extensive search revealed only one article about the use of histrelin in IVF,46 while deslorelin has never been applied in human IVF. Except for its combination with treatment of endometriosis, goserelin is not routinely used in ART, partly because it is only available as a depot preparation. Depot preparations also on the market for triptorelin and leuprolide are not to be used as first choice, as discussed earlier. Thirteen prospective randomized trials were traced in the literature comparing different agonists with each other.47–58 The problem with those studies is that the optimal dosage has not been determined for any of the applied individual agonists. Therefore, the value of these articles is limited with respect to elucidating the question as to which compound should be used. All the agonists seem effective and the differences in the studies can be explained by a dosage incompatibility. These studies make absolutely clear that proper dosefinding studies for the use of GnRH agonists in ART are still urgently needed. In fact, it is rather strange that they still have not been performed, more than 10 years after the introduction of the agonists in IVF. It is obvious that the dose required for the prevention of premature LH surges during COS cycles in ART will be different from that to treat carcinoma of the

prostate, which requires complete chemical castration (see below).

What is the optimal dose? Finding the right dose in the treatment of infertility disorders has been notoriously difficult for obscure reasons. Proper dose-finding studies for the use of gonadotropins are lacking and it therefore took until the middle of the 1980s before an adequate treatment protocol, with a maximum of effect and a minimum of side effects, was introduced by Polson et al.59 There is only one prospective, randomized, double-blind, placebo-controlled dose-finding study performed in IVF for the GnRH agonist triptorelin. This study demonstrated that the dosage needed for the suppression of the LH surge is much smaller, namely only 15–50% of the dosage needed for the treatment of a malignant disease.31 It is very likely that dose-finding studies for the other agonists will give similar results.

What is the optimal scheme? Many treatment schedules with the use of GnRH agonists in ART have been designed. The duration and initiation of agonist administration before the start of the actual ovarian stimulation varies widely. Initiation of the agonist treatment may be in either the early follicular or the midluteal phase of the preceding cycle. The cycle may be spontaneous or induced by progestogen and/or estrogen compounds. There is still much debate about the optimal GnRH agonist protocol. Tan published in 1994 a review article stating that the ‘long protocol’ was superior to the short and ultrashort protocols.60 Moreover, a major advantage of the long GnRH agonist protocol is its contribution to the planning of the ovum pick-up, since both the initiation of exogenous gonadotropins after pituitary desensitization and the administration of hCG can be delayed, without any detrimental effect on IVF outcome.61,62 A meta-analysis comparing ultrashort, short, and long IVF protocols showed a higher number of oocytes retrieved and higher pregnancy rates in the long protocol, although more ampules of gonadotropins were needed.63 In terms of gonadotropin suppression and number of retrieved oocytes, the midluteal phase of the preceding cycle is the optimal moment for the initiation of the GnRH agonist, in comparison to the follicular, early or late luteal phases.64–66 However, a problem with (prospective randomized) clinical studies is that certain groups of patients, for example the poor responders (with or without elevated basal FSH) or patients with polycystic ovary syndrome, are often excluded. There is a possibility that, especially in the excluded groups, other schemes are preferable. An unwanted side effect of starting the GnRH agonist in the luteal or follicular phase in the long protocol is the induction of the formation of functional cysts. Keltz

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et al observed both a poor stimulation outcome and a reduction in pregnancy rates in a cycle with cyst formation.67 However, Feldberg et al could not confirm this finding.68 Ovarian cyst formation was reduced when pretreatment with an oral contraceptive was applied.69 Damario et al showed the beneficial effect of this strategy in high responder patients with respect to cancellation rates and pregnancy rates.70 A long GnRH agonist protocol in combination with an oral contraceptive seems to be advantageous, in prevention of functional ovarian cysts and especially for the larger IVF centers for programming of IVF cycles. Another practical advantage of including an oral contraceptive is the fact that the coincidence of GnRH agonist use and early pregnancy is prevented. The mean desensitization phase with an agonist in the long protocols is about 3 weeks. Several investigators have tried to shorten this long duration of administration, leading to the so-called ‘early cessation protocol.’71–74 Increased human menopausal gonadotropin (hMG)/FSH requirement and cancellation rates were reported after early cessation in 137 normal IVF patients,74 but the opposite was found in a recent study which included 230 normo-ovulating IVF patients,71 although pregnancy rates were the same in both studies.74 The paradoxical drop of serum LH following early cessation, which leads to significantly lower estradiol levels on the day of hCG, may have a deleterious effect on IVF outcome.71,74 The early discontinuation protocol may improve ovarian response based on a hypothetical effect on the ovary, and was therefore additionally tested in poor responders. Although the number of retrieved oocytes was significantly higher and the amount of required gonadotropins was reduced after early cessation in comparison to the long protocol, this new approach reported no further advantages in these patients in terms of pregnancy and implantation rates.72,73 In conclusion, the currently available data do not favor an ‘early cessation’ protocol, but this approach might have some beneficial effects in poor responders. To prevent any detrimental effect of the profound suppression of circulating serum gonadotropins after cessation of GnRH agonist therapy, the opposite regimens have recently been developed in which the GnRH agonist administration is continued during the luteal phase, the so-called ‘continuous-long protocol.’ In a large prospective randomized study (n = 319) comparing this continued long protocol vs the standard long protocol, higher implantation and pregnancy rates were found in the continuous-long protocol.75 Since the use of a long protocol in poor responders has been found to result in reduced ovarian responses to hormonal stimulation, the short GnRH agonist protocol has been proposed as providing better stimulation for these patients. In the ‘short or flare-up protocol,’ GnRH agonist therapy is started at cycle day 2 and gonadotropins treatment is started 1 day later. The immediate stimulatory action of the GnRH agonist

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serves as the initial stimulus for follicular recruitment (so-called flare-up). Adequate follicular maturation is on average reached in 12 days, which should allow enough time for sufficient pituitary desensitization in order to prevent any premature LH surges. The initial stimulatory effect of GnRH agonist on pituitary hormone levels may improve the ovarian response.76 On the other hand, this short protocol might increase gonadotropins in the early phase, which induces enhanced ovarian androgen release. This is associated with declined oocyte quality and reduced ongoing pregnancy rates compared to the long protocol.77 Nevertheless, experience to date shows that the short protocol has an important role in the treatment of poor responders.78 Other investigators even promoted an ‘ultrashort protocol’ in ‘poor responders,’ in which the agonist is given during a period of 3 days in the early follicular phase. At the second day of agonist administration, stimulation with gonadotropin administration (high dosages) is started.79–82 In very high responders, in patients at risk of ovarian hyperstimulation syndrome (OHSS), gonadotropin was discontinued while continuing the GnRH agonist; this so-called ‘coasting’ might prevent the development of severe OHSS.83,84 This strategy allows a delay of a variable number of days in administering hCG injection until safe estradiol levels are attained. However, sufficient randomized controlled trials comparing coasting with no coasting are lacking.85 Only one prospective comparative trial in 60 IVF patients showed a similar incidence of moderate and severe OHSS whether coasting was applied or not.86 In Table 39.2 the most important advantages and disadvantages of the different GnRH agonist protocols are summarized. After the clinical availability of GnRH antagonists, an additional indication for the use of GnRH agonists became of interest. GnRH analogs may be used as an alternative way for hCG to trigger the endogenous LH and FSH surges and subsequent final maturation of the oocytes and ovulation.87,88 Since hCG is believed to contribute to the occurrence of OHSS owing to its prolonged circulating half-life time compared with native LH, this strategy seems to be an attractive alternative to prevent OHSS. In the early 1990s, it was already shown that single-dose GnRH agonists administrated in COH–IVF patients were able to induce an endogenous rise in both LH and FSH levels, leading to follicular maturation and pregnancy.89,90 Mean serum LH and FSH levels rose over 4–12 hours and were elevated for 24–34 hours after GnRH agonist, in comparison to approximately 6 days of elevated hCG levels after 5000 IU of hCG administration. The capacity of a single administration of GnRH analog to trigger follicular rupture in anovulatory women or in preparation for IUI has been well established. This seems to induce lower OHSS rates with comparable or even improved results, despite short luteal phases, in comparison to hCG cycles.87,88,91 Interest in this approach

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Table 39.2

Summary of advantages and disadvantages of the different GnRH agonist protocols

GnRH agonist protocol

Route of administration

Administration days of cycle (CD)

Duration of administration

Advantages

Disadvantages

Ultrashort protocol Short protocol

In/sc In/sc

3 days 8–12 days

Patient’s comfort Patient’s comfort

Low PR No programming

Long follicular

In/sc

28–35 days

Long luteal

In/sc

Menstrual early cessation Follicular early cessation Long follicular (depot)

In/sc

7–12 days

Programming, good PR Programming, good PR Inconclusive

Long duration of administration Long duration of administration Low estradiol levels

13–20 days

Inconclusive

Low estradiol levels

Depot

CD 2,3–4,5 CD 2,3 until day of hCG CD 2 until day of hCG CD 21 until day of hCG CD 21 until menses CD 21 until stim. day 6,7 CD 2

Once

Patient’s comfort

Long luteal (depot)

Depot

CD 21

Once

Patient’s comfort

Ultralong

In/sc/depot

CD 2 or 21

8–12 weeks, depot 2 or 3 times

Only for special cases

(Too) long duration of action (Too) long duration of action Side effects due to estrogen deficiency

In/sc

was lost during the 1990s, because GnRH agonists were introduced in ovarian hyperstimulation protocols to prevent premature luteinization by pituitary desensitization, precluding stimulation of the endogenous LH surge. However, interest has returned following the introduction of GnRH antagonist protocols in which the pituitary responsiveness is preserved. This new concept of triggering final oocyte maturation after GnRH antagonist treatment by a single GnRH agonist injection was successfully tested in COH patients for intrauterine insemination (IUI)92 and in high responders for IVF.93 None of these patients developed OHSS. The efficacy and success of this new treatment regimen was established in a prospective multicenter trial, in which 47 patients were randomized to receive either 0.2 mg of triptorelin, 0.5 mg of leuprorelin or 10 000 IU of hCG.94 The LH surges peaked at 4 hours after agonist administration and returned to baseline after 24 hours; the luteal phase steroid levels were also closer to the physiologic range compared to the hCG groups. In terms of triggering the final stages of oocyte maturation, similar outcomes were observed in all groups, as demonstrated by the similar fertilization rates and oocyte quality.94 A prospective randomized study in 105 stimulated IUI cycles treated with a GnRH antagonist, in patients with clomiphene-resistant polycystic ovary syndrome (PCOS), showed significantly more clinical pregnancies statistically after ovulation triggering by a GnRH agonist in comparison to hCG (28.2 vs 17% per completed cycle, respectively).95 Thus, this new approach of ovulation triggering seems to be an attractive alternative for hCG in ART if administered in GnRH antagonist-treated cycles, with lower OHSS and similar or improved IVF outcome.

21–28 days

Conclusions GnRH agonists are widely used in IVF to control the endogenous LH surge and achieve augmentation of multifollicular development. Disadvantages, such as the necessity for luteal support, increased total gonadotropin dose per treatment cycle and consequently higher costs, appear to be outweighed by the observed increase in ability to control the cycle, higher yield of good-quality oocytes and embryos, and consequent improvement of pregnancy rates. The introduction of GnRH agonists in IVF is not an example of excellent research, since proper dose-finding studies are still awaited. Further research in finding the right dose and protocol can still improve the clinical benefits of the GnRH agonists. Daily administered shortacting preparations deserve preference to the depot formulations. Intranasal administration best fits a patient’s comfort considerations, while the subcutaneous route may be advocated for research purposes. The long GnRH agonist protocols give the highest pregnancy rates in the normal responders. There is some evidence that the short flare-up protocol is the treatment of choice for patients with diminished ovarian reserve (poor responders). Dose reduction might be the key point in optimizing pregnancy rates. Finally, GnRH agonists can be used to induce final maturation and ovulation as an alternative to hCG in ART.

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37. Albuquerque LE, Saconato H, Maciel MC. Depot versus daily administration of gonadotrophin releasing hormone agonist protocols for pituitary desensitization in assisted reproduction cycles. The Cochrane Library 2003; Issue 1. Oxford: Update Software. 38. Weissman A, Shoham Z. Favourable pregnancy outcome after administration of a long-acting gonadotrophin-releasing hormone agonist in the mid-luteal phase. Hum Reprod 1993; 8: 496–7. 39. Balasch J, Martinez F, Jove I, et al. Inadvertent gonadotrophin-releasing hormone agonist (GnRHa) administration in the luteal phase may improve fecundity in in vitro fertilization patients. Hum Reprod 1993; 8: 1148–51. 40. Ron-El R, Lahat E, Golan A, et al. Development of children born after ovarian superovulation induced by long-acting gonadotropin-releasing hormone agonist and menotropins, and by in vitro fertilization. J Pediatr 1994; 125: 734–7. 41. Cahill DJ, Fountain SA, Fox R, et al. Outcome of inadvertent administration of a gonadotrophinreleasing hormone agonist (buserelin) in early pregnancy. Hum Reprod 1994; 9: 1243–6. 42. Gartner B, Moreno C, Marinaro A, et al. Accidental exposure to daily long-acting gonadotrophin-releasing hormone analogue administration and pregnancy in an in vitro fertilization cycle. Hum Reprod 1997; 12: 2557–9. 43. Taskin O, Gokdeniz R, Atmaca R, et al. Normal pregnancy outcome after inadvertent exposure to longacting gonadotrophin-releasing hormone agonist in early pregnancy. Hum Reprod 1999; 14: 1368–71. 44. Lahat E, Raziel A, Friedler S, et al. Long-term follow-up of children born after inadvertent administration of a gonadotrophin-releasing hormone agonist in early pregnancy. Hum Reprod 1999; 14: 2656–60. 45. Surrey ES, Silverberg KM, Surrey MW, et al. Effect of prolonged gonadotropin-releasing hormone agonist therapy on the outcome of in vitro fertilization– embryo transfer in patients with endometriosis. Fertil Steril 2002; 78: 699–704. 46. Ziegler D de, Cedars MI, Randle D, et al. Suppression of the ovary using a gonadotropin releasing-hormone agonist prior to stimulation for oocyte retrieval. Fertil Steril 1987; 48: 807–10. 47. Balasch J, Jove IC, Moreno V, et al. The comparison of two gonadotropin-releasing hormone agonists in an in vitro fertilization program. Fertil Steril 1992; 58: 991–4. 48. Poarinaud J, Oustry P, Perineau M, et al. Randomized trial of three luteinizing hormonereleasing hormone analogues used for ovarian stimulation in an in vitro fertilization program. Fertil Steril 1992; 57: 1265–8. 49. Penzias AS, Shamma FN, Gutmann JN, et al. Nafarelin versus leuprolide in ovulation induction for in vitro fertilization: a randomized clinical trial. Obstet Gynecol 1992; 79: 739–42. 50. Tapanainen J, Hovatta O, Juntunen K, et al. Subcutaneous goserelin versus intranasal buserelin for pituitary down-regulation in patients undergoing IVF: a randomized comparative study. Hum Reprod 1993; 8: 2052–5.

51. Dantas ZN, Vicino M, Balmaceda JP, et al. Comparison between nafarelin and leuprolide acetate for in vitro fertilization: preliminary clinical study. Fertil Steril 1994; 61: 705–8. 52. Goldman JA, Dicker D, Feldberg D, et al. A prospective randomized comparison of two gonadotrophinreleasing hormone agonists, nafarelin acetate and buserelin acetate, in in vitro fertilization–embryo transfer. Hum Reprod 1994; 9: 226–8. 53. Tarlatzis BC, Grimbizis G, Pournaropoulos F, et al. Evaluation of two gonadotropin-releasing hormone (GnRH) analogues (leuprolide and buserelin) in short and long protocols for assisted reproduction techniques. J Assist Reprod Genet 1994; 11: 85–91. 54. Lockwood GM, Pinkerton SM, Barlow DH. A prospective randomized single-blind comparative trial of nafarelin acetate with buserelin in long-protocol gonadotrophin-releasing hormone analogue controlled in vitro fertilization cycles. Hum Reprod 1995; 10: 293–8. 55. Tanos V, Friedler S, Shushan A, et al. Comparison between nafarelin acetate and D-Trp6–LHRH for temporary pituitary suppression in in vitro fertilization (IVF) patients: a prospective crossover study. J Assist Reprod Genet 1995; 12: 715–19. 56. Oyesanya OA, Teo SK, Quah E, et al. Pituitary downregulation prior to in vitro fertilization and embryo transfer: a comparison between a single dose of Zoladex depot and multiple daily doses of Suprefact. Hum Reprod 1995; 10: 1042–4. 57. Avrech OM, Goldman GA, Pinkas H, et al. Intranasal nafarelin versus buserelin (short protocol) for controlled ovarian hyperstimulation before in vitro fertilization: a prospective clinical trial. Gynecol Endocrinol 1996; 10: 165–70. 58. Corson SL, Gutmann JN, Batzer FR, et al. A doubleblind comparison of nafarelin and leuprolide acetate for down-regulation in IVF cycles. Int J Fertil Menopausal Stud 1996; 41: 446–9. 59. Polson DW, Mason HD, Saldahna MB, et al. Ovulation of a single dominant follicle during treatment with low-dose pulsatile follicle stimulating hormone in women with polycystic ovary syndrome. Clin Endocrinol (Oxf) 1987; 26: 205–12. 60. Tan SL. Luteinizing hormone-releasing hormone agonists for ovarian stimulation in assisted reproduction. Curr Opin Obstet Gynecol 1994; 6: 166–72. 61. Chang SY, Lee CL, Wang ML, et al. No detrimental effects in delaying initiation of gonadotropin administration after pituitary desensitization with gonadotropin-releasing hormone agonist. Fertil Steril 1993; 59: 183–6. 62. Dimitry ES, Oskarsson T, Conaghan J, et al. Beneficial effects of a 24 h delay in human chorionic gonadotrophin administration during in vitro fertilization treatment cycles. Hum Reprod 1991; 6: 944– 6. 63. Daya S. Gonadotrophin-releasing hormone agonist protocols for pituitary desensitization in in vitro fertilization and gamete intrafallopian transfer cycles. The Cochrane Library 2001; Issue 1. Oxford: Update Software. 64. Pellicer A, Simon C, Miro F, et al. Ovarian response and outcome of in vitro fertilization in patients

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65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

treated with gonadotrophin-releasing hormone analogues in different phases of the menstrual cycle. Hum Reprod 1989; 4: 285–9. Kondaveeti-Gordon U, Harrison RF, Barry-Kinsella C, et al. A randomized prospective study of early follicular or midluteal initiation of long protocol gonadotropin-releasing hormone in an in vitro fertilization program. Fertil Steril 1996; 66: 582–6. San Roman GA, Surrey ES, Judd HL, et al. A prospective randomized comparison of luteal phase versus concurrent follicular phase initiation of gonadotropin-releasing hormone agonist for in vitro fertilization. Fertil Steril 1992; 58: 744–9. Keltz MD, Jones EE, Duleba AJ, et al. Baseline cyst formation after luteal phase gonadotropin-releasing hormone agonist administration is linked to poor in vitro fertilization outcome. Fertil Steril 1995; 64: 568–72. Feldberg D, Ashkenazi J, Dicker D, et al. Ovarian cyst formation: a complication of gonadotropinreleasing hormone agonist therapy. Fertil Steril 1989; 51: 42–5. Biljan MM, Mahutte NG, Dean N, et al. Pretreatment with an oral contraceptive is effective in reducing the incidence of functional ovarian cyst formation during pituitary suppression by gonadotropinreleasing hormone analogues. J Assist Reprod Genet 1998; 15: 599–604. Damario MA, Barmat L, Liu HC, et al. Dual suppression with oral contraceptives and gonadotrophin releasing-hormone agonists improves in vitro fertilization outcome in high responder patients. Hum Reprod 1997; 12: 2359–65. Cédrin-Durnerin I, Bidart JM, Robert P, et al. Consequences on gonadotrophin secretion of an early discontinuation of gonadotrophin-releasing hormone agonist administration in short-term protocol for in vitro fertilization. Hum Reprod 2000; 15: 1009–14. Dirnfeld M, Fruchter O, Yshai D, et al. Cessation of gonadotropin-releasing hormone analogue (GnRH-a) upon down-regulation versus conventional long GnRH-a protocol in poor responders undergoing in vitro fertilization. Fertil Steril 1999; 72: 406–11. Garcia-Velasco JA, Isaza V, Requena A, et al. High doses of gonadotrophins combined with stop versus non-stop protocol of GnRH analogue administration in low responder IVF patients: a prospective, randomized, controlled trial. Hum Reprod 2000; 15: 2292–6. Fujii S, Sagara M, Kudo H, et al. A prospective randomized comparison between long and discontinuous-long protocols of gonadotropin-releasing hormone agonist for in vitro fertilization. Fertil Steril 1997; 67: 1166–8. Fujii S, Sato S, Fukui A, et al. Continuous administration of gonadotrophin-releasing hormone agonist during the luteal phase in IVF. Hum Reprod 2001; 16: 1671–5. Padilla SL, Dugan K, Maruschak V, et al. Use of the flare-up protocol with high dose human follicle stimulating hormone and human menopausal gonadotropins for in vitro fertilization in poor responders. Fertil Steril 1996; 65: 796–9.

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77. Loumaye E, Coen G, Pampfer S, et al. Use of a gonadotropin-releasing hormone agonist during ovarian stimulation leads to significant concentrations of peptide in follicular fluids. Fertil Steril 1989; 52: 256–63. 78. Fasouliotis SJ, Simon A, Laufer N. Evaluation and treatment of low responders in assisted reproductive technology: a challenge to meet. J Assist Reprod Genet 2000; 17: 357–73. 79. Acharya U, Irvine S, Hamilton M, et al. Prospective study of short and ultrashort regimens of gonadotropin-releasing hormone agonist in an in vitro fertilization program. Fertil Steril 1992; 58: 1169–73. 80. Scott RT, Navot D. Enhancement of ovarian responsiveness with microdoses of gonadotropinreleasing hormone agonist during ovulation induction for in vitro fertilization. Fertil Steril 1994; 61: 880–5. 81. Feldberg D, Farhi J, Ashkenazi J, et al. Minidose gonadotropin-releasing hormone agonist is the treatment of choice in poor responders with high folliclestimulating hormone levels. Fertil Steril 1994; 62: 343–6. 82. Surrey ES, Bower J, Hill DM, et al. Clinical and endocrine effects of a microdose GnRH agonist flare regimen administered to poor responders who are undergoing in vitro fertilization. Fertil Steril 1998; 69: 419–24. 83. Sher G, Zouves C, Feinman M, et al. ‘Prolonged coasting’: an effective method for preventing severe ovarian hyperstimulation syndrome in patients undergoing in vitro fertilization. Hum Reprod 1995; 10: 3107–9. 84. Fluker MR, Hooper WM, Yuzpe AA. Withholding gonadotropins (“coasting”) to minimize the risk of ovarian hyperstimulation during superovulation and in vitro fertilization–embryo transfer cycles. Fertil Steril 1999; 71: 294–301. 85. D’Angelo A, Amso N. “Coasting” (withholding gonadotrophins) for preventing ovarian hyperstimulation syndrome. Cochrane Database Syst Rev 2002; (3): CD002811. 86. Egbase PE, Sharhan MA, Grudzinskas JG. Early unilateral follicular aspiration compared with coasting for the prevention of severe ovarian hyperstimulation syndrome: a prospective randomized study. Hum Reprod 1999; 14: 1421–5. 87. Emperaire JC, Ruffie A. Triggering ovulation with endogenous luteinizing hormone may prevent the ovarian hyperstimulation syndrome. Hum Reprod 1991; 6: 506–10. 88. Lanzone A, Fulghesu AM, Villa P, et al. Gonadotropin-releasing hormone agonist versus human chorionic gonadotropin as a trigger of ovulation in polycystic ovarian disease gonadotropin hyperstimulated cycles. Fertil Steril 1994; 62: 35–41. 89. Gonen Y, Balakier H, Powell W, et al. Use of gonadotropin-releasing hormone agonist to trigger follicular maturation for in vitro fertilization. J Clin Endocrinol Metab 1990; 71: 918–22. 90. Itskovitz J, Boldes R, Levron J, et al. Induction of preovulatory luteinizing hormone surge and prevention of ovarian hyperstimulation syndrome by gonadotropin-releasing hormone agonist. Fertil Steril 1991; 56: 213–20.

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91. Romeu A, Monzo A, Peiro T, et al. Endogenous LH surge versus hCG as ovulation trigger after low-dose highly purified FSH in IUI: a comparison of 761 cycles. J Assist Reprod Genet 1997; 14: 518–24. 92. Olivennes F, Fanchin R, Bouchard P, et al. Triggering of ovulation by a gonadotropin-releasing hormone (GnRH) agonist in patients pretreated with a GnRH antagonist. Fertil Steril 1996; 66: 151–3. 93. Itskovitz-Eldor J, Kol S, Mannaerts B. Use of a single bolus of GnRH agonist triptorelin to trigger ovulation after GnRH antagonist ganirelix treatment in women undergoing ovarian stimulation for assisted reproduction, with special reference to the prevention of ovarian

hyperstimulation syndrome: preliminary report: short communication. Hum Reprod 2000; 15: 1965–8. 94. Fauser BC, de Jong D, Olivennes F, et al. Endocrine profiles after triggering of final oocyte maturation with GnRH agonist after cotreatment with the GnRH antagonist ganirelix during ovarian hyperstimulation for in vitro fertilization. J Clin Endocrinol Metab 2002; 87: 709–15. 95. Egbase PE, Grudzinskas JG, Al Sharhan M, Ashkenazi L. HCG or GnRH agonist to trigger ovulation in GnRH antagonist-treated intrauterine insemination cycles: a prospective randomized study. Hum Reprod 2002; 17: 2–O–006.

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40 GnRH antagonists Michael Ludwig

Introduction Ovarian stimulation has a long history, which goes back for more than 80 years. It started with the control of follicular growth – either to induce follicular growth in otherwise anovulatory cycles, or to induce multifollicular growth to increase the chance of conception.1 In the 1980s, the first reports were published on the use of gonadotropin-releasing hormone (GnRH) agonists.2 From then on, it was possible not only to control folliculogenesis but also to control pituitary function: to suppress endogenous luteinizing hormone (LH) for prevention of premature LH surges. This allowed, for the first time, an optimal timing of ovulation induction with human chorionic gonadotropin (hCG) and oocyte pick-up. Originally, GnRH analogs were designed with the idea of a substance that binds to pituitary GnRH receptors and blocks GnRH action at this site. GnRH agonists, however, still have an intrinsic action and therefore lead to pituitary suppression after only a short period of LH and follicle-stimulating hormone (FSH) release – the so-called flare-up effect. It took nearly 10 years more to develop substances, called GnRH antagonists, which were able to block pituitary function without any intrinsic effect, and which could be used for clinical studies in the field of ovarian stimulation.

The development of GnRH antagonists The initial problem with these substances was their histamine release capability, and hence the problem of allergic reactions. When this problem was solved,

the drugs could not be reconstituted because of their low solubility.3 Finally, four drugs were developed which were used for preclinical and clinical studies in ovarian stimulation for infertility treatment: 1. 2. 3. 4

Nal-Glu. Cetrorelix (Cetrotide, Serono International SA, Geneva, Switzerland). Ganirelix (Orgalutran/Antagon, Organon, Oss, The Netherlands). Antide.

Since 1999, two of them, cetrorelix and ganirelix, have been commercially available. The biochemical structure of the drugs is shown in Table 40.1, and shows the high complexity of changes that needed to be made – compared to e.g. the GnRH agonist triptorelin – to achieve the goal of antagonistic actions.

GnRH antagonists in clinical studies Introduction This chapter will focus mainly on two GnRH antagonists – cetrorelix and ganirelix. These are the only preparations available on the market at the present time. They are different: cetrorelix has two different dosages (Cetrotide 0.25 mg, Cetrotide 3 mg) and ganirelix only one (0.25 mg Orgalutran/Antagon). Therefore, cetrorelix can be used in two different protocols – the single-dose and the multiple-dose protocols – ganirelix only in the multiple-dose protocol.

Table 40.1 Structure of GnRH and their analogs. To have agonistic actions only changes on positions 6 and 10 had to be made (triptorelin). For the GnRH antagonists, the GnRH molecule has to be changed in a much more sophisticated way Name

Amino acid sequence

GnRH Triptorelin Nal-Glu Antide Cetrorelix Ganirelix

pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2 pGlu-His-Trp-Ser-Tyr-DTrp-Leu-Arg-Pro-GlyNH2 NACD2Nal-D4C7Phe-D3Pal-Ser-Arg-DGlut(AA)-Leu-Arg-Pro-DAlaNH2 NACD2Nal-D4ClPhe-D3Pal-Ser-Lys(Nic)-DDLys(Nic)-Leu-Lys(Isp)Pro-DAlaNH2 NACD2Nal-D4ClPhe-D3Pal-Ser-Tyr-DCit-Leu-Arg-Pro-DAlaNH2 NACD2Nal-D4ClPhe-D3Pal-Ser-Tyr-DHarg(Et2)-Leu-Harg(Et2)-Pro-DAlaNH2

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75 IU gonadotropins Cetrotide 3 mg

hCG

Oocyte pick-up

Embryo transfer

Cetrotide 0.25 mg Orgalutran/Antagon

2 2 4 6 8 10 12 Days of spontaneous menstrual cycle

Fig 40.1 The single-dose GnRH antagonist protocol. Ovarian stimulation using gonadotropins starts on day 2 or 3 of the spontaneous menstrual cycle. In a fixed protocol the GnRH antagonist is administered in a single dose on day 7 of gonadotropins. A single injection of Cetrotide 3 mg lasts for 96 hours to prevent a premature LH surge. In case that hCG cannot be administered within this time frame, additional dosages of Cetrotide 0.25 mg should be given daily.

Some data on Nal-Glu will also be presented, since it was the first GnRH antagonist to be used in ovarian stimulation protocols. Antide was used in an important study regarding luteal phase support, and therefore will also be discussed. The idea with the development of GnRH antagonists was to have an ovarian stimulation protocol available which is as close to the normal cycle as possible. This could not be achieved with the GnRH agonist long protocol since the normal cycle was switched off by this procedure. Other ways to use GnRH agonists, such as the short protocol, used the normal menstrual cycle; these protocols, however, have been shown to be less effective than the long protocol.4 The most convenient way for the patient is to start with spontaneous menstrual bleeding, using the natural resources and endogenous gonadotropins as efficiently as possible and suppressing pituitary function as late as necessary. To reach this goal, different protocols have been developed over the years in parallel, the singledose and the multiple-dose GnRH antagonist protocols.

The single-dose protocol The single-dose protocol uses a high dose of cetrorelix as an intermediate pseudo-depot preparation; this leads to suppression of pituitary function for a couple of days (Fig 40.1). In 1991 a French group published their results with an ovarian stimulation protocol using clomiphene citrate from cycle days 2 to 6 and an overlapping gonadotropin stimulation with human menopausal gonadotropin (hMG) on days 4, 6, and 8. Gonadotropins were individualized from day 9 onwards. On an individual basis, 5 mg of the GnRH antagonist Nal-Glu was administered, when estradiol levels exceeded 600 pg/ml. This was repeated 48 hours later in case hCG could not be administered up to that point.5 A couple of years later a series of 17 patients were treated using gonadotropins and 5 mg of the GnRH

4

6

8

Oocyte pick-up

hCG

10

Embryo transfer

12

Days of spontaneous menstrual cycle

Fig 40.2 The multiple-dose GnRH antagonist protocol. Ovarian stimulation using gonadotropins starts on day 2 or 3 of the spontaneous menstrual cycle. In a fixed protocol the GnRH antagonist is administered for the first time on day 6 of gonadotropins. This is continued up to and including the day of hCG.

antagonist cetrorelix. In cases where estradiol, in follicles of more than 14 mm diameter, exceeded 150–200 pg/ml, the GnRH antagonist was administered. A second injection was carried out 48 hours later since no hCG could be administered up to that time.6 The same group tried to simplify this approach by fixing the administration of 3 mg cetrorelix on day 8 of the cycle. This was trialed in a series of 11 patients. A second injection of the GnRH antagonist was scheduled for 72 hours later, if necessary.7 However, it was observed that only those patients who had low estradiol levels on day 8 needed a second dose of cetrorelix. Already in that publication it was proposed that tailoring of GnRH antagonist administration would have avoided the necessity for second dose administration. Finally, the group performed a dose-finding study and could identify 3 mg cetrorelix as the minimal effective dose in the single-dose GnRH antagonist protocol. In cases where a 2 mg dose was given, premature LH surges were observed.8

The multiple-dose protocol When the first results with the single-dose protocol were published,5 others tried to use a multiple-dose approach with Nal-Glu in 10 healthy volunteers.9 After administration of Nal-Glu during 4 days in the late follicular phase, Dittkoff et al observed a stop in follicular growth, a drop in estradiol levels – and a suppression of endogenous LH. This first publication led to the development of the multiple-dose GnRH antagonist protocol (Fig 40.2). In a first series for clinical application 20 patients were treated for an in vitro fertilization (IVF) procedure with multiple doses of 3 mg and 1 mg of cetrorelix. No LH surge was observed.10 In a second dose-finding series with a nonrandomized approach, doses of 3 mg, 1 mg, and 0.5 mg of cetrorelix were used in parallel. Again, no premature LH surge was observed.11 In another randomized dose-finding study with 0.5 mg, 0.25 mg, and 0.1 mg cetrorelix, a dose of 0.25 mg was identified as the minimal effective dose in the multiple-dose protocol.12

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For ganirelix, one dose-finding study used a worldwide, multicentric, prospective randomized approach comparing 2 mg, 1 mg, 0.5 mg, 0.25 mg, 0.125 mg and 0.0625 mg in a multiple-dose GnRH agonist protocol. For this agent, 0.25 mg daily was identified as the minimal effective dose.13

Table 40.2 Results of the meta-analysis which compared the GnRH antagonist protocols and the GnRH agonist long protocol. Shown are odds ratios (OR) and 95% confidence intervals (CI)

GnRH antagonist protocols vs GnRH agonist long protocol Following the successful treatment of several series of patients in the dose-finding studies, it could be assumed that a successful protocol had been developed. An open, noncontrolled prospective multicentric study included 322 patients with oocyte pick-up, treated in the multiple-dose protocol with Cetrotide 0.25 mg. In this study about 75% of oocytes had a metaphase II status, fertilization rate was 59.2%, and in 92.2% embryo transfer could be performed. The clinical pregnancy rate per embryo transfer was 23.6%, and in 19.5% per embryo transfer ongoing pregnancies could be registered.14 All these results led to the conclusion that the use of GnRH antagonist leads to protocols which were ready for daily clinical use. For a direct comparison, however, only prospective, randomized studies are helpful. These should compare the new protocol with the established gold standard, which is used most widely in the world – the GnRH agonist long protocol. Several of these studies have been done so far.15–21 Two early meta-analyses attempted to look for the common result of these studies; both came to quite similar conclusions.22,23 The main results were: 1.

2.

A significantly lower pregnancy rate for all studies together,22,23 which can be confirmed by both meta-analyses in the case of ganirelix,22,23 but not for cetrorelix.23 A significantly lower risk of ovarian hyperstimulation syndrome (OHSS) overall and OHSS °III,22,23 which can be confirmed for cetrorelix alone;23 for ganirelix the risk seems not to be reduced compared to the GnRH long protocol results.22,23

The risk of a premature LH surge seems somewhat higher in the GnRH antagonist protocols. This difference was not significant but may become significant in case of a higher numbers of patients. Furthermore, the length of ovarian stimulation was shortened by about 1 day (odds ratio [OR] = −1.12; 95% confidence interval [CI] −1.45 to −0.80).22 A significantly lower number of gonadotropin ampules have to be used in case of the GnRH antagonist protocols (OR = 3.34; 95% CI 5.21–1.47).22 Meanwhile, more studies have been published and a recent meta-analysis has summarized all these results.24 This study included 22 published studies with 3176 subjects treated in either the GnRH agonist

541

Parameter

OR

95% CI

Live birth rate Miscarriage rate OHSS (hospital admission) OHSS °III LH surge

0.86 1.03 0.46 0.47 4.05

0.72–1.02 0.52–2.04 0.26–0.82 0.18–1.25 1.53–10.72

Data according to Kolibianakis et al.24

long protocol or the GnRH antagonist protocols (Table 40.2). Additionally to the data shown in the table, the number of cumulus–oocyte–complexes was significantly lower in the GnRH antagonist protocol as compared to the GnRH agonist protocol (−1.19, 95% CI −1.82 to −0.56). Two problems have to be discussed with respect to the observed findings: • •

How can the lower risk of OHSS be explained? Why is there a difference overall regarding pregnancy rates?

Regarding OHSS, there are two more interesting observations made by both meta-analyses. First, the number of retrieved oocytes was significantly lower. Furthermore, the estradiol levels on the day of hCG were lower in the GnRH antagonist cycles, as reported in two earlier meta-analysis, with a difference of ~570 pg/ml (95% CI ~662–477)22 and ~650 pg/ml (95% CI ~743–557),23 respectively. Both results are consistent with a reduction in the risk of OHSS, since the number of oocytes retrieved as well as the estradiol levels are well-known risk factors for the development of an OHSS. In another paper Ludwig et al, have also shown a more physiologic way of follicular maturation in GnRH antagonist protocols, with the development of less small follicles as compared to the GnRH agonist long protocol.25 Regarding pregnancy rates, the discussion is more complicated. In fact, the most recent meta-analysis by Kolibianakis et al24 shows no significant difference between the protocols at all; however, this may be just a matter of time and the inclusion of more subjects from future studies. There might be a difference between the two compounds, which by pharmacokinetic mechanisms leads to differences in follicular maturation and/or endometrial growth and differentiation. Comparable data for this theory are not yet available. However, the data from the ganirelix dosefinding study might point in this direction, since with the highest doses of ganirelix the pregnancy rates dropped.13 On the other hand, with higher doses of cetrorelix in the dose-finding studies, satisfying pregnancy rates were achieved.10,11

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An important difference between the studies, however, is that all ganirelix studies were done exclusively with recombinant human (rh) FSH, but two of three cetrorelix studies were done with hMG (Table 40.2). A suppression of endogenous LH production during follicular maturation might therefore be the mechanism responsible for these observations. There is one large study, which was done using a single 3 mg dose of cetrorelix and rhFSH, which does not support this suggestion.21 In that study, pregnancy rates in the GnRH antagonist and GnRH agonist groups were quite comparable to each other (OR = 1.00, 95% CI 0.69–1.46):21 if there is a negative effect from the use of rhFSH in a GnRH antagonist protocol – due to the missing LH activity – it should be observed after administration of a 3 mg dose even more compared to the lower dosages in the multiple-dose protocols. The main difference between this study and the others was that the GnRH antagonist was started on an individual basis – tailored to the individual patient’s response, with a follicular diameter of 14 mm. Most other studies started the GnRH antagonist in a fixed protocol, which might, in some cases, lead to an early suppression of endogenous LH and thereby to detrimental effects on follicular growth and the endometrium, thereby explaining the slightly reduced pregnancy rate. Further studies have to be done to evaluate this question further. Some data are already included in this chapter (see below). For the moment, however, one might conclude that the GnRH antagonist protocols provide: • •

•

A safer way of ovarian stimulation, with fewer cases of OHSS. A comparable and satisfying pregnancy rate, especially in the case of an individualized approach. The possibility of using rhFSH as well as hMG for ovarian stimulation.

Daily use of GnRH antagonists in clinical practice At present (e.g. in Germany) about 20% of cycles for IVF treatment are done using the GnRH antagonist protocols. Some centers have already switched their standard protocol to the GnRH antagonists, and perform this in more than 90% of their treatment cycles. Two studies are available at the moment, which compare – after switching from the GnRH agonist long protocol to the GnRH antagonist protocol – the results of the two protocols in a larger series of patients.26,27 The results of these comparison studies are shown in Tables 40.3 and 40.4. Both groups could demonstrate a similar outcome of treatment – one more proof for the reliability of these protocols.

Table 40.3 Use of Cetrotide 0.25 mg in a multiple-dose protocol in daily clinical practice. The data were compared to data from the long agonist protocol cycles performed in the same year (1999). The authors switched their routine stimulation procedure from the long agonist to the multipledose antagonist protocol in May that year. No statistics were applied Cetrotide 0.25 mg Long agonist multiple-dose protocol protocol Cycles (n) Embryo transfers (n) Male infertility Cumulative embryo score∗ Embryos per transfer Clinical pregnancy rate Ongoing pregnancy rate

136 134 51.5% 22.66 ± 14.05

348 344 63.1% 23.00 ± 13.20

2.43 ± 0.67 20.1% 17.2%

2.52 ± 0.88 19.2% 16.6%

∗

mean ± standard deviation. Data from Ludwig et al.26

Tailoring GnRH antagonists to individual patients’ needs Introduction To respect individual patient needs is always an integral part of ovarian stimulation protocols. The process starts with the choice of the optimal protocol for the individual patient and proceeds with the choice of a certain gonadotropin starting dose. A dose adaptation may be carried out during gonadotropin administration and right up to the point when the decision is made to give hCG for final follicular maturation and ovulation induction. Tailoring to individual patient needs has to be learned – and new protocols might add new aspects of tailoring, which may not previously have been possible with other protocols. Some of these aspects will be discussed in the following paragraphs.

Use of an oral contraceptive pill for programming Originally, with the GnRH agonist long protocol, the use of an oral contraceptive pill had different indications: •

• •

•

Induction of menstrual bleeding in cases of oligo- or amenorrhea to provide an optimal endometrial quality and fix the day of GnRH agonist administration. Programming the cycle to schedule oocyte pick-up and embryo transfer in a more flexible way. Suppression of endogenous LH before start of treatment in cases of high basal LH, as for example, with polycystic ovary syndrome. Suppression of endogenous LH and FSH to avoid ovarian cyst formation prior to gonadotropin initiation.

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Table 40.4 Routine use of the Cetrotide 3 mg single-dose protocol in daily clinical practice. Data from a retrospective case–control study. Controls were matched according to age, basal FSH, and number of previous trials

Cycles (n) Age (years) Basal FSH (U/l) Number of trials Duration of stimulation (days) Estradiol level (pg/ml) Number of oocytes Clinical pregnancy rate (%) Ongoing pregnancy rate (%)

Cetrotide 3 mg single-dose protocol

Long agonist protocol

219 33.4 ± 3.7 6.2 ± 1.5 2.6 ± 1.5 12.1 ± 2.1 2163 ± 998 9.6 ± 4.9 26.0% 19.6

219 33.4 ± 3.9 6.2 ± 1.3 2.4 ± 1.5 11.6 ± 1.6 2587 ± 952 10.6 ± 5.1 27.8% 22.4

p

< 0.01 0.04

Data are mean ± standard deviation if not otherwise defined. Data according to Samama et al.27

Table 40.5 Use of an oral contraceptive pill in a prospective, randomized study before starting a multiple-dose GnRH antagonist protocol Oral contraceptive pill No programming Patients Ampules FSH Days FSH Mean number of oocytes Mean number of embryos transferred Clinical pregnancies/ embryo transfer

75 22.13 ± 6.5 11.4 ± 2.7 5.8

75 20.1 ± 4.7 10.62 ± 2.0 6.3

2.5

2.3

39.7%

41.2%

Data according to Obruca et al.28

It was suggested that cycle programming might be different with the use of a GnRH antagonist protocol. In a recent prospective, randomized study, however, comparable results were achieved with and without an oral contraceptive pill (Table 40.5).28 In another recent prospective randomized study,29 however, the authors described after pretreatment with an oral contraceptive pill: • • •

a lower concentration of FSH, LH, and estradiol when starting the cycle a longer stimulation period (11.6 vs 8.7 days) more oocytes retrieved (13.5 vs 10.2).

It might be – but future studies have again to address this question – that pretreatment with an oral contraceptive pill could help to overcome some disadvantages of the GnRH antagonist protocols. Cycle programming might have more advantages with respect to endogenous LH levels, which are discussed in conjunction with endometrial quality.

Tailoring to body weight Data regarding cetrorelix plasma and follicular fluid levels have shown that these levels are quite low. The

substance disappears from circulation within a few hours or days.30 Therefore, it was discussed whether in obese women the minimal effective dose of a GnRH antagonist might be too low, and on the other hand, in lean women too high. For ganirelix this individualization of dosage has been integrated in the approval for clinical use. Regarding cetrorelix, a recent evaluation of available data could not confirm any correlation of body weight and cycle outcome in either the multiple-dose or the single-dose protocol.31 Tailoring to body weight, therefore, may be necessary in ganirelix but not in cetrorelix cycles.

Tailoring to individual ovarian response In the very beginning especially, the studies for ovarian stimulation using the GnRH antagonist single-dose protocol were done with respect to individual ovarian responsiveness. The single dose of cetrorelix was administered when a certain estradiol level5 or a certain follicular diameter, or both, was achieved.6 Already in the mid-1990s it was suggested that tailoring the administration of the GnRH antagonist to individual patient needs would avoid unnecessary injections.7 Thus we performed a prospective, randomized study to compare a fixed multiple-dose protocol to a flexible, tailored multiple-dose protocol and a flexible, tailored single-dose protocol.32 Patients were treated with a starting dose of 150 IU rhFSH, and monitoring started on day 6 of the cycle. In the flexible protocols the GnRH antagonist was administered in a multiple- or single-dose approach when the leading follicle reached a diameter of 14 mm. The study was powered for the number of Cetrotide 0.25 mg vials needed in the two protocols – the flexible and the fixed – as well as for the number of monitoring visits. For both end parameters, a difference of 2 was assumed to be of clinical relevance. Results of this study are shown in Table 40.6. It becomes apparent that by using the flexible, tailored approach the same number of monitoring visits was

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Table 40.6 Results of a prospective, randomized study to compare a flexible and a fixed approach for the use of GnRH antagonists in ovarian stimulation for IVF. For a better overview, only the results for the multiple-dose protocols are shown. In the tailored, flexible protocol the GnRH antagonist was started with a follicle size of 14 mm. In the fixed protocol, the standard approach was used (start day 6 of gonadotropins) Group Patients with embryo transfer Number of monitoring visits Total number of Cetrotide vials (0.25 mg) Total IU of rhFSH Increase of rhFSH dose with start of Cetrotide (% of cycles) (n) Estradiol on hCG day (pg/ml) Number of COC retrieved Positive fetal heartbeats beyond 12 weeks of gestation (ongoing pregnancies) (n)

Fixed protocol

Flexible, tailored protocol

18 2.83 ± 0.77 6.81 ± 1.61 2232 ± 624 40 (8)

18 2.43 ± 0.61 4.59 ± 1.651 1838 ± 5762 6 (12)

1041 ± 459 6.15 ± 4.18³ 3

1737 ± 11603 10.97 ± 7.07 4

1

p <0.05. p <0.01. 3 p = 0.046. Data according to Ludwig et al.32 2

necessary. However, the number of Cetrotide 0.25 mg vials was significantly reduced by about 2, and significantly less rhFSH was needed. The latter was due to the fact that more often the dose was increased in the fixed protocol (6% vs 40%). However, despite this higher dose of gonadotropins, significantly higher levels of estradiol were achieved and significantly more oocytes could be retrieved by oocyte pick-up (10.97±7.07 vs 6.15±4.18, p=0.046) (Table 40.7). The study could not be used to estimate the pregnancy rate, or to give an idea whether pregnancy rates could be increased by that approach, as the numbers were too small. However, the study clearly demonstrated that an improved ovarian response could be obtained by tailoring the ovarian stimulation protocol to individual patient needs: i.e. to start the GnRH antagonist not in a fixed manner on day 6 of gonadotropins but according to the leading follicle size. Meanwhile different studies have dealt with the question of advantages and disadvantages of a fixed or flexible GnRH antagonist protocol. A meta-analysis of these studies has shown that less GnRH antagonist ampules (−1.2) and less gonadotropins (−95.5 IU) were necessary in the flexible protocol.33 However, a tendency towards less pregnancy was also present in the flexible protocol, especially with a delay of the GnRH antagonist beyond day 8 of ovarian stimulation. With the initiation of the GnRH antagonist, an increase in FSH dose does not optimize the outcome for the patient according to a recent prospective, randomized trial.34 Therefore, tailoring should not start too late. Most studies used a follicular size of about 14 mm. It may be important to start with a smaller size of follicles, like 11–12 mm, to achieve optimal results without the disadvantages described above.

Future studies might show whether implantation and pregnancy rates can also be positively influenced by this approach.

Endometrial quality in GnRH antagonist protocols Another prospective, nonrandomized study, also using the flexible approach of GnRH antagonist administration, showed results which, on the first view, might be somewhat conflicting with our own.35 The authors evaluated endometrial status on the day of oocyte pick-up by endometrial biopsy. A correlation was made between the course of ovarian stimulation, basic hormonal parameters, and the start of the GnRH antagonist. The day of oocyte retrieval was set as day zero. The authors described a mean advancement of endometrial differentiation 2.5 ±0.1 days. In a multiple regression analysis the authors could identify basal LH at initiation of rhFSH stimulation and through the duration of rhFSH treatment before the start of the GnRH antagonist as a significant factor which contributed to the advancement of endometrial differentiation. No pregnancies were achieved when endometrium was advanced for more than 3 days, which was the case in 6 patients.35 As with previous studies in the GnRH agonist long protocol, these results also indicate the detrimental effect of the premature luteinization and progesterone rise. This negative effect has already been discussed by others for the GnRH antagonists.36 Therefore, it may not be the individualization of GnRH antagonist start in this study, but the necessity to prepare these cycles by an oral contraceptive pill. This might be able to suppress endogenous LH to a level that avoids the negative effect on endometrial maturation. Fanchin et al in 2003 reported on a prospective, randomized study which included a study group

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Table 40.7 A prospective, randomized study using the preparation of the cycle from day 20 of the previous cycle with 4 mg estradiol/day. Compared to the spontaneous cycle group, the outcome was improved and showed a better synchronization of the follicular cohort with more mature oocytes and embryos. There was a nonsignificant trend towards higher pregnancy rates (data not shown in the abstract) Parameters Number of follicles > 10 mm (day 8) Follicle size (day 8) CV follicle size (day 8) GnRH antagonist start (days) Day of hCG (days) Metaphase II oocytes

Estradiol

No estradiol

16.4 ± 6.7 9.9 ± 2.5 0.22 8.1 ± 1.0 11.9 ± 1.2 9.3 ± 4.4

16.8 ± 5.8 11.0 ± 3.4 0.26 7.5 ± 1.4 11.0 ± 1.3 7.3 ± 3.3

p

0.001 0.02 0.001 0.001 0.03

Data according to Fanchin et al.37

Table 40.8 Results in GnRH antagonist cycles with or without preparation using an oral contraceptive pill. Retrospective data analysis from one single center Oral contraceptive

Used

Not used

n Pregnancy rate per embryo transfer Pregnancy rate per attempt Implantation rate

136 41.7% (45/108)

86 41.4% (29/70)

33.1% (45/136)

33.7% (29/86)

18.7% (58/310)

21.2% (43/203)

Data according to Shapiro.38

prepared with 17β-estradiol (4 mg/day) from day 20 of the previous cycle up to day 2 of the treatment cycle. The control group started ovarian stimulation in a spontaneous menstrual cycle.37 The authors were able to report on a better synchronization of the follicular cohort. This led to a longer duration of ovarian stimulation and a later start of the GnRH antagonist (with a follicle size of >13 mm), since the follicles were smaller on day 8, but also had a lower size variability. This led to more follicles >15 mm, more mature oocytes, and more available embryos (Table 40.7). Whether the trend towards higher pregnancy rates was due only to the better ovarian response or also to a better endometrial quality has to be further elucidated. These data, however, also highlight the necessity for cycle preparation.37 On the other hand, data from a retrospective series showed similar, but high, pregnancy rates independently whether an oral contraceptive was used before hand or not (Table 40.8).38 Only a further prospective, randomized study will be able to evaluate this problem further – by comparing endometrial advancement in an individualized GnRH antagonist protocol with and without a previous cycle with an oral contraceptive pill. Up to that time, especially in patients with known high basal LH levels, an oral contraceptive pill should be discussed as a helpful tool.

The need for additional LH activity The discussion on differences regarding implantation and pregnancy rates has shown that a drop in endogenous LH might be responsible for this result. However, we have also shown the data from a large prospective, randomized study using rhFSH and Cetrotide 3 mg in a tailored way, where pregnancy rates did not show any difference compared to the GnRH agonist long protocol.21 If an LH drop has a detrimental effect, then it should occur with a high dose of a GnRH antagonist and rhFSH treatment. Since this was not the case, these data indicate an advantage to tailoring compared to a fixed approach. Furthermore, Olivennes demonstrated data which also supported this point of view. In a retrospective analysis he could not find any detrimental effect on the outcome of IVF cycles when estradiol continued to either rise or fall with the administration of a single GnRH antagonist dose. These results were confirmed in other independent studies (Table 40.9). The debate as to whether additional LH activity is needed or not must therefore be done in a very differentiated way: there might be some patients, e.g. old patients, low responders, etc., who would benefit from additional hormones. As shown recently for the long protocol in a prospective randomized study, administration of LH to all patients results in a worse instead of a better cycle outcome.39,40 Meanwhile, for GnRH antagonist protocols, prospective randomized studies as well as retrospective data analysis have been done. Four of them are mentioned here. In a small study by Ludwig et al in 2003, which was conducted to detect a difference in oocyte numbers to a power of 2, patients were treated in a multiple-dose GnRH antagonist protocol using Cetrotide 0.25 mg daily and a fixed dose of 150 IU rhFSH.41 They were randomized to receive an additional 75 IU rhLH from the start day of GnRH antagonist treatment. The GnRH antagonist was administered for the first time when the leading follicle size reached 14 mm. There were no differences in outcome parameters.41 A similar approach was taken by Cédrin Durnerin et al in 2003 in a multicenter

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Table 40.9 Effect of a drop or increase of peripheral estradiol levels after the administration of Cetrotide 3 mg in ovarian stimulation protocols for IVF. There was no difference in clinical outcome between the two groups (a) Increase of estradiol

Decrease of estradiol

Cycles (n) Retrieved oocytes Mature oocytes Embryos available Ongoing pregnancy rate

59 9.7 ± 4.7 6.8 ± 4.1 4.7 ± 3.6 20%

45 8.2 ± 4.6 7.3 ± 4.4 5.1 ± 3.3 24%

(b) Estradiol levels

Increase

Plateau

Decrease

124 46.0% 21.8%

27 44.4% 19.3%

10 30.0% 20.8%

Increase

Plateau

Decrease

239 37.1 ± 4.2 30.7%

16 37.8 ± 5.2 46.2%

6 36.0 ± 5.0 50.0%

n Pregnancy rate per embryo transfer Implantation rates (c) Estradiol levels n Age (years) Clinical pregnancy rates

Numbers are mean ± standard deviation, if not otherwise defined. Data according to François Olivennes (pers comm) (a). Others could confirm these results in retrospective series in the multiple-dose protocol using Orgalutran/Antagon38 (b) or Cetrotide 0.25 mg daily63 (c).

prospective, randomized study. These workers used a single-dose protocol (Cetrotide 3 mg, started in a tai\lored approach with a leading follicle size of 13–15 mm). The rhFSH dose was adjusted to individual patients’ responses. No differences were detected regarding the outcome parameters – independently, regardless of whether 75 IU rhLH were added or not from the start of GnRH antagonist treatment.42 The data from these two studies are shown in Table 40.10. Bosch et al in 2003 analyzed a prospective series of 96 patients undergoing their first IVF or intracytoplasmic sperm injection (ICSI) cycle.43 LH was measured in serum on gonadotropin day 3, the day that the GnRH antagonist Cetrotide 0.25 mg was started, 2 days after the start of the GnRH antagonist, and on the day of hCG administration. Three groups of patients were set up retrospectively, based on the serum LH levels of each day. They were grouped to have LH levels either below the 25th percentile, between the 25th and 75th percentiles, or above the 75th percentile. Differences in cycle outcome with regard to estradiol levels on the day of hCG, number of oocytes retrieved, implantation, and pregnancy rates were not observed between the different LH groups. It was concluded that profound suppression of LH levels after GnRH antagonist start did not have any influence on cycle outcome.43 A fourth study questioned whether the relative change of LH and progesterone after GnRH anagonist initiation might have an influence on the chance to conceive. In that study artificially different LH levels were created using the GnRH antagonist Antide

(iturelix) in a dose of 2 mg, 1 mg, 0.5 mg, 0.5 mg/0.5 ml, and 0.25 mg daily. Again, ovarian stimulation was done using rhFSH. The GnRH antagonist was started in a fixed manner on gonadotropin day 6 in a prospective randomized way for the five different dosages. From the area under the curve for LH, the conclusion was drawn that there might be a certain LH window, which had to be considered for all pregnancies. Neither excessively high nor excessively low LH levels were associated with pregnancies (Fig 40.3).44 To conclude, LH supplementation is not indicated in all GnRH antagonist cycles. It may be that it is even worse for cycle outcome when excessively high LH levels are achieved. Therefore, from clinical experience, I recommend LH addition for those patients who do not show sufficient follicular growth dynamics on gonadotropin day 6 (i.e. only follicles of 10–11 mm size) as well as for those who did not respond well in a previous cycle. Furthermore, LH addition might be helpful in older patients, but the age level still has to be defined. These recommendations still have to be evaluated in future studies.

Timing of hCG administration Looking through hundreds of papers addressing ovarian stimulation, there are different sizes of follicles reported, according to which hCG should be administered. These range from 17 up to 22 mm for one or several follicles. Additionally, sometimes estradiol levels are defined which have to be met before hCG can be given.

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Table 40.10 Use of rhLH addition in rhFSH-treated GnRH antagonist cycles. Shown are the results of two prospective, randomized studies. One was done using the multiple-dose protocol (a), the other one using the single-dose protocol (b) (a) rhFSH only (n = 19)

rhFSH + rhLH (n = 13)

11.89 ± 2.26 1813 ± 473 9.32 ± 5.94

11.54 ± 2.03 1753 ± 393 8.38 ± 7.54

rhFSH

rhFSH + rhLH

80 8.9 ± 1.6 1613 ± 522 1048 ± 691 10.0 ± 4.7 26 (32.9)

95 8.8 ± 1.7 1565 ± 549 1474 ± 791 9.9 ± 4.7 28 (29.5)

Gonadotropin days (n) FSH (total dose) (IU) Oocytes (n) (b) Outcome n Duration of stimulation (days) FSH (total dose) (IU) Estradiol (pg/ml)∗ Oocytes retrieved Pregnancies (%) ∗

p <0.001. Data according to Ludwig et al.41 and Cédrin-Durnerin et al.42

LH

GnRH antagonist start

AUC of LH change window after start of the GnRH antagonist

AUC: area under the curve

Fig 40.3 The optimal LH window, calculated from a prospective, randomized study using the GnRH antagonist Antide. It seems to be that the LH change after GnRH antagonist initiation has a relevant impact on the outcome of the cycle. Fixed limits for this window, however, cannot be given at present. Data according to Huirne et al.44

Since the long GnRH agonist protocol and the GnRH antagonist protocol are different in many respects, one timing of hCG administration might be critical. This could be of importance when the results regarding longer exposure to the GnRH antagonist or higher LH levels are considered.35 Until recently only one prospective, randomized study addressed this question. In this study, patients were randomized to receive hCG (10 000 IU) on the day when one follicle reached 17 mm in size, or 2 days later (Table 40.11).45 Doing this, the authors observed a significant improvement in cycle outcome, with a greatly improved implantation rate in the early hCG group and a trend towards optimized ongoing pregnancy rates. Whether this could lead to an improvement in embryo or endometrial quality is still a matter of debate. This is an important step in achieving an optimal cycle outcome in GnRH antagonist cycles.

Replacement of hCG by a GnRH agonist for ovulation induction In individual patients at higher risk for OHSS it may be helpful to replace the administration of hCG with a

GnRH agonist in a GnRH antagonist protocol. This has been tried in several studies up to now. The results are heterogeneous and sometimes a worse outcome has been reported. Overall, a recent meta-analysis reported less oocytes with the use of a GnRH agonist. However, pregnancy rates overall were significantly lower (OR = 0.21, 95% CI 0.05–0.84).46 Currently, the use of GnRH agonists for triggering ovulation in high-responder patients cannot be recommended as a standard procedure. Patients treated in this way should be informed of a worse outcome regarding pregnancy rates.

Luteal phase support in GnRH antagonist protocols There is no longer any debate as to whether in GnRH agonist-treated cycles, especially long protocol cycles, the luteal phase has to be performed.47 Several prospective, randomized studies have proved that progesterone is as effective as hCG injections. The risk of OHSS is lowest with progesterone administration alone.47,48 In GnRH antagonist cycles it was suggested that owing to the short duration of GnRH antagonist action and a short washout phase30 no luteal phase support is clearly indicated. However, Albano et al showed that in those seven cycles where luteal phase support was not performed, early bleeding occurred and no pregnancies could be established.49,50 Meanwhile, a prospective, randomized study was performed using the GnRH antagonist Antide in combination with rhFSH. In this study, ovulation was induced using either rhCG (250 µg), rhLH (1 mg), or a GnRH agonist (0.2 mg triptorelin). No luteal phase support was applied in any of the cases. The study was cancelled when it became clear that the luteal

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Table 40.11 Timing of hCG administration. hCG was administered either with a leading follicle size of 17 mm (early hCG group) or 2 days later (late hCG group). The outcome was significantly better in the early hCG group

Age (years) Previous cycles Basal FSH (IU/l) FSH starting dose (IU) COC retrieved Embryos/transfer Ongoing implantation rate (%) Ongoing pregnancy rate (%)

Early hCG

Late hCG

p

32.4 ± 0.3 1.6 ± 0.2 7.1 ± 0.3 195 ± 7.5 12.8 ± 0.6 2.04 ± 0.1 22.9 37.6

32.9 ± 0.3 1.5 ± 1.3 7.7 ± 0.3 208 ± 6.3 10.9 ± 0.6 2.04 ± 0.1 14.1 27.8

ns ns ns ns 0.03 ns 0.01 0.09

ns: not significant. Data according to Kolibianakis et al.45

Table 40.12 Necessity for luteal phase support in GnRH antagonist-treated cycles. Shown are the results of a prospective, randomized study in which ovulation was induced using either rhCG, rhLH, or a GnRH agonist64

n No patients achieving ET Pregnancy (%) Ongoing pregnancy (%) Duration luteal phase (days)

rhCG

rLH

GnRH agonist

p Value

11 9 2 (18) 2 (18) 13 (12–15)

13 11 1 (8) 0 (0) 10 (4–16)

15 14 2 (13) 1 (7) 9 (6–15)

0.4 0.7 0.3 0.005

phase length and pregnancy rates were significantly reduced compared to the expected rates. Overall, a pregnancy rate of 14.7% was achieved (Table 40.12). To conclude, there is enough evidence now that in GnRH antagonist-treated cycles luteal phase support must be applied to achieve satisfying results. The luteal phase insufficiency seems to depend not on a prolonged duration of GnRH antagonist activity, but on the supraphysiologic levels of sex steroids which lead to prolonged pituitary suppression by a feedback mechanism.

Alternative GnRH antagonist protocols Introduction Besides the above-mentioned and now well-established protocols, other protocols are theoretically possible. At this point, only those will be described which have already been used in clinical practice.

‘Natural’ cycles Originally, Edwards and Steptoe suggested that the optimal protocol might be the natural cycle – to allow oocyte maturation in an optimal way.51 This opinion was shared by others. It took a couple of years from the start of IVF to learn that in fact ovarian stimulation can improve the outcome of IVF cycles.52

On the other hand, ovarian stimulation increases the risk of side effects, especially the risk of OHSS. Even when this risk can be reduced by the use of GnRH antagonist protocols, it might be even lower with alternative protocols. In this field, the work from Rongieres-Bertrand and Olivennes is of outstanding importance.53 These authors reported on 44 IVF cycles in 33 subjects, who were known to be or expected to be good responders. These were monitored by transvaginal ultrasound up to the time when estradiol levels reached 100–150 pg/ml with a leading follicle size of 14 mm. At that time the patients received 0.5 or 1 mg cetrorelix and follicular maturation was supported by 150 IU hMG daily. Because of the cancellation of cycles before hCG, the lack of possibility of retrieving oocytes, and total fertilization failure after successful oocyte retrieval, only 22 embryo transfers could be performed (55%). However, seven pregnancies were obtained with this minimal ovarian stimulation procedure. Therefore, despite being suboptimal, this protocol provided a first step towards an easy-to-use and low-cost process, especially for those patients who might be at high risk of developing OHSS.

Clomiphene citrate As outlined above, the first ovarian stimulation protocols in the early 1980s were done using clomiphene

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citrate alone or in combination with gonadotropins. These protocols were later abandoned, since the use of GnRH analogs allowed a better timing of oocyte retrieval and the maturation of more oocytes within one cycle. The introduction of GnRH antagonists has changed this again, since now the combination of clomiphene citrate, gonadotropins, and GnRH antagonists allows minimal ovarian stimulation with the theoretical advantage of suppressing a premature endogenous LH surge. Engel et al published a prospective trial with several steps of an overlapping stimulation procedure using clomiphene citrate and gonadotropins.31 Clomiphene citrate was given from days 3 to 7 of a spontaneous cycle, and gonadotropins were started either subsequently or overlapping in different dosages. The GnRH antagonist was administered in a daily dose starting on a fixed scheme on day 6 of ovarian stimulation. Overall, they reached a pregnancy rate of 19.6% in 107 cycles. The rate of premature LH surges, however, was unacceptably high at 21.5%. Despite an acceptable pregnancy rate, the risk makes the protocol highly unreliable.31 Williams et al,54 in a retrospective controlled study observed a high pregnancy rate using a similar approach (37%), which was comparable to that of a conventional GnRH agonist long protocol (41%). A premature LH surge was observed in only 5%.54 Finally, Fiedler and Ludwig reported on their experience with a huge number of cycles, which used clomiphene citrate from day 5 onward. Gonadotropins were started overlapping on the last day of clomiphene citrate, and the GnRH antagonist was started in a multiple-dose fashion on an individual basis, depending on follicular size.55 Taken together, this approach seems valuable in good responder patients, especially in those who are at high risk of OHSS – and even more in those who do not to have supernumerary oocytes or embryos cryopreserved. However, the ideal protocol for the combination of clomiphene citrate, gonadotropins, and a GnRH antagonist has still to be defined.

The low responder patient The GnRH antagonist protocols may be the ideal protocols for low responder patients, since they allow late suppression of endogenous gonadotropins but no occurrence of a flare-up effect.56,57 Up to now, however, no large enough prospective, randomized trials have been published to prove this beneficial effect. Therefore, GnRH antagonists might not be more helpful under these circumstances than the other protocols that have been described in recent years.58 Also, a recent meta-analysis on this topic only could find a higher number of oocytes retrieved in poor responder patients as compared to the GnRH agonist long protocol (0.41, 95% CI 0.0–0.83) – but other parameters, especially clinical pregnancy rates,

549

did not differ, even compared to the GnRH agonist short protocol.59 At this point, it should also be mentioned that it might be patient preselection that leads to poor results in running IVF programs: preferentially, patients are treated with GnRH antagonists when the decision is made to use protocols known to respond badly to ovarian stimulation. As a result, overall results using GnRH antagonists are worse than the established standard – and the substances may not work as well as the alternatives. Only those programs that switch from one protocol to another for a certain period will be able to really evaluate the protocols.26,27

Outcome of pregnancies and children born after GnRH antagonist use The ultimate goal of ovarian stimulation for IVF is the birth of a healthy child. The use of new drugs of course has to be proven to be safe for the mother, the ongoing pregnancy, as well as for the fetus and child. Three studies have been published on this subject and the use of GnRH antagonists.60–62 Two of these studies deal principally with a similar set of data,61,63 therefore, only the later study, which includes all data, will be presented here. The data are shown in Table 40.13 and demonstrate a good safety profile for the two available GnRH antagonists, cetrorelix and ganirelix. Data regarding a follow-up study of children born were provided by Ludwig et al.60 They looked for the development of body weight and length and collected data on these children during the first 2 years of life. No abnormal development was observed in these children.60

Conclusion The introduction of GnRH antagonists in the field of ovarian stimulation has led to several theoretical advantages, which have subsequently proved to be correct. These are: •

• • •

a more convenient method of ovarian stimulation with a shorter period of stimulation and no preceding pituitary suppression a more physiologic way of ovarian stimulation, integrated in a spontaneous menstrual cycle pituitary suppression is started when a premature LH surge threatens the risk of OHSS can be substantially reduced.

With a standard, fixed ovarian stimulation procedure, pregnancy rates were slightly but not significantly reduced compared to the GnRH agonist long protocol. However, more data have been collected which indicate that tailoring of these new protocols to individual patient needs might improve the outcome of ovarian stimulation and also implantation and pregnancy rates.

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Table 40.13 Data regarding the health of children born following the use of GnRH antagonists. The total cohort of pregnancies for cetrorelix (Ludwig et al)60 included 208 pregnancies; to make comparison with the data from Boerrigter et al62 possible, only data from liveborn children were included in this table Ludwig et al.60 Parameter Included pregnancies (n) singletons (%) twins (%) triplets (%) Stillbirth (%) Mean gestational age (total) (weeks) Term birth, n (%) Mean birthweight (g) Weight ≥ 1500 g (%) Major malformations (%)

Boerrigter et al.62

Cetrorelix

Ganirelix

GnRH agonist

163 120 (73.6) 40 (24.5) 3 (1.8) 2 (1.0) 38.0 ± 2.8 86 (63.1) 2843 ± 756 15 (7.2)∗ 3.1

340 258 (75.9) 82 (24.1) 10 (2.9) 5 (1.2) 38.0 ± 3.1 306 (73.0) 2834 ± 768 25 (6.1) 3.8

134 91 (67.9) 43 (32.1) 7 (5.2) 2 (1.1) 37.4 ± 3.2 107 (59.8) 2716 ± 821 18 (10.1) 3.3

Data are shown in mean ± standard deviation if not otherwise defined. ∗ In the paper from Ludwig et al,60 the weight limit was <1500 g. Data according to Ludwig et al60 and Boerrigter et al.62

One aspect of these tailoring methods is the possibility of treating patients either by the single-dose or the multiple-dose GnRH antagonist protocol, depending on their known or expected ovarian response. Furthermore: •

•

•

• •

cycle preparation using an oral contraceptive pill or estradiol seems to be important to improve follicle cohort synchronization GnRH antagonist should be started according to the individual patient’s response, i.e. with a follicle size of 11–12 mm rhLH addition to rhFSH stimulation protocols may be important for single patients but is not necessary in all cycles and may even worsen the outcome timing of hCG may be a critical step and should happen at about 17–18 mm follicle size luteal phase support is necessary to achieve optimal outcome of the cycles.

Further studies are needed to develop these possibilities and to allow the evaluation of alternative protocols which allow a more convenient approach to individual good responder patients.

4.

5.

6.

7.

8.

9.

References 1. Ludwig M, Felberbaum RE, Diedrich K, Lunenfeld B. Ovarian stimulation: from basic science to clinical application. Reprod Biomed Online 2002; 5(Suppl 1): 73–86. 2. Porter RN, Smith W, Craft IL, et al. Induction of ovulation for in vitro fertilisation using buserelin and gonadotropins. Lancet 1984; 2: 1284–5. 3. Rivier J. Novel antagonists of GnRH: a compendium of their physicochemical properties, activities, relative potencies and efficacy in humans. In: Lunenfeld

10.

11.

B, Insler V, eds. GnRH Analogues – the State of the Art 1993. New York: Parthenon, 1993: 13–26. Daya S. Gonadotropin releasing hormone agonist protocols for pituitary desensitization in in vitro fertilization and gamete intrafallopian transfer cycles. Cochrane Database Syst Rev 2000; 2: CD001299. Frydman F, Conel C, De Ziegler D, et al. Prevention of premature luteinizing hormone and progesterone rise with a gonadotropin-releasing hormone antagonist, Nal-Glu, in controlled ovarian hyperstimulation. Fertil Steril 1991; 56: 923–7. Olivennes F, Fanchin R, Bouchard P, et al. The single or dual administration of the LHRH antagonist cetrorelix prevents premature LH surges in an IVFET program. Fertil Steril 2001; 62: 468–76. Olivennes F, Fanchin R, Bouchard P, et al. Scheduled administration of a gonadotrophinreleasing hormone antagonist (Cetrorelix) on day 8 of in vitro fertilization cycles: a pilot study. Hum Reprod 1995; 10: 1382–6. Olivennes F, Alvarez S, Bouchard P, et al. The use of a GnRH antagonist (Cetrorelix) in a single dose protocol in IVF-embryo transfer: a dose finding study of 3 versus 2 mg. Hum Reprod 1998; 13: 2411– 14. Ditkoff EC, Cassidenti DL, Paulson RJ, et al. The gonadotropin-releasing hormone antagonist (NalGlu) acutely blocks the luteinizing hormone surge but allows for resumption of folliculogenesis in normal women. Am J Obstet Gynecol 1991; 165(6 Pt 1): 1811–17. Diedrich K, Diedrich C, Santos E, et al. Suppression of the endogenous luteinizing hormone surge by the gonadotropin-releasing hormone antagonist cetrorelix during ovarian stimulation. Hum Reprod 1994; 9: 788–91. Felberbaum R, Reissmann T, Küpker W, et al. Hormone profiles under ovarian stimulation with

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human menopausal gonadotropin (hMG) and concomitant administration of the gonadotropin releasing hormone (GnRH)-antagonist cetrorelix at different dosages. J Assist Reprod Genet 1996; 13: 216–22. Albano C, Smitz J, Camus M, et al. Comparison of different doses of gonadotropin-releasing hormone antagonist cetrorelix during controlled ovarian hyperstimulation. Fertil Steril 1997; 67: 917–22. The Ganirelix Dose-finding Study Group. A double-blind, randomized, dose-finding study to assess the efficacy of the gonadotrophin-releasing hormone antagonist ganirelix (Org 37462) to prevent premature luteinizing hormone surges in women undergoing ovarian stimulation with recombinant follicle stimulating hormone (Puregon). Hum Reprod 1998; 13: 3023–31. Felberbaum RE, Albano C, Ludwig M, et al. Controlled ovarian stimulation for assisted reproduction with HMG and concomitant midcycle administration of the LHRH-antagonist cetrorelix (Cetrotide®) according to the multiple dose protocol – results of a prospective noncontrolled phase III study. Hum Reprod 2000; 15: 1015–20. Albano C, Felberbaum RE, Smitz J, et al. Controlled ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the LHRH-antagonist cetrorelix (Cetrotide) and the LHRHagonist buserelin. Hum Reprod 2000; 15: 526–31. Olivennes F, Belaisch-Allart J, Emperaire JC, et al. A prospective randomized controlled study in IVF-ET with a single dose of a LH-RH antagonist (cetrorelix) or a depot formula of a LH-RH agonist (triptorelin). Fertil Steril 2000; 73: 314–20. Mannaerts BMJ, Borm G. Treatment with the gonadotrophin-releasing hormone antagonist ganirelix in women undergoing ovarian stimulation with recombinant follicle stimulating hormone is effective, safe and convenient: results of a controlled, randomized, multicenter trial. European Orgalutran Study Group. Hum Reprod 2000; 15: 1490–8. Fluker M, Grifo J, Leader A, et al. The North American Ganirelix Study Group. Efficacy and safety of ganirelix acetate versus leuprolide acetate in women undergoing controlled ovarian hyperstimulation. Fertil Steril 2001; 75: 38–45. The European and Middle East Orgalutran® Study Group. Comparable clinical outcome using the GnRH antagonist ganirelix or a long protocol of the GnRH agonist triptorelin for the prevention of premature LH surges in women undergoing ovarian stimulation. Hum Reprod 2001; 16: 644–51. Roulier R, Chabert-Orsini V, Sitri MC, Barry B. Utilisation des antagonistes de la LHRH (Cetrotide® 3 mg) en pratique courante dans une population non électionnée: étude prospective, randomisée, comparative versus agonistes retard de la GnRH. Federation Française d’Etudes de la Reproduction, September 2001, Palais des Congrès, Lyon, France. Roulier R, Chabert-Orsini V, Sitri MC, et al. Depot GnRH agonist versus the single dose GnRH antagonist regimen (cetrorelix, 3 mg) in patients undergoing assisted reproduction treatment. Reprod Biomed Online 2003; 7: 185–9. Al-Inany H, Aboulghar M. GnRH antagonists in assisted conception: a Cochrane Review. Hum Reprod 2002; 17: 874–85.

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23. Ludwig M, Katalinic A, Diedrich K. Use of GnRH antagonists in ovarian stimulation for ART compared to the long protocol: a meta-analysis. Arch Gynecol Obstet 2001; 265: 175–82. 24. Kolibianakis EM, Collins J, Tarlatzis BC, et al. Among patients treated for IVF with gonadotrophins and GnRH analogues, is the probability of live birth dependent on the type of analogue used? A systematic review and meta-analysis. Hum Reprod Update 2006; 12: 651–71. 25. Ludwig M, Felberbaum RE, Devroey P, et al. Significant reduction of the incidence of ovarian hyperstimulation syndrome (OHSS) by using the LHRH antagonist Cetrorelix (Cetrotide) in controlled ovarian stimulation for assisted reproduction. Arch Gynecol Obstet 2000; 264: 29–32. 26. Ludwig M, Felberbaum RE, Diedrich K. LHRH antagonist protocols in IVF do not lead to worse results than the long LHRH agonist protocol. Rev Gynecol Obstet 2000; 7: 249–50. 27. Samama M, Olivennes F, Fanchin R, et al. One year treatment with GnRH antagonist single dose cetrorelix protocol in controlled ovarian hyperstimulation: a comparative study with GnRH agonist treatment. Hum Reprod 2002; 17(Abstract book): 151. 28. Obruca A, Fischl FH, Huber JC. Programming oocyte retrieval using oral contraceptive pretreatment before ovarian stimulation with a GnRH antagonist (Cetrotide) protocol. Hum Reprod (Abstract book) 2001; 16: 89. 29. Huirne JAF, van Loenen ACD, Donnez J, et al. Effect of an oral contraceptive pill on follicular development in IVF/ICSI patients receiving a GnRH antagonist: a randomized study. Reprod Biomed Online 13: 235–45. 30. Ludwig M, Albano C, Olivennes F, et al. Plasma and follicular fluid concentrations of LHRH antagonist cetrorelix (Cetrotide®) in controlled ovarian stimulation for IVF. Arch Gynecol Obstet 2001; 266: 12–17. 31. Engel J, Ludwig M, Felberbaum RE, et al. Use of cetrorelix in combination with clomiphene citrate and gonadotrophins: a suitable approach to ‘friendly IVF’? Hum Reprod 2002; 17: 2022–6. 32. Ludwig M, Katalinic A, Banz C, et al. Tailoring the GnRH antagonist cetrorelix acetate to individual patients’ needs in ovarian stimulation for IVF: results of a prospective, randomized study. Hum Reprod 2002; 17: 2842–5. 33. Al-Inany H, Aboulghar MA, Mansour RT, Serour GI. Optimizing GnRH antagonist administration: metaanalysis of fixed versus flexible protocol. Reprod Biomed Online 2005; 10: 567–70. 34. Propst AM, Bates GW, Robinson RD, et al. A randomized controlled trial of increasing recombinant follicle-stimulating hormone after initiating a gonadotropin-releasing hormone antagonist for in vitro fertilization-embryo transfer. Fertil Steril 2006; 86: 58–63. 35. Kolibianakis E, Bourgain C, Albano C, et al. Effect of ovarian stimulation with recombinant follicle- stimulating hormone, gonadotropin releasing hormone antagonists, and human chorionic gonadotropin on endometrial maturation on the day of oocyte pick-up. Fertil Steril 2002; 78: 1025–9. 36. Crespo J, Escudero E, Bosch E, et al. When to start the GnRH antagonist in IVF? Preliminary results. Hum Reprod 2002; 17 (Abstract book): 34–5.

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37. Fanchin R, Salomon L, Castelo-Branco A, et al. Luteal estradiol administration coordinates FSHinduced follicular growth and improves the outcome of GnRH antagonist COH protocols. Hum Reprod (Abstract book) 2003; 18: 2–3. 38. Shapiro DB. GnRH antagonists in normal-responder patients. Fertil Steril 2003; 80(Suppl 1): S8–15. 39. Balasch J, Fábregues F, Creus M, et al. Pure and highly purified follicle-stimulating hormone alone or in combination with human menopausal gonadotrophin for ovarian stimulation after pituitary suppression in in vitro fertilization. Hum Reprod 1996; 11: 2400–4. 40. Tesarik J, Mendoza C. Effects of exogenous LH administration during ovarian stimulation of pituitary down-regulated young oocyte donors on oocyte yield and developmental competence. Hum Reprod 2002; 7: 3129–37. 41. Ludwig M, Katalinic A, Schultze-Mosgau A, et al. LH supplementation in GnRH antagonist protocols: preliminary results from a prospective, randomised study. Hum Reprod (Abstract book) 2003; 18: 3. 42. Cédrin-Durnerin I, Grange-Dujardin D, Laffy A, et al. Is there a need for recombinant LH in GnRH antagonist treated cycles? Hum Reprod (Abstract book) 2003; 18: 1–2. 43. Bosch E, Escudero E, Crespo J, et al. Serum LH in ovarian stimulation with GnRH antagonists. Hum Reprod (Abstract book) 2003; 18: 50. 44. Huirne J, Loenen ACD, Schats R, et al. LH window for embryo implantation in GnRH antagonist treated IVF patients. Hum Reprod (Abstract book) 2003; 18: 72–3. 45. Kolibianakis E, Albano C, Tournaye H, et al. Timing of HCG administration for ovulation triggering in GnRH antagonist cycles. A randomized controlled trial. Hum Reprod (Abstract book) 2003; 18: 2. 46. Griesinger G, Diedrich K, Devroey P, Kolibianakis EM. GnRH agonist for triggering final oocyte maturation in the GnRH antagonist ovarian hyperstimulation protocol: a systematic review and meta-analysis. Human Reprod Update 2006; 12: 159–68. 47. Ludwig M, Diedrich K. Evaluation of an optimal luteal phase support protocol in IVF. Acta Obstet Gynecol Scand 2001; 80: 452–66. 48. Ludwig M, Finas A, Katalinic A, et al. Prospective, randomized study to evaluate the success rates using hCG, vaginal progesterone or a combination of both for luteal phase support. Acta Obstet Gynecol Scand 2001; 80: 574–82. 49. Albano C, Smitz J, Tournaye H, et al. Luteal phase and clinical outcome after human menopausal gonadotrophin/gonadotrophin releasing hormone antagonist treatment for ovarian stimulation in in vitro fertilization/intracytoplasmic sperm injection cycles. Hum Reprod 1999; 14: 1426–30. 50. Albano C, Grimbizis G, Smitz J, et al. The luteal phase of nonsupplemented cycles after ovarian superovulation with human menopausal gonadotropin and the gonadotropin-releasing hormone antagonist cetrorelix. Fertil Steril 1998; 70: 357–9. 51. Edwards RG, Steptoe PC. A Matter of Life. New York: Morrow, 1980.

52. Trounson AO, Leeton JF, Wood C, et al. Pregnancies in humans by fertilization in vitro and embryo transfer in the controlled ovulatory cycle. Science 1981; 212: 681–2. 53. Rongieres-Bertrand C, Olivennes F, Righini C, et al. Revival of the natural cycles in in vitro fertilization with the use of a new gonadotrophin-releasing hormone antagonist (Cetrorelix): a pilot study with minimal stimulation. Hum Reprod 1999; 14: 683–8. 54. Williams SC, Gibbons WE, Muasher SJ. Ovarian hyperstimulation for in vitro fertilization using sequential clomiphene citrate and gonadotropin with or without the addition of a gonadotropin-releasing hormone antagonist. Fertil Steril 2002; 78: 1068–72. 55. Fiedler K, Ludwig M. Use of clomifen citrate in in vitro fertilization (IVF) and IVF/intracytoplasmic sperm injection cycles. Fertil Steril 2003; 80: 1521–3. 56. Akman MA, Erden HF, Tosun SB, et al. Addition of GnRH antagonist in cycles of poor responders undergoing IVF. Hum Reprod 2000; 15: 2145–7. 57. Akman MA, Erden HF, Tosun SB, et al. Comparison of agonistic flare-up-protocol and antagonistic multiple dose protocol in ovarian stimulation of poor responders: results of a prospective randomized trial. Hum Reprod 2001; 16: 868–70. 58. Surrey ES, Schoolcraft WB. Evaluating strategies for improving ovarian response of the poor responder undergoing assisted reproductive techniques. Fertil Steril 2000; 73: 667–76. 59. Griesinger G, Diedrich K, Tarlatzis BC, Kolibianakis EM. GnRH-antagonists in ovarian stimulation for IVF in patients with poor response to gonadotrophins, polycystic ovary syndrome, and risk of ovarian hyperstimulation: a meta-analysis. Reprod Biomed Online 2006; 13: 628–38. 60. Ludwig M, Riethmüller-Winzen H, Felberbaum RE, et al. Health of 227 children born after controlled ovarian stimulation for in vitro fertilization using the luteinizing hormone-releasing hormone antagonist cetrorelix. Fertil Steril 2001; 75: 18–22. 61. Olivennes F, Mannaerts B, Struijs M, et al. Perinatal outcome of pregnancy after GnRH antagonist (ganirelix) treatment during ovarian stimulation for conventional IVF or ICSI: a preliminary report. Hum Reprod 2001; 16: 1588–91. 62. Boerrigter PJ, De Bie JJ, Mannaerts BM, et al. Obstetrical and neonatal outcome after controlled ovarian stimulation for IVF using the GnRH antagonist ganirelix. Hum Reprod 2002; 17: 2027–34. 63. Kiminami A, Yoshida A, Kakinuma A, et al. Change in estradiol on the day after initiation of the GnRH antagonist Cetrorelix in IVF cycles does not affect pregnancy outcome. Hum Reprod (Abstract book) 2003; 18: 106. 64. Beckers NGM, Macklon NS, Eijkemans MJ, et al. Nonsupplemented luteal phase characteristics after the administration of recombinant human chorionic gonadotropin, recombinant luteinizing hormone, or gonadotropin-releasing hormone (GnRH) agonist to induce final oocyte maturation in in vitro fertilization patients after ovarian stimulation with recombinant follicle stimulating hormone and GnRH antagonist cotreatment. J Clin Endocrinol Metab 2003; 88: 4186–92.

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41 Monitoring IVF cycles Matts Wikland, Torbjörn Hillensjö

Introduction Historically, monitoring of ovarian response, by means of measuring ovarian hormones, came into use for ovulation induction owing to complications of gonadotropin therapy. In ovulation induction cycles with gonadotropins, Klopper and co-workers showed that success rates and complication rates were not dependant on monitoring as such, but on the treatment protocol used. If more gonadotropins are given, successes increase, as do complications such as ovarian hyperstimulation syndrome (OHSS) and multiple births. Monitoring merely gives us the possibility of deciding how far we want to go.1 This may be true for ovulation induction cycles but not for in vitro fertilization (IVF) cycles, where the number of transferred embryos can be restricted and thereby at least the risk for multiple births can be minimized, and probably also the severity of OHSS if no embryos are transferred. Owing to the dramatic increase of IVF cycles worldwide, and all the different ovarian stimulation protocols used, different ways of monitoring have been tested. Of all the methods described for monitoring IVF cycles, ultrasound imaging of the uteroovarian response to gonadotropins have become the clinically most useful. The method was first evaluated in the natural cycle, but it was soon realized that it was in stimulated cycles where it could really be useful.2,3 One problem though, was that the size (mean diameter as well as the volume) of the mature follicle seems to vary greatly.4,5 In order to overcome this problem, several studies have been performed to determine the value of combining serum estradiol (E2) measurements and ultrasound monitoring of follicular maturation in stimulated cycles.6–9 This combination of ultrasound and hormonal monitoring seemed to be important in protocols with clomiphene citrate and gonadotropins alone where the endogenous luteinizing hormone (LH) peak could not be controlled. With the introduction of gonadotropin-releasing hormone (GnRH) analogs combined with gonadotropins, the risk for high tonic levels of LH or premature LH peaks has disappeared.10 Thus, with the use of GnRH analogs, or lately GnRH antagonists, there seems to be less need for extensive hormonal monitoring of IVF cycles.11 Ultrasound alone or in certain cases combined with

one or two serum E2 measurements seems to be sufficient in the majority of women entering an IVF cycle.12 Nowadays, in most IVF units, ultrasound imaging has become a very important method for monitoring and power Doppler analysis of perifollicular blood flow seems to be a promising clinically useful method.

Why monitor the cycle Ovarian stimulation with gonadotropins in assisted reproductive technologies (ART) cycles is performed for one reason only, and that is to achieve as many mature healthy oocytes as possible. The more mature oocytes that can be retrieved in one cycle, the better the chance of having several good embryos, of which one can be transferred and the others frozen for future use, one at a time. With such a philosophy, the number of started stimulated cycles could be reduced probably to a minimum before a full-term pregnancy is achieved. Recent data from a large database analysis clearly indicate that the more high-quality embryos in one stimulated cycle the better the chance of achieving a pregnancy.13 With the protocols used today for controlled ovarian hyperstimulation (COH), in our opinion, there are five reasons for monitoring the cycle: 1. 2. 3. 4. 5.

Beforehand, predict the ovarian response to gonadotropins. Monitor the effect of pituitary down-regulation. During the stimulation, evaluate whether the dose of gonadotropin is adequate. Avoid OHSS. Find the optimal time to give human chorionic gonadotropin (hCG).

Thus, monitoring before starting a COH may identify poor responders as well as women at risk for OHSS.14– 16 Furthermore, if a protocol with a GnRH analog has been used, the pituitary down-regulation has to be verified before starting with gonadotropins. Since multiple follicular developments play a major roll in the successful outcome of IVF, ovarian stimulation with follicle-stimulating hormone (FSH) alone is, in the majority of cases, the first choice. However, identifying adequate follicular development during such a

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stimulation, and finally optimizing the time of hCG administration, also requires monitoring, preferably by a simple method, since it sometimes has to be repeated over a short period of time. Ideally, the method should be noninvasive. Unfortunately, there is no such method. All methods are indirect. The fact that there is no method by which the oocyte maturation can be monitored directly in vivo, and the huge number of IVF cycles performed today, means that monitoring has to be simple and of course reliable. In this respect, vaginal ultrasound has over the years proved to be the most practical way of monitoring ovarian stimulation for IVF since it gives the actual response of the uterus and the ovaries.

Prediction of ovarian response to FSH Prediction of ovarian responses prior to stimulation is important since it helps us to choose an optimal starting dose of FSH. Traditionally, the ovarian reserve has been evaluated by means of basal day 3 FSH measurement or a clomiphene citrate challenge test.17 However, measuring the number of small antral follicles in both ovaries by vaginal ultrasound has proved to be a reliable predictor of the ovarian reserve.18 Ng and coworkers were able to show that the number of antral follicles, as measured by vaginal ultrasound, was even superior to basal day 3 FSH as well as body mass index (BMI) for predicting the number of oocytes retrieved for IVF.18 They demonstrated that in women with fewer antral follicles, a longer duration and higher dose of gonadotropin were required but still significantly less oocytes were retrieved. They also showed that if fewer than six antral follicles were found in a cycle prior to the start of stimulation, there was an increased risk that cycles would be cancelled before egg collection. Furthermore, those women at risk for OHSS can be identified. Women with typical PCO (polycystic ovary)-like ovaries, as well as those with multifollicular ovaries (MFO), can easily be identified. It seems that women with more than 10 antral follicles have an increased risk of OHSS. In our program we routinely perform a vaginal scan in the early follicular phase of a cycle prior to the IVF cycle. The purpose is to identify those who could be poor as well as high responders. By doing so, it seems easier to identify an optimal starting dose of FSH and in certain cases also decide upon the type of protocol to be used, particularly in poor responders where sometimes an antagonist protocol can be beneficial.

Monitoring pituitary down-regulation As mentioned above, today the most used protocol for IVF cycles is long pituitary down-regulation with a GnRH agonist. When using such a protocol, one has to verify the down-regulation before starting with FSH. If the GnRH agonist is started in the late luteal phase a menstrual bleeding normally indicates that the estrogen

is low and FSH can be started. However, measuring suppression of ovarian/pituitary hormones in blood will clearly confirm down-regulation. A simpler and quicker way, in our opinion, is to perform a vaginal scan and measure the endometrial thickness, which should be less than 4 mm, and the number of small follicles (<8 mm), which should be less than four. In cycles where the GnRH agonist has been started in the early follicular phase, hormone analysis is believed to be mandatory for confirming pituitary suppression. However, even in this group it has clearly been demonstrated that ultrasound imaging is enough to verify down-regulation.19 It has also been shown that color flow Doppler velocimetry of the utero-ovarian arteries can be used for verifying pituitary desensitization in these women. Dada and co-workers did show that the ovarian artery resistance index was the best Doppler predictor of pituitary suppression and a mean discriminatory cut-off value of 0.9 was found to have the highest specificity and positive predictive value20 in women who had stared with the GnRH in the early follicular phase. It is no doubt that uteroovarian scanning is useful for evaluating pituitary suppression before administering FSH.

Monitoring follicular maturation There are five methods that can be used clinically for monitoring the follicular maturation in IVF cycles: 1. 2. 3. 4. 5.

Serum E2. Ultrasound measurements of follicular growth and endometrial thickness. Ultrasound and serum E2 combined. Perifollicular blood flow by means of power Doppler imaging. Perifollicular blood flow using three-dimensional ultrasound.

There is an extensive literature regarding the use of all the above methods for monitoring the ovarian response in ART. Even though no study has shown that either serum E2 or ultrasound alone is superior to the other for monitoring follicular maturation in IVF cycles, there are data in the literature showing that ultrasound imaging (UI) of the follicular and endometrial growth in the majority of cases is sufficient.21 It has been said that ultrasound should be used for timing and then E2 to avoid complications.22 Which of the two methods the clinician relies on most for the decision to increase, decrease, or stop the gonadotropin seems to be very much dependent on experience and/or routines used at the clinic. Furthermore, there is no consensus about how often the monitoring has to be done during ovarian stimulation. The number of times for monitoring seems to be arbitrarily chosen and thus varies considerably between different clinics. Thus, there are simple as well as complicated methods for monitoring ART cycles by means of serum E2 and/or ultrasound.

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However, irrespective of the method chosen, there seems to be no difference in the outcome of the IVF cycle.

Monitoring with serum E2 alone Serum E2 as the only method for monitoring ART cycles stimulated with gonadotropins was mainly used in the early days of IVF. The method for monitoring was based on experience from monitoring of ovulation induction cycles. Some groups have tried to identify a certain serum E2 level that should be reached before hCG should be given.23 Others have claimed that the number of days E2 increased was important, and thus gave hCG based on that.24 Even though some groups are still using E2 measurements as the sole monitoring method, the majority of groups using hormone measurements for monitoring also use ultrasound.

Monitoring with ultrasound Ultrasound monitoring of follicular diameter and endometrial thickness is a noninvasive method. It can be performed by the clinician and gives an actual status of the number and size of growing follicles. The endometrial thickness as measured by ultrasound can be used as a bioassay of the total follicular estrogen production. Vaginal ultrasound scanning of the uteroovarian response to FSH stimulation is a simple and reliable method that for many years has proved to be the most practical way of monitoring ART cycles. Since the end of 1991 our group has utilized a monitoring system of ultrasound alone in the majority of IVF cycles. In women not at risk of OHSS, or poor responders (normally identified before the treatment cycle), only one ultrasound scan was performed on stimulation day 9 or 10. If the patient on that day had three follicles of 18 mm (mean of two diameters), fewer than 15 follicles, and an endometrial thickness of ≥7 mm, hCG was given and oocyte pick-up performed 36–38 hours later. If the follicles did not fulfill these criteria on the day for the scan, a follicular growth of 2 mm/24 hours was predicted and hCG given according to that. The 18 mm diameter as well as the number of follicles were chosen arbitrarily. Retrospectively comparing 361 ART cycles performed during the year 1989, when several ultrasound and serum estradiol measurements were used for monitoring each cycle, with 500 cycles performed during 1991 using the above simplified method, the take-home baby rate was 17% and 26%, respectively.25 In another retrospective analysis of our data with the same simplified method of monitoring the follicular maturation by ultrasound only once during the stimulation by gonadotropins, the take-home baby rate per started cycle was 31% and 1.8% of OHSS.11 Between 1991 and 2002 this simplified monitoring system was used in our IVF program for 7325 cycles.

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During that period, the take-home baby rate per started cycle was 25%, and mild to moderate OHSS occurred in 2.7% of the cycles. These figures show that it is possible to use a simple monitoring by ultrasound and still achieve a good pregnancy. Our experience has also been confirmed by others.14 Recently the first multicenter prospective randomized study comparing ultrasound and hormonal plus ultrasound monitoring of IVF cycles showed that the addition of E2/follicle criteria to ultrasound monitoring of IVF cycles in normal responders seldom changes the timing of hCG, and does not increase pregnancy rate or the risk of OHSS.21 In GnRH antagonist protocols, ultrasound monitoring has been recommended to start on FSH stimulation day 6, since that has been the day for starting with the GnRH antagonist.26 However, in our IVF program, the antagonist is started when the largest follicle is ≥12 mm, irrespective of the day in the cycle. In normal responders, it means that the first scan can be performed on stimulation day 7–8. The most important advantage of monitoring the IVF cycle only once (or occasionally twice) by ultrasound, is that the woman has to spend less time in the clinic. The simplified monitoring will thus bring down the costs of the treatment. The disadvantage of using a simple monitoring system such as described above is that there is no possibility of increasing the dose of gonadotropins early in the cycle. However, whether such an early increase in the FSH dose has any significance for the outcome of the IVF cycle seems unlikely.

Monitoring by estrogens and ultrasound Combining ultrasound and estrogens seems mainly to be important in women at risk for OHSS.27,28 A situation where adding the hormone analysis can be valuable is when ultrasound monitoring shows adequate follicular growth but inadequate endometrial growth. This could indicate a low estrogen production/follicle due to a low endogenous LH level. If that is the case, adding recombinant LH could be beneficial. From a more practical point of view, and particularly in women who for some reason are believed to respond poorly, it can be valuable to analyze the level of serum E2 on FSH day 5. Arbitrarily we have found that if the serum E2 is <700 pmol/l, the FSH dose can safely be increased by 75–150 units and the scan performed on stimulation day 9 or 10. This is a simple way of early discovery that the starting dose has been sufficient.

Monitoring by color Doppler and three-dimensional ultrasound A Doppler duplex system combining pulsed Doppler and gray-scale ultrasound has made it possible to noninvasively

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study ovarian blood flow and use that as a measurement of ovarian angiogenesis. From animal studies it is well known that there is a correlation between follicular vascularity and oocyte maturation. In a classic clinical study, Nargund and co-workers showed a significant increased oocyte recovery from follicles with a high peak systolic velocity as measured by pulsed Doppler and gray-scale ultrasound. Furthermore, they found that oocytes from poorly vascularized follicles produced morphologically poor embryos as compared to oocytes from highly vascularized follicles.29 Later, in a very elegant study, Van Blerkom and co-workers showed by means of color Doppler imaging (CDI) that follicles with normal perifollicular blood flow contained oocytes free of cytoplasmatic or chromosomal/spindle defects.30 However, the CDI is a time-consuming and difficult method that cannot be used in the daily clinical setting. In 1994, a new color Doppler technique called power Doppler imaging (PDI) was described.31 The PDI technique has many advantages as compared to CDI: it is more sensitive, enables flow with lower volumes and velocity to be displayed, and can thus display areas where the mean velocity is zero. Thus, PDI has proved to be a technique that can be used for measuring perifollicular blood flow and is simple enough to be used in the daily clinical setting. Chui and co-workers adopted the PDI technique in their IVF program and showed that high-grade follicular vascularity resulted in oocytes/embryos that had an increased potential for becoming a full-term pregnancy.32 Further studies using PDI for monitoring perifollicular blood flow have shown that the technique can be used clinically for identifying follicles with the oocytes that seem to have a better chance of resulting in good embryos.33 Recently, a combination of power Doppler angiography (PDA) and three-dimensional ultrasonography (3DUS) has been introduced for predicting and monitoring ovarian response in IVF-ET (embryo transfer) cycles.34,35 However, whether this technique really improves the monitoring of the cycle is still unclear. Furthermore, the technique is complicated and time consuming, making it less practical for the daily monitoring of ART cycles.

Conclusion Two-dimensional ultrasound scanning of follicular size is still the method of choice for monitoring IVF cycles, irrespective of the protocol used for COH. It is the most practical, and is still reliable enough for monitoring ovarian stimulation with gonadotropins. Combining ultrasound monitoring of follicular size with E2 is particularly valuable for monitoring poor responders as well as those at risk for OHSS.

References 1. Klopper A, Aiman J, Besser M. Ovarian steroidogenesis resulting from treatment with menopausal gonadotropin. Eur J Obstet Gynecol Reprod Biol 1974; 4: 25–30.

2. Hackeloer BJ, Nitsche S, Daume E, Sturm G, Bucholz R. Ultraschaldarstellung von ovarveranderungen bei gonadotropinstimulierung, Geburtsh Fruenheilk 1977; 37: 185–9. 3. Ylöstalo P, Lindgren P, Nillius SJ. Ultrasonic measurement of ovarian follicles, ovarian and uterine size during induction of ovulation with human gonadotropins. Acta Endocrinol 1981; 98: 592–8. 4. Vargyas JM, Marrs RP, Kletzky OA, Mishell DR. Correlation of ultrasonic measurement of ovarian follicle size and serum estradiol levels in ovulatory patients following clomiphene citrate for in vitro fertilization. Am J Obstet Gynecol 1982; 144: 569–73. 5. Wittmaack FM, Kreger DO, Blasco L, et al. Effect of follicular size on oocyte retrieval, fertilization, cleavage, and embryo quality in in vitro fertilization cycles: a 6-year data collection. Fertil Steril 1994; 62: 1205–10. 6. Cabau A, Bessis R. Monotoring of ovulation induction with human menopausal gonadotropin and human chorionic gonadotropin by ultrasound. Fertil Steril 1981; 36: 178–82. 7. McArdle C, Seibel M, Hann LE, Weinstein F, Taymor M. The diagnosis of ovarian hyperstimulation (OHS): the impact of ultrasound. Fertil Steril 1983; 39: 464–7. 8. Salam MN, Marinho AO, Collins WP, Rodeck CH, Campbell S. Monitoring gonadotropin therapy by real-time ultrasonic scanning of ovarian follicles. Br J Obstet Gynaecol 1982; 89: 155–9. 9. Venturoli S, Fabbri R, Paradisi R, et al. Induction of ovulation with human urinary follicle stimulating hormone: endocrine pattern and ultrasound monitoring. Eur J Obstet Gynecol Reprod Biol 1983; 16: 135–45. 10. Messinis IE, Tempelton AA, Baird DT. Endogenous luteinizing hormone surge during superovulation induction with sequential use of clomiphene citrate and pulsatile human menopausal gonadotrophin. J Clin Endocrin Metab 1985; 61: 1076–81. 11. Wikland M, Borg J, Hamberger L, Svalander P. Simplification of IVF. Minimal monitoring and the use of subcutaneous highly purified FSH administration for ovulation induction. Hum Reprod 1994; 9: 1430–6. 12. Bergh C, Howles C, Borg K, et al. Recombinant human follicle stimulating hormone (r-hFSH; Gonal-F) versus highly purified urinary FSH (Metrodin HP): results of a randomized comparative study in women undergoing assisted reproductive techniques 1997; 12: 2133–9. 13. Templeton A, Morris JK. Reducing the risk of multiple births by transfer of two embryos after in vitro fertilization. N Engl J Med 1998; 27: 573–7. 14. Forman R, Robinson J, Egan J, et al. Follicular monitoring and outcome of in vitro fertilization in gonadotropin-releasing hormone agonist-treated cycles. Fertil Steril 1991; 55: 567–73. 15. Shoham Z, Di Carlo C, Patel A, Conway G , Jacobs H. Is it possible to run a successful ovulation program based solely on ultrasound monitoring? The importance of endometrial measurements. Fertil Steril 1991; 56: 836–41. 16. Tomas C, Nuojua-Huttunen S, Martikainen H. Pretreatment transvaginal ultrasound examination predicts ovarian responsiveness to gonadotrophins in in-vitro fertilization. Hum Reprod 1997; 12: 220–3.

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Monitoring IVF cycles 17. Scott RT, Hofmann GE. Prognostic assessment of ovarian reserve. Fertil Steril 1995; 63: 1–11. 18. Ng EH, Tang OS, Ho PC. The significance of the number of antral follicles prior to stimulation in predicting ovarian responses in an IVF programme. Hum Reprod 2000; 15: 1937–42. 19. Barash A, Weissman A, Manor M, et al. Prospective evaluation of endometrial thickness as a predictor of pituitary down-regulation after gonadotropin-releasing hormone analogue administration in an in vitro fertilization program. Fertil Steril 1998; 69: 496–9. 20. Dada T, Salaha O, Allgar V. Sharma V, Utero-ovarian blood flow characteristics of pituitary desensitisation. Hum Reprod 2002; 16: 1663–70. 21. Lass A. UK Timing of hCG Group. Monitoring of in vitro fertilization–embryo transfer cycles by ultrasound versus ultrasound and hormonal levels: a prospective, multicenter, randomised study. Fertil Steril 2003; 80: 80–5. 22. Schoemaker J, Meer M, Weissenbruch M. Re-evaluation of the role of estrogens as a marker for ovulation induction. In: Lunenfeld B, ed. FSH Alone in Ovulation Induction. London: Parthenon, 1993; 23–7. 23. Wramsby H, Sundstrom P, Liedholm P. Pregnancy rate in relation to number of cleaved eggs replaced after in-vitro fertilization in stimulated cycles monitored by serum levels of estradiol and progesterone as sole index. Hum Reprod 1987; 2: 325–8. 24. Levran D, Lopata A, Nayudu PL, et al. Analysis of the outcome of in vitro fertilization in relation to the timing of human chorionic gonadotropin administration by the duration of estradiol rise in stimulated cycles. Fertil Steril 1985; 44: 335–41. 25. Wikland M, Vaginal ultrasound in assisted reproduction. In: Hamberger H, Wikland M, eds. Assisted Reproduction. Baillière’s Clin Obstet Gynaecol 1992; 2: 283–96. 26. Ganarelix Dose-finding Study Group. A double-blind, randomized, dose-finding study to assess the efficacy of the GnRH-antagonist ganarelix (Org 37462) to prevent premature luteinizing hormone surges in women undergoing controlled ovarian hyperstimulation with recombinant follicle stimulating hormone. Hum Reprod 1998; 13: 3023–31.

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27. Forman R, Frydman R, Egan D. Severe ovarian hyperstimulation syndrome using agonists of gonadotropinreleasing hormone for in vitro fertilization: a European series and a proposal for prevention. Fertil Steril 1990; 55: 502. 28. Waldenstrom U, Kahn J, Marsk L, Nilsson S. High pregnancy rates and successful prevention of severe ovarian hyperstimulation syndrome by ‘prolonged coasting’ of very hyperstimulated patients: a multicentre study. Hum Reprod 1999; 14: 294–7. 29. Nargund G, Bourne T, Doyle P, et al. Associations between ultrasound indices of follicular blood flow and oocyte recovery and preimplantation embryo quality. Hum Reprod 1996; 11: 109–13. 30. Van Blerkom J, Antczak M, Schrader R. The developmental potential of human oocytes is related to the dissolved oxygen content of follicular fluid: association with vascular endothelial growth factor levels and perifollicular blood flow characteristics. Hum Reprod 1997; 12: 1047–55. 31. Rubin JM, Bude RO Carson PL, Bree RL, Adler RS. Power Doppler US: potential useful alternative to mean frequency-based color Doppler US. Radiology 1994; 190: 853–6. 32. Chui DK, Pugh ND, Walker SM, Gregory M, Shaw RW. Follicular vascularity – the predictive value of transvaginal ultrasonography in an in vitro-fertilization program: a preliminary study. Hum Reprod 1997; 12: 191–6. 33. Bhal PS, Pugh ND, Gregory L, O´Brien S, Shaw RW. Perifollicular vascularity as a potential variable affecting outcome in stimulated intrauterine insemination treatment cycles: a study using transvaginal power Doppler. Hum Reprod 2001; 16: 1682–9. 34. Vlaisavljevic V, Reljic M, Gavric Lovrec V, Zazula D, Sergent N. Measurement of perifollicular blood flow of the dominant follicle using three-dimensional power Doppler. Ultrasound Obstet Gynecol 2003; 22: 520–6. 35. Mercé LT, Barco MJ, Bau S, Troyano JM. Prediction of ovarian response and IVF/ICSI outcome by threedimensional ultrasonography and power Doppler angiography. Eur J Obstet Gynecol Reprod Biol 2007; 132: 93–100.

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42 Oocyte collection Gab Kovacs

History The very first human pregnancy using in vitro fertilization (IVF) in the world (although transient) was achieved by the Monash University team in 1973,1 using laparotomy for obtaining the oocyte. Meanwhile, Morgenstern and Soupart in 1972 had described an experimental procedure for both abdominal and vaginal approaches to oocyte recovery, using a special oocyte recovery unit (ORU), in conjunction with gynecological surgery.2 As this was very traumatic, and laparoscopy was just being applied to gynecology, the laparoscopic approach became routine by the late 1970s.3,4 The expertise of Patrick Steptoe in laparoscopy and his successful partnership with Bob Edwards resulted in the birth of Louise Brown in 1978.5 It was the laparoscopic approach, with modification of the collection needle by Carl Wood’s team,6 that was used in the stimulated/controlled cycles which resulted in the next eight births from the Monash team, which converted IVF from a research tool to a clinical treatment. It was also used by the Jones’ team when they used human menopausal gonadotropins to achieve the first pregnancies in the USA.7 During the early 1980s, IVF became applied worldwide, using laparoscopic oocyte collection. The pioneering work of Susan Lenz in Copenhagen,8 and Wilfred Feichtinger in Vienna9 changed oocyte collection to a transvaginal ultrasound-guided technique. With its efficacy being proven equivalent to laparoscopy by a study published by the Monash team,10 most of the world’s IVF units abandoned laparoscopy.

Anesthesia/analgesia The change to transvaginal ultrasound-guided oocyte collection, meant that relaxant analgesia was no longer required. Currently, there is great variation in the type of analgesia used for oocyte collection. In China, oocyte collection is undertaken without any analgesia, whereas in many places some intravenous sedation or even general anesthesia is administered. This depends on several factors, including cultural expectations, the facility used for the oocyte collection, and the medical financial rebate system. A balance has to be reached, with minimal risk and cost, but without causing the

women unacceptable discomfort. A recent survey of anesthetic practice employed for oocyte collection in the UK11 found that intravenous sedation was the preferred method of sedation, being used in 62.4% of units. General anesthesia was the primary method in 24.6% of units; sedation was performed by nonanesthetic doctors in 46% and by nurses in 8.2%. All aspects of the use of anesthesia and sedation for oocyte collection are discussed in detail in Chapter 50.

The equipment In the early days, manual suction with a needle, plastic tubing and syringe were used.2 Berger and colleagues3 devised a special aspiration unit, with a 20-gauge 10inch needle connected by polyethyelene tube to a 10mm Vacutainer, which then connected to a vacuum bottle with an adjustable pressure gauge. The suction was turned on or off by a thumb valve. The technique then was modified with the use of a suction pump operated by a foot pump.6 Today, sophisticated suction pumps with adjustable aspiration pressure are widely available commercially (Fig 42.1).

The suction There has been surprisingly little study undertaken on the physical aspects of oocyte recovery. We published the findings of experiments on bovine eggs carried out in the laboratories of Cook Medical Technology in Brisbane.12 Some of the observations of these studies are outlined below. In this study, we measured the velocity and flow rates of oocytes through the collection system, and observed the damaging effect of nonlaminar flow to the oocyte.

Application of vacuum to the follicle Vacuum applied after the needle entry into the follicle After application of the vacuum, the pressure within the system equilibrates, resulting in a steady flow rate until the fluid volume decreases and the follicle collapses, so that the follicular wall blocks the lumen of the needle. The time for the system to equilibrate depended on the vacuum pressure, the diameter of the

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Table 42.1 follicles

The diameter-to-volume ratio of typical

Follicle diameter (mm)

Fig 42.1

Vaccum pump.

needle, and the volume of the follicle. Maximum flow was achieved when the pressure was at a steady state. Should air be sucked into the system, by entering around where the needle pierces the follicle wall, frothing with nonlaminar flow results, which I call the ‘cappuccino effect.’ This has a deleterious effect on the oocyte, as it is thrown around the collection system.

Vacuum deactivated before the needle was withdrawn from the follicle If the pressure was deactivated while the needle was still in the follicle (and there were no leaks), the pressure within the needle and collecting tube dropped, and there was often backflow towards the follicle. This can result in the oocyte being sucked back and possibly lost. The amount of backflow depends on how much air enters the system and how much higher the collection tube is above the patient’s pelvis.

The vacuum profiles within the aspiration system It was estimated that using the system at 150 kPa it took 5 seconds for the system to stabilize. The pressure within the follicle, before penetration, varies, depending on the size (maturity), shape, and position of the follicle. The internal pressure increases, correlating with size. However, due to the pressure caused by the needle deforming the surface of the follicle at the time of puncture, the pressure within the follicle may be much higher (up to 60 mmHg). The more blunt the needle, the higher the resultant pressure. This may result in follicular fluid being lost, as it spurts out during the process. If the pressure is already applied, some or most of this fluid will be aspirated as it escapes along the outer wall of the follicle. There is a pressure gradient down the collection system, so that the pressure at the tip of the needle is only 5% of the pressure at the pump. The oocyte is therefore exposed to ever-increasing pressures as it travels along the needle, the collection tube, and the collecting test tube. Excessive pressure can cause the ovum to swell and the zona to crack.

Follicle volume (ml)

6

0.1

7

0.2

8

0.3

9

0.4

10

0.5

11

0.7

12

0.9

13

1.1

14

1.4

15

1.8

16

2.1

17

2.6

18

3.0

19

3.6

20

4.2

The typical dead space of needle and collecting tubule is 1.0–1.2 ml.

Follicle and needle volumes Table 42.1 lists the respective volumes contained in follicles between 6 and 20 mm in diameter. A 6-mm follicle only contains 0.1 ml, so that 10–12 follicles need to be emptied before the dead space of 1.0–1.2 ml in a standard needle and collecting tube is filled, and fluid reaches the collection test tube.

Application of the vacuum Following the penetration of the follicle by the needle and the application of suction, the pressure within the follicle, the needle, and the collecting tube equilibrates. If there is a tight seal around the needle – i.e. the needle was sharp and was introduced precisely through the follicular wall and the hand is kept still so that tearing does not result – when the suction pressure is reduced, there will be backflow of fluid into the follicle. This can result in the oocyte being lost. On the other hand, if the needle is withdrawn while the suction is still applied, there is sudden change of pressure at the needle tip from the high vacuum of the follicle to atmospheric pressure, with a rapid surge of fluid towards the collection tube. If the oocyte is contained in the terminal portion of the fluid, it is subjected to increased speeds of travel as well as turbulence, resulting in loss of the cumulus mass and even fracture of the zona pellucida.

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Damage within the follicle During aspiration, the oocyte has to accelerate from a resting state to the velocity of the fluid within the needle. If this is too rapid, the cumulus may be stripped off. The higher the aspiration pressure, the greater the risk, and the smaller the follicle, the higher the pressure that is needed. This may be very relevant in the collection of immature oocytes for in vitro maturation.

Damage to oocytes Even with laminar flow there are significant differences in velocity of the follicular fluid within the center of the needle compared to the periphery. This can result in ‘drag’ on the outer layers of cumulus, resulting in potential damage. The longer the needle, the smaller its internal diameter, the greater the pressure required to maintain the same velocity. It was found that when a 17gauge collection needle was used, all oocytes lost their cumulus mass when the aspiration pressure reached 20 kPa (150 mmHg). It is therefore recommended that pressures be kept <120 mmHg: the higher the speed of travel, the more chance of damage to the oocyte. Apart from the speed of travel, turbulent nonlaminar flow can also damage the oocyte, either stripping its cumulus mass or fracturing the zona. It is believed that an intact cumulus may be important in preventing damage to oocytes.

Fig 42.2

Collection tubes in a test tube warmer.

deviated from the projected path, as observed by ultrasound. It is the clinical protocol at Monash IVF that if ≤4 follicles are present, a DLN should be used and the follicles flushed. If >4 follicles are present, an SLN is used and the follicles are sequentially aspirated. Flushing of follicles requires the use of a DLN, as with an SLN with a dead space of 1.0–1.2 ml, the oocyte is likely to be flushed up and down within the system.

Avoiding turbulent flow

Technique Flushing or rapid oocyte collection When transvaginal oocyte collection was first undertaken, the technique of laparoscopic harvesting was transferred to the transvaginal approach. Follicles were initially aspirated, and then repeatedly flushed to try and recover as many oocytes as possible. This, however, is time consuming and also uses large quantities of culture medium. It was soon recognized that most oocytes can be recovered by just aspirating, and that the follicular fluid from the next follicle will often flush the oocyte into collection tube. This was called the ROC technique (rapid oocyte recovery). Scott and colleagues from Norfolk Virginia13 compared oocyte recovery rates with either a singlelumen needle (SLN; n = 22) or a double-lumen needle (DLN; n = 22) and the technical aspects of their use: 210 and 212 follicles were aspirated with each type of needle, respectively. Follicular diameters were measured ultrasonically at the time of aspiration and recorded. One or more washes were performed when using the DLN and the SLN was withdrawn each time to recover the fluid in the dead space of the needle. The oocyte recovery rates and the incidence of fractured zonae were the same for both needles. Although there were no differences between the two needles in the number of oocytes provided for IVF, the DLN needle was more flexible and frequently

When aspirating follicles, it is important to recognize that in order to fill the dead space between the needle tip and the aspiration tube, somewhere between 1 and 2 ml of follicular fluid is needed. As described above, in order to avoid damage to the cumulus–oocyte mass during aspiration, one should avoid nonlaminar flow within the collection tube.

Temperature control Another important point is to deliver oocytes to the laboratory in the best condition, which includes being aware of the effect of cooling. Redding and colleagues from New Zealand14 investigated the effects of IVF aspiration on the temperature, pH, and dissolved oxygen of bovine follicular fluid. They found that the temperature of follicular fluid dropped by 7.7 ± 1.3°C upon aspiration, dissolved oxygen levels rose by 5 ± 2 vol%, and the pH increased by 0.04 ± 0.01. They concluded that these changes could be detrimental to the oocyte’s health, and efforts should be made to minimize these changes. The collection tubes are therefore kept in a test tube warmer while they are waiting to be connected to the collection system (Fig 42.2).

The approach Any ultrasound machine with the capacity to use a transvaginal probe with a needle guide can be used.

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Fig 42.3

Ultrasound picture as seen on the screen.

The ovaries are visualized and ovarian follicles are then aspirated in a systematic fashion. It is my habit to always commence with the right ovary, and then to aspirate follicles sequentially. It is best to keep the needle within the ovary if possible, to minimize the amount of trauma to the ovarian capsule. When all follicles within the right ovary are aspirated, the needle is withdrawn from the vagina and the needle is flushed with medium to clear any blood. The pressure is retested, and the left ovary is then aspirated (Fig 42.3).

Complications Whereas complications are discussed in detail in Chapter 56, a brief synopsis only is provided here. Transvaginal oocyte collection has become the method of choice during the last two decades. However, although complications are rare, several possible complications of transvaginal oocyte collection have been reported. The commonest operative complications are: • hemorrhage • trauma to pelvic structures • pelvic infection, tubo-ovarian or pelvic abscess. Rarely reported complications include: • • • • • •

ovarian torsion rupture of ovarian endometriosis appendicitis ureteral obstruction vertebral osteomyelitis anesthetic complications.

The incidence of postoperative acute abdomen was reported by Dicker and colleagues in 1993 from Israel:15 they reported 14 cases, out of 3656 patients

undergoing the procedure, presenting with a clinical picture of acute abdomen; in nine patients, tuboovarian and pelvic abscess were diagnosed; in three cases, severe intra-abdominal bleeding occurred, with one patient requiring laparotomy and hemostasis; and ruptured endometriotic cysts caused acute abdomen in two patients. In 1993, Tureck from Philadelphia published a retrospective analysis of 674 patients who underwent transvaginal retrieval of oocytes during a 3-year period: 10 (1.5%) patients required hospital admission because of perioperative complications, 9 of these patients needing intravenous antibiotics and one patient requiring admission and observation for an expanding broad-ligament hematoma. Hemorrhage can result in vaginal bleeding at and after the oocyte collection (overt bleeding) or intraabdominal bleeding (covert bleeding). Bennett and colleagues,16 from a 4-year prospective study carried out at King’s College London of 2670 consecutive procedures, reported that vaginal hemorrhage occurred in 229 (8.6%) of the cases, with a significant loss (classified as more than 100 ml) in 22 (0.8%) patients. Hemorrhage from the ovary with hemoperitoneum formation was seen on two occasions and necessitated emergency laparotomy in one instance. A single case of pelvic hematoma formation from a punctured iliac vessel was also recorded; this settled without intervention. As early as the 1990s it was recognized that preexisting endometrioma was a risk factor for pelvic infection after oocyte collection, with Younis and colleagues from Israel in 1997 reporting on three infertile women with ovarian endometriomata, who presented with late manifestation of severe pelvic abscess 40, 24, and 22 days after oocyte collection, respectively.17 Severe endometriosis with ovarian endometriomata seems to be a significant risk factor for pelvic abscess development. Late manifestation of pelvic abscess supports the notion that the presence of old blood in an endometrioma provides a culture medium for bacteria to grow slowly after transvaginal inoculation. More vigorous antibiotic prophylaxis and better vaginal preparation was recommended when oocyte pick-up is performed in patients with endometriosis. Overall, the risk of significant pelvic infection is between 1:200 and 1:500. Consequently, prophylactic antibiotics are not indicated, unless an endometrioma is entered, or there is a past history of pelvic infection, and then it is our policy to administer a single dose of intravenous antibiotic, e.g. gentamicin.

Very uncommon complications Ureteric obstruction There is a case report from Greenville, South Carolina, USA, of a case of acute ureteral obstruction following seemingly uncomplicated oocyte retrieval. Prompt diagnosis and ureteral stenting led to rapid patient recovery, with no long-term urinary tract sequelae.18

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Vertebral osteomyelitis The most bizarre complication reported after oocyte collection is vertebral osteomyelitis, which was reported from Tel Aviv by Almog and colleagues.19 They reported a case of vertebral osteomyelitis as a complication of transvaginal oocyte retrieval in a 41year-old woman who underwent IVF-ET (embryo transfer) treatment. After she returned with severe low back pain, vertebral osteomyelitis was diagnosed and treated with antibiotics.

Cullen’s sign (periumbilical hematoma) Bentov and colleagues20 described two cases of periumbilical hematoma (Cullen’s sign) following ultrasound-guided transvaginal oocyte retrieval. Spontaneous resolution of the symptoms occurred within 2 weeks. They concluded that the appearance of a periumbilical hematoma (Cullen’s sign) following ultrasound-guided transvaginal oocyte retrieval reflects a retroperitoneal hematoma of a benign course.

Troubleshooting It is important that before commencing oocyte collection, the system is tested by aspirating some culture medium. This also provides a column of fluid into which to collect the follicular fluid, thus encouraging laminar flow. Should suction then subsequently decrease or stop, the following steps should be undertaken: •

•

• • • • •

Ensure that the suction pump is turned on and that the suction pedal is functioning (many aspiration pumps have a light that goes on, and some have audible signals when the pump is activated). Check that all connections of tubing between the aspiration tube and the pump are tightly connected. Exclude any cracks in the aspiration test tube. Ensure that the collection tubing is not kinked or damaged. Rotate the needle within the follicle to ensure that it is not blocked by follicular wall tissue. If still no suction, remove the needle and perform a ‘retrograde flush’ to clear any blockage. Before reinserting the needle, recheck by aspirating some culture medium.

Failure to get oocytes – check hCG given Sometimes several follicles are aspirated, and no oocytes are recovered. If the fluid collected is very clear and devoid of cells (granulosa and cumulus), suspicion may be raised that the patient has not had her trigger human chorionic gonadotropin (hCG). It is suggested that before follicles from the second ovary

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are aspirated, some of the follicular fluid is tested with a urinary pregnancy test strip. As these turn blue (react positively) when the concentration exceeds 25 miU/ml, if the hCG was administered, there should be sufficient hCG in the follicle to give a positive result. If the test is negative, it is possible to abandon the collection, administer hCG, and defer the collection from the other ovary until about 36 hours later. Although the number of oocytes collected will be limited to one ovary, it is still possible to salvage the cycle.

Pretreatment of pathology It has long been suggested that tubal disease, and in particular hydrosalpinx, has a detrimental effect on the outcome of IVF. To determine whether surgical removal of hydrosalpinges improved outcome, Johnson and colleagues21 undertook a Cochrane analysis of all trials, comparing a surgical treatment for tubal disease with a control group generated by randomization. The studied outcomes were live birth (and ongoing pregnancy), pregnancy, ectopic pregnancy, miscarriage, multiple pregnancy, and complications. Three randomized controlled trials involving 295 couples were included in this review. The odds of ongoing pregnancy and live birth were increased with laparoscopic salpingectomy for hydrosalpinges prior to IVF. The odds of pregnancy were also increased, but there was no significant difference in the odds of ectopic pregnancy. They recommended that laparoscopic salpingectomy should be considered for all women with hydrosalpinges prior to IVF treatment. They also concluded that the role of surgery for tubal disease in the absence of a hydrosalpinx is unclear and merits further evaluation.

Endometriosis Al-Fadhli and colleagues22 reported a study that evaluated the effects of different stages of endometriosis on the outcome of treatment in an IVF program. They found that the presence of endometriosis, including stages III and IV, does not affect IVF outcome. However, women with endometriosis required more gonadotropin than those with no endometriosis. Women with an obliterated cul-de-sac have fewer oocytes retrieved.

Assessing clinical competence It is recommended that prior to undertaking oocyte collections, a structured training program is carried out. One approach is that the instructor aspirates one side and, having collected some eggs, the trainee should do the other side. The number of supervised collections probably varies between 20 and 40 before trainees should be credentialed to perform collections on their own. Ongoing assessment of clinical competence should then be regularly reassessed. Our clinical

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indicator is the oocyte collection rate: the number of oocytes aspirated per follicle (>13 mm) on the prehCG scan. The collection rates are then compared between clinicians working within the unit. Other indicators that could be recorded are the time taken for the oocyte collection and the complication rate, although the incidence of bleeding and infection is so low that it is probably meaningless unless a very large number of oocyte collections are available for analysis.

References 1. De Kretzer D, Dennis P, Hudson B, et al. Transfer of a human zygote. Lancet 1973; ii: 728–9. 2. Morgenstern LL, Soupart P. Oocyte recovery from the human ovary. Fertil Steril 1972; 23: 751–8. 3. Berger MJ, Smith DM, Taymor ML, Thompson RS. Laparoscopic recovery of mature human oocytes. Fertil Steril 1975; 26: 513–22. 4. Steptoe PC, Edwards RG. Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotrophins. Lancet 1970; i: 683–9. 5. Steptoe PC, Edwards RG. Birth after reimplantation of a human embryo. Lancet 1978; ii: 366. 6. Renou P, Trounson AO, Wood C, Leeton JF. The collection of human oocytes for in vitro fertilization. I. An instrument for maximizing oocyte recovery. Fertil Steril 1981; 35: 409–12. 7. Jones HW, Acosta AA, Garcia J. A technique for the aspiration of oocytes from human ovarian follicles. Fertil Steril 1982; 37: 26–9. 8. Lenz S. Ultrasonic-guided follicle puncture under local anesthesia. J In Vitro Fert Embryo Transf 1984; 1: 239–43. 9. Feichtinger W, Kemeter P. Laparoscopic or ultrasonically guided follicle aspiration for in vitro fertilization? J In Vitro Fert Embryo Transf 1984; 1244–9. 10. Kovacs GT, King C, Cameron I, et al. A comparison of vaginal ultrasonic-guided and laparoscopic retrieval of occytes for in vitro fertilization. Asia Oceania J Obstet Gynaecol 1990; 16: 39–43. 11. Yasmin E, Dresner M, Balen A. Sedation and anaesthesia for transvaginal oocyte collection: an

12.

13.

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15.

16.

17.

18.

19.

20

21.

22.

evaluation of practice in the UK. Hum Reprod 2004; 19: 2942–5. Horne R, Bishop CJ, Reeves G, Wood C, Kovacs GT. Aspiration of oocytes for in-vitro fertilization. Hum Reprod Update 1996; 2: 77–85. Scott RT, Hofmann GE, Muasher SJ, et al. A prospective randomized comparison of single- and doublelumen needles for transvaginal follicular aspiration. J In Vitro Fert Embryo Transf 1989; 6: 98–100. Redding GP, Bronlund JE, Hart AL. The effects of IVF aspiration on the temperature, dissolved oxygen levels, and pH of follicular fluid. J Assist Reprod Genet 2006; 23: 37–40. Dicker D, Ashkenazi J, Feldberg D, et al. Severe abdominal complications after transvaginal ultrasonographically guided retrieval of oocytes for in vitro fertilization and embryo transfer. Fertil Steril 199; 59: 1313–15. Bennett SJ, Waterstone JJ, Cheng WC, Parsons J. Complications of transvaginal ultrasound-directed follicle aspiration: a review of 2670 consecutive procedures. J Assist Reprod Genet 1993; 10: 72–7. Younis JS, Ezra Y, Laufer N, Ohel G. Late manifestation of pelvic abscess following oocyte retrieval, for in vitro fertilization, in patients with severe endometriosis and ovarian endometriomata. J Assist Reprod Genet 1997; 14: 343–6. Miller PB, Price T, Nichols JE Jr, Hill L. Acute ureteral obstruction following transvaginal oocyte retrieval for IVF. Hum Reprod 2002; 17: 137–8. Almog B, Rimon E, Yovel I, et al. Vertebral osteomyelitis: a rare complication of transvaginal ultrasound-guided oocyte retrieval. Fertil Steril 2000; 73: 1250–2 Bentov Y, Levitas E, Silberstein T, Potashnik G. Cullen’s sign following ultrasound-guided transvaginal oocyte retrieval. Fertil Steril 2006; 85: 227. Johnson NP, Mak W, Sowter MC. Surgical treatment for tubal disease in women due to undergo in vitro fertilisation. Cochrane Database Syst Rev 2004; 3: CD002125. Al-Fadhli R, Kelly SM, Tulandi T, Tanr SL. Effects of different stages of endometriosis on the outcome of in vitro fertilization. J Obstet Gynaecol Can 2006; 28: 888–91.

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43 The luteal phase: luteal support protocols James P Toner

This review discusses the special need for luteal support in assisted reproduction and the options currently available to provide this support. Differences between natural cycles and those seen in assisted reproductive technologies (ART) (stimulated and programmed) are discussed, followed by a review of the components of support (estradiol [E] and progesterone [P]), their timing, and route of replacement. Lastly, standard protocols for replacement are provided. Progesterone and estradiol are required for successful pregnancy, both to prepare the uterus for embryo implantation and to stabilize the endometrium during pregnancy. The success of donor egg programs which replace only these two hormones has amply demonstrated the sufficiency of this approach.1,2 In the normal luteal phase of a nonpregnant woman, E and P production peaks about 4 days after ovulation and continues at this level for about a week, until falling several days before the next menses (Fig 43.1).3 During this time, P is secreted in a pulsatile fashion every 1–4 hours, with measured levels ranging between 4 and 20 ng/ml during peak production. This P production is enormous: it is 40-fold more than the maximal E production, some 25 mg daily vs 0.6 mg for E. In normal cycles, P and E production wanes about 10 days after ovulation. Menses follows that event about 4 days later unless a pregnancy occurs. A dip in ovarian P production can occur even during cycles of pregnancy, but in that case it is quickly reversed, with P

production restored by human chorionic gonadotropin (hCG) stimulation of the corpus luteum. A shift from ovarian to placental production of gonadal steroids occurs over a period of weeks. In one study, placental P production was detected as early as 50 days of gestational age (36 days after embryo transfer) in hormone-replaced cycles of donor egg recipients.4 This timing accords well with the observed effects of surgical removal of the corpus luteum in early pregnancy. Lutectomy led to miscarriage in almost every case if performed before 7 weeks of gestational age, and almost never if performed after that time.5

The special problem of the luteal phase after ovarian stimulation In stimulated cycles typical of in vitro fertilization (IVF) therapy, the luteal phase is different from the normal one in two important ways. First, since ovarian stimulation produces multiple corpora lutea, the levels of both E and P in the early part of the luteal phase are supraphysiological. Secondly, and perhaps what is more important, the duration of ovarian steroid production in stimulated cycles is usually shorter than normal by 1–3 days. This truncated luteal phase has been noted since the earliest days of IVF (see Fig 43.2),6 and created concern that an early menses might prevent a successful implantation,

1500

100 E2 P4

50 500

0

0 –12

–8

–4

0

4

8

12

nmol /l

pmol/l

1000

Fig 43.1 E and P levels in normal cycles.3 Note that decline in E and P production begins 9 days after the LH peak (presumably about 8 or 9 days after ovulation). P levels have fallen to about half their peak 10 days after the LH peak, and thereafter fall steadily back to baseline, which is reached about 15 days after the LH peak. This decline is similar for E, and produces menses at about 14 days after the LH peak.

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SM

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50

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20 24 8 16 24 R hCG

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6

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10

Retrieval Hour

Luteal cycle day

since menses were on occasion observed to occur as early as 10 days after egg retrieval. Moreover, the decline of serum E and P levels is also more abrupt than the rate of fall in natural cycles (compare Figs 43.1 and 43.2). This early and rapid fall was the reason luteal support was adopted in the early days of IVF therapy. With the advent of gonadotropin-releasing hormone (GnRH) agonist use in the late 1980s, the problem of the short luteal phase became even more common. A recent study in GnRH antagonist cycles has documented inadequate luteal phases; the impairment was most profound when GnRH agonist was used to induce final follicular maturation, intermediate when recombinant luteinizing hormone (rLH) was used, and least when hCG was used.7 Multiple studies show the importance of some form of luteal support in such cycles.8–11 Three comprehensive meta-analyses now affirm this observation.12–14 The supraphysiological E and P levels after ovarian stimulation may also have effects on uterine receptivity, even when luteal length is adequate. Endometrial histology is advanced, especially in high-responding cycles.15–18 Increased uterine contractions in highresponding women at the time of embryo transfer19 have been associated with lower pregnancy rates.20 The duration of required supplementation is not entirely clarified. In IVF cycles, pregnancy itself (via hCG production) leads to a rise in endogenous estradiol and progesterone production, and this may be sufficient to obviate the need for continued supplementation past

X 12

14

Fig 43.2 Luteal P levels in IVF cycles. These cycles were reported in an early book on IVF, and show very clearly the early fall in P levels and its consequence: early menses in unsupported cycles. In the five cycles shown here, P levels had fallen abruptly by day 9 after egg retrieval in all cases, and all but one bled before 14 days had elapsed after egg retrieval, one as early as day 10. This observation led to the wide use of P supplementation.6 The few studies omitting P supplementation have lower pregnancy rates.6

the point of pregnancy, as one recent study has suggested.21 This approach is reported by one group to lead to more early pregnancy losses but no fewer live births.22 However, most clinicians empirically continue the supplementation through about 10 weeks’ gestation (or 8 weeks from egg retrieval).

The special problem of programmed cycles While the success of donor egg therapy has clearly demonstrated that simple E and P replacement alone is sufficient to produce the conditions needed for pregnancy, the endometrial development is not entirely normal with this approach. The typical observation is that the stroma is more advanced than the glands, leading to a ‘dyssynchrony’ of histological appearance. Since the normal cycle has a small amount of preovulatory P production, it is possible that the ‘dyssynchrony’ is due to the omission of this P in the typical programmed cycle regimens. In fact De Ziegler suggests that providing a small amount of P in programmed cycles eliminates this disparity.15 Observations in hormone-replaced and stimulated cycles taken together would suggest that endometrial glandular development is most related to the duration of P exposure, whereas stromal development is most related to the P dose.15 Whether correction of this disparity increases pregnancy rates is untested.

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Timing of luteal support In stimulated IVF cycles, steroid production during the first week after egg retrieval is likely to be well timed and more than sufficient, so the start of exogenous steroid support is not apt to be critical within this window. However, a recent trial in routine IVF that compared starting progesterone 3 vs 6 days after retrieval found higher pregnancy rates with the earlier start.23 Vaginal progesterone supplementation before embryo transfer may, however, be useful in quieting uterine contractions and thereby reducing embryo displacement.24 In programmed cycles, however, the timing is critical, since the only source of sufficient P is exogenous. Navot et al2 showed that 2-day-old embryos transferred on the 2nd to 4th days of P therapy produced pregnancies, while transfers out of that window did not. A more recent and larger study confirmed the importance of the timing of P therapy.26 In this study, embryos from donor eggs were transferred into recipients 44–48 hours after egg retrieval. Recipients had a standard form of E and P replacement, but the day on which P was started was variable. The highest pregnancy rates occurred when 2day-old embryos were transferred on the 4th or 5th day of P therapy, a little longer than observed by Navot.2 This corresponds to beginning P on the day the donor was given hCG or the day afterwards, but before egg retrieval. It suggests that a longer exposure than is natural might improve the odds for pregnancy (Table 43.1).

Elements of luteal support • Progesterone supplementation. P is the sine qua non of the luteal phase. It is so central to endometrial preparation and pregnancy support that many practitioners view it as the only important hormone in luteal physiology. It needs to be exogenously provided in all programmed cycles, and in most stimulated cycles (unless hCG is substituted) to achieve appropriate pregnancy rates and support. • Estradiol supplementation. Conventional luteal support regimens in IVF have replaced only P, although both E and P are normally secreted in tandem, and both fall prematurely in most cases after ovarian stimulation cycles. While E does not directly mediate luteinization, some E is probably required to stimulate P receptor replenishment so that P can act. In support of the notion that luteal E may also be beneficial is the result of a meta-analysis which found that hCG injections (which stimulate both E and P production) are a superior form of luteal support compared to P alone, while P alone is in turn better than no such luteal support.11,26 A recent trial of hCG administration when midluteal estradiol levels were low in IVF cycles also demonstrated increased pregnancy rates with such use.27 However, given the increased risk of ovarian hyperstimulation syndrome (OHSS) that hCG produces, the use of hCG to provide combined E and P secretion has not been widely adopted.

567

Table 43.1 Relationship between the duration of P treatment before embryo transfer and the subsequent pregnancy rate38 # days after P started

‘Cycle day’

n

Implantation rate (%)

Pregnancy rate (%)

2 3 4 5 6

16 17 18 19 20

18 25 40 60 49

0 3.5 14.1 15.8 5.6

0 12 40 48 20

If the superiority of hCG over P is due to its ability to stimulate E production, then simple replacement of E in addition to P might be helpful. Two recent studies support this hypothesis. In one study,28 high-responding patients (>2500 pg/ml E at time of hCG) pretreated with a long GnRH agonist protocol were randomized to receive either P alone (50 mg p.v. [vaginally] bid and 50 mg i.m. [intramuscularly] qd) or both E (2 mg p.o. [orally] bid) and P. Patients who received both E and P had higher pregnancy rates (40% vs 26%), higher implantation rates (15% vs 10%), and lower miscarriage rates (11% vs 17%) than those who took no E. In the other study,29 more positive pregnancy tests (38.5%) were observed when estradiol patches were used than with progesterone alone (13.5%). A Cochrane meta-analysis also suggests a benefit of E supplementation.12

Route of support Possible routes of P delivery include transdermal, oral, intramuscular, transvaginal, sublingual, nasal, and rectal. Of these, only three – oral, intramuscular, and transvaginal – have been widely used, and only two – intramuscular and transvaginal – are satisfactory methods at this time. • Oral route. The development of the micronization process allowed for much improved absorption of oral P. However, the systemic levels of P are too low after oral administration30 (Fig 43.3) to provide adequate endometrial support. The first passage of P through the liver after oral ingestion leads to massive metabolism: at best only 10% of the administered dose circulates as active progesterone.31 Any effort to increase the oral dose sufficiently to achieve the requisite serum P levels produces a degree of somnolence unacceptable to most patients. Recent clinical trials of oral supplementation of IVF cycles confirm the inadequacy of this route (Table 43.2). Note that women who took only oral P supplements in their IVF cycles had a lower pregnancy and implantation rate, and a higher miscarriage rate, than those who took either vaginal or intramuscular P.

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16 14 12 10 8 6 4 2 0 0

12

24

36

48

60

Crinone 90 mg vaginally vs Prometrium 100 mg orally Vag − true

Oral − true

Vag − RIA

Oral − RIA

Fig 43.3 Serum levels of P after oral vs vaginal administration (Prometrium or Crinone), as measured by standard radioimmunoassay (RIA) or liquid chromatography–mass spectrometry (true). Note that the oral use has a short halflife, and is overreported by RIA. Alternatively, vaginal use yields a longer half-life and is a more accurate representation of the true amount of P in the circulation.

Table 43.2 The effect of oral vs nonoral P supplementation on clinical outcomes in IVF cycles. Note the poorer outcomes after oral use in both studies Study

Licciardi et al, 199910 Friedler et al, 19998

Regimens

Clinical pregnancy rate (%)

i.m. 50 mg qd Oral 200 mg tid Vag 100 mg bid Oral 200 mg qid

Therefore, oral progesterone supplementation should not be relied upon for luteal support of pregnancy. • Intramuscular route. Intramuscular administration of P in oil has been the standard route of delivery in the United States for ART cycles. The i.m. route delivers P at relatively high efficiency and without the metabolism encountered with the oral route as a result of hepatic ‘first-pass’ metabolism. However, the i.m. method also has several drawbacks. It is uncomfortable for the patient and on occasion produces serious side effects, such as sterile abscess formation and allergic response. Recovering from these side effects can take many weeks, because the half-life of the oil vehicle in the muscle is long. Large, tender wheals develop at each injection site, and soon no more space is readily available for injection. In most cases, these allergic reactions are due to specific components of the oil vehicle and can be avoided by switching to a different type of vegetable oil as the base. Other limitations of this route are the need to use and dispose of needles, the need to administer the injection daily (or train someone else to do it), and the cost of both equipment and personnel.

58 46 47 33

Implantation rate (%)

41 18 31 11

Miscarriage rate (%)

N/a N/a 13 40

The usual i.m. dosing is from 25 to 100 mg daily, sometimes in divided doses. This regimen produces peak serum P levels that can be well above the physiologic range. Endometrial architecture has generally shown appropriate ‘in-phase’ development, and pregnancy and miscarriage rates have seemed to be ‘normal.’4,15 It is useful to note that i.m. P at the usual doses is able to delay menses in most women. • Vaginal route. The vaginal route offers several important advantages over i.m. dosing: 1. It is convenient and acceptable to patients. 2. It does not hurt or require any special equipment or training to administer. 3. It rarely produces allergic reactions. Formulations used in the vagina to date have included Endometrin (micronized P tablets), pharmacistformulated suppositories (usually in a paraffin base), Silastic rings, Crinone 8% (a gel), and Prometrium (a gelatin capsule). All these products have higher patient acceptability than injections. Initial trials comparing various routes of P administration in ART cycles supported the advantages of vaginal therapy. Devroey and colleagues in a series of

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studies32–34 demonstrated that vaginal therapy was at least as good as (if not better than) i.m. replacement, and clearly better than oral. This was borne out in endometrial histology, but what is more important in pregnancy and miscarriage rates, and in both IVF and donor egg cycles. Paradoxically, this superiority in clinical outcomes was observed even though serum progesterone levels were abnormally low35 (Fig 43.4). This led to the demonstration of a ‘targeted drug delivery’ from vagina to uterus.36–38 Based on these findings, the group in Belgium adopted vaginal therapy and has continued to use it ever since (as Utrogestan [Prometrium in the United States] at a dose of 200 mg three times daily). In the United States, the first FDA-approved system for pregnancy support is Crinone 8%. It is a bioadhesive vaginal gel containing 90 mg micronized P in an emulsion system designed to adhere to the vaginal mucosa, and thus achieve a controlled and sustained delivery. Dose-ranging studies suggest that once-daily administration of 90 mg is about four times more than the dose required for satisfactory endometrial development. Higher doses of micronized P capsules (Prometrium or Utrogestan, 200 mg three times daily) given vaginally produce the same endometrial effects. Advantages of Crinone over other vaginal therapies are a longer half-life (Fig 43.5) and lower

Fig 43.4 Comparison of serum P levels and endometrial development during three types of P replacement. Note that oral P replacement led to low serum P levels and uniformly inadequate endometrial development, while vaginal P replacement had equally low serum P levels but the best endometrial development.

100%

% maximum serum P4

IM (50 bid)

75%

50%

25%

0% 0

4

8

Oral capsule

12 16 Hours since P

20

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24

28

Crinone

Fig 43.5 Comparison of serum levels after P administration by different means. For comparison, the peak serum level is normalized for each method. Note the rapid fall after oral use, and the prolonged levels after Crinone use.

patient-to-patient variability in absorption (Fig 43.6). Clinical trials of Crinone have been encouraging to date. While not all reports have been favorable,39,40 other published experiences have been. In donor egg cycles at the

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Table 43.3 Interim analysis of multicenter trial of Crinone 8% in IVF. Sixteen centers contributing at least 40 cycles each from 1998 were evaluated. Note that the pregnancy rates were slightly higher than those same centers reported to SART as their 1997 data. Also note the low rate of miscarriage once a sac was documented (<10%)53

12

P 4 max

10 8 6

Age

4 2 0 Crinone 8% (90 mg) P4 vaginal (100 mg)

P4 oral (100 mg)

<35 35–39 40+ Total SART 1997

n

Clinical pregnancy (%)

Ongoing pregnancy (%)

605 437 142 1184 4801

39.7 34.6 16.9 35.1 33.6

35.0 30.7 14.8 31.0 –

Fig 43.6 Comparison of peak serum P levels and the variability among patients after P administration by different methods. Note that the lowest variability was associated with Crinone use.

Jones Institute, reassuring pregnancy and miscarriage rates were seen at both the twice- and once-daily dosing levels compared to i.m. therapy.41,42 A head-to-head comparison of luteal support with Crinone 8% vs Utrogest demonstrated statistical equivalence in IVF cycles for most clinical endpoints.43-45 Trends in pregnancy and miscarriage rate favored Crinone 8%, as did endpoints related to patient convenience. The newest option for vaginal P replacement in the US is Endometrin, an effervescing tablet containing micronized P. Initial studies have demonstrated pharmacokinetics sufficient to induce normal endometrial development at the 100 mg twice daily dose.46 In a very large clinical study, pregnancy and miscarriage rates were no different from those seen with Crinone.47,48 Prometrium is a gelatin capsule filled with micronized P in peanut oil. Some women report a light staining or discharge with its use, and prefer to wear a panty-shield to protect their undergarments. This discharge does not alter the efficacy of the product. Vaginal replacement may also be possible by way of progesterone-impregnated rings. Zegers-Hochschild et al49 report on a ring that supplies continuous release of 10–20 nmol/l progesterone daily for up to 90 days in IVF and donor egg cycles. In their randomized trial, pregnancy rates were as high with vaginal as with intramuscular therapy. Another option may be intramuscular 17α-hydroxyprogesterone caproate, which can be given intramuscularly twice weekly.50 Pregnancy and miscarriage rates were no different with this approach than with daily intramuscular injections, but were higher than vaginal at a different clinic.51 Theoretical concerns about teratogenesis when not using progesterone per se led to less wide use of this approach, and studies are insufficient at this time to answer the question satisfactorily for most. A randomized controlled trial 52 and a large, multicenter experience are also reassuring53 (Table 43.3).

Peculiarities of vaginal progesterone therapy • Bleeding. While most US practitioners with experience in i.m. P therapy have come to expect that menses will be delayed as long as P therapy continues,54 the experience with vaginal P therapy is very different: it does not seem to be able to delay the onset of menses past normal. On the other hand, there is no evidence that this bleeding causes miscarriage or lower pregnancy rates. The timing of the bleeding is ‘physiological;’ i.e. it comes when it normally does in natural cycles that are not conception cycles. Roman and colleagues55 have retrospectively analyzed bleeding patterns in their IVF patients (Fig 43.7). They find very little ‘early’ bleeding, but that most patients bleed at or within 3 days after the days of ‘expected’ menses. Only 3 of 52 pregnant patients had any bleeding; thus, bleeding seems to signal the lack of pregnancy. • Accumulation/leakage. Crinone is formulated in an inert base (polycarbophil) which attaches to and combines with the vaginal epithelium. This provides its extended duration of action. However, the base material is not absorbed, and in some women accumulates into a cheesy material. Though it does not interfere with absorption of later doses, some women confuse it with yeast infections. Unless the ‘discharge’ is associated with pruritus or erythema, there is no need to institute antimonilial therapy. If the accumulation is objectionable to a particular woman, it can be removed through a speculum or with a finger. • Intercourse. There is no prohibition from coital activity during the use of vaginal P products. Although there may be some P absorption by the male through penile exposure, it carries no known or suspected risks.

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expected

16 14

Frequency

12 10 8 6 4 2 0 11

12

13

14

15

16

17

18

19

20

21

Days after hCG

Progesterone options Based on these considerations, the following regimens appear to be equally effective in endometrial preparation and hence pregnancy support: 1. 2. 3. 4.

Progesterone in oil 50 mg intramuscularly, once daily. Crinone 8% vaginally, once or twice daily. Prometrium 200 mg vaginally, 3 or 4 times daily. Endometrin 100 mg vaginally, 2 or 3 times daily.

I believe practitioners can choose whichever of these options appeals to them and their patients.

Luteal support protocols Support in stimulated ART cycles Nearly all centers provide luteal support after ovarian stimulation (at least in the latter half of the luteal phase). This practice is supported by the evidence. It is customary to start support shortly after egg retrieval. The evidence that hCG (or estradiol replacement) is superior to P supplementation alone is not conclusive but is sensible; so it seems prudent at this time to consider adding E also. Therefore, I would recommend the following options (Fig 43.8): • P and E replacement beginning the day of (or up to 3 days after) egg retrieval and continuing until pregnancy testing some 14 days after egg retrieval, or • hCG every 3–5 days throughout luteal phase (in cycles at low risk of OHSS).

22

Fig 43.7 Histogram of the frequency of first bleeding after various luteal days in IVF cycles supplemented with vaginal P (Utrogen 200 mg tid). Bleeding only occasionally began earlier than a full luteal phase, and only rarely in pregnant patients. Most bleeding signaled the lack of pregnancy, and came at or shortly after the ‘expected’ menstrual time.

be varied from as short as 7 days to as long as 35 days without ill effects.56,57 Most centers strive to start the E replacement a few days before the donor starts her FSH injections. The start of P has been varied, from the day the donor receives her hCG until the day of egg retrieval; similar pregnancy rates were observed across this range of days. In our program, we have traditionally begun P the day after egg retrieval (Fig 43.9). Any of the methods of P replacement listed above would suffice to produce an adequate luteal phase.

Support in thaw-transfer cycles One of the areas of greatest confusion is the management of thaw cycles. Some of this confusion stems from the mix of embryo types and transfer days available, and some to confusion surrounding naming conventions. In thaw-transfer cycles, we give P for 3 days before ‘day 3’ embryos (8 cells ideally) and for 5 days before ‘day 5’ embryos (blastocysts ideally) – see Fig 43.10. We expect to achieve synchrony between embryos and endometrium when: • We thaw embryos frozen 1 day after retrieval (prezygotes), 1 day after starting P, and transfer them 2 or more days later. • We thaw embryos frozen 2 days after retrieval (2–4 cells), 2 days after starting P, and transfer them on that day (or later). • We thaw embryos frozen 3 days after retrieval (4–8 cells), 3 days after starting P, and transfer them on that day (or later). • We thaw embryos frozen 5 or 6 days after retrieval (blastocysts), 5 days after starting P, and transfer them on that day.

Support in recipient cycles In donor egg cycles, the recipient must be synchronized with the donor to assure a receptive endometrium at the appropriate time. The ‘follicular’ phase of these cycles can

Other considerations Acupuncture has been reported to improve pregnancy rates in IVF.58 In this study, acupuncture treatment

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P4 (and E2) supplementation

14

Fig 43.8 In cycles of controlled ovarian hyperstimulation for IVF, progesterone (P4) supplementation is begun as early as the day of egg retrieval, and as late as 6 days afterwards, to support the luteal phase. Some programs also provide estradiol (E2) supplementation at the same time.

28

Fig 43.9 In programmed cycles using fresh embryos from donor eggs, progesterone (P4) replacement is begun as early as 2 days before the egg donor’s retrieval, and as late as the egg retrieval day itself. Estradiol (E2) replacement is provided throughout.

28

Fig 43.10 In programmed cycles using frozen embryos, the timing of progesterone (P4) replacement depends on the stage at which the embryos were frozen. As a rule, progesterone is given for at least as many days as the stage frozen, and in some programs, as many as 2 additional days. Thus, progesterone is given from 5 to 7 days before the thawing of blastocysts, and from 3 to 5 days before day 3 embryos are thawed. Estradiol (E2) replacement is provided throughout.

Optional start timing

−14

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−10

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just before and just after embryo transfer was associated with a significantly higher pregnancy rate (42.5% vs 26.3%). Since this is the only study yet available, it is not clear whether this effect is real, and what the mechanism of effect would be.

Summary P and E play central roles in preparation for and maintenance of human pregnancy. Until the luteoplacental shift occurs at about 7 weeks of gestational age, the ovary’s production of these hormones is critical to pregnancy maintenance. Beyond 7 weeks’ gestation, the placenta normally makes enough E and P to obviate any dependence on ovarian or exogenously supplied hormone. In most cases of contemporary ART, P supplementation is common practice. Various routes of administration have been developed and tried, but most have proved to have limitations. In many clinics i.m. delivery of progesterone has remained the principal practice, but is somewhat painful for patients and occasionally leads to sterile abscess formation or serious and prolonged allergic reaction. The vaginal route of P delivery has recently emerged as a promising alternative. Apart from its high patient acceptability, it also seems to offer more ‘targeted’ delivery of progesterone to the uterus.

References 1. De Ziegler D, Cornel C, Bergeron C, et al. Controlled preparation of the endometrium with exogenous estradiol and progesterone in women having functioning ovaries. Fertil Steril 1991; 56: 851–5. 2. Navot D, Laufer N, Kopolovic J, et al. Artificially induced endometrial cycles and establishment of pregnancies in the absence of ovaries. N Engl J Med 1986; 314: 806–11. 3. Roseff SJ, Bangah ML, Kettel LM, et al. Dynamic changes in circulating inhibin levels during the luteal-follicular transition of the human menstrual cycle. J Clin Endocrinol Metab 1989; 69: 1033. 4. Scott R, Navot D, Liu H-C, Rosenwaks Z. A human in vivo model for the luteoplacental shift. Fertil Steril 1991; 56: 481–4. 5. Csapo AI, Pulkkinen MO, Rutter B, et al. The significance of the human corpus luteum in pregnancy maintenance. Am J Obstet Gynecol 1972; 112: 1061–7. 6. Jones HW Jr, Jones GS, Hodgen GD, Rosenwaks Z, eds. IVF-Norfolk. Baltimore: Williams & Wilkins, 1986: 232. 7. Beckers NG, Macklon NS, Eijkemans MJ, et al. Nonsupplemented luteal phase characteristics after the administration of recombinant human chorionic gonadotropin, recombinant luteinizing hormone, or gonadotropin-releasing hormone (GnRH) agonist to induce final oocyte maturation in inhibitor fertilization patients after ovarian stimulation with recombinant follicle-stimulating hormone and GnRH antagonist cotreatment. J Clin Endocrinol Metab 2003; 88: 4186–92.

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8. Friedler S, Raziel A, Schachter M, et al. Luteal support with micronized progesterone following in vitro fertilization using a down-regulation protocol with gonadotrophin-releasing hormone agonist: a comparative study between vaginal and oral administration. Hum Reprod 1999; 14: 1944–8. 9. Hutchinson-Williams K, DeCherney AH, Lavy G, et al. Luteal rescue in in vitro fertilization-embryo transfer. Fertil Steril 1990; 53: 495–9. 10. Licciardi RL, Kwiatkowski A, Noyes NL, et al. Oral vs. intramuscular progesterone for in vitro fertilization: a prospective randomized study. Fertil Steril 1999; 71: 614–18. 11. Soliman S, Daya S, Collins J, Hughes EG. The role of luteal phase support in infertility treatment: a meta-analysis of randomized trials. Fertil Steril 1994; 61: 1068–76. 12. Daya S, Gunby J. Luteal phase support in assisted reproduction cycles. Cochrane Database Syst Rev 2004; CD004830. 13. Pritts EA, Atwood AK. Luteal phase support in infertility treatment: a meta-analysis of the randomized trials. Hum Reprod 2002; 17: 2287–99. 14. Fatemi HM, Popovic-Todorovic B, Papanikolaou E, Donoso P, Devroey P. An update of luteal phase support in stimulated IVF cycles. Hum Reprod Update 2007; 13: 581–90. 15. De Ziegler D, Fanchin R, Massonneau M, et al. Hormonal control of endometrial receptivity. The egg donation model and controlled ovarian hyperstimulation. Ann NY Acad Sci 1994; 734: 209–20. 16. Kolb BA, Paulson J. The luteal phase of cycles utilizing controlled ovarian hyperstimulation and the possible impact of this hyperstimulation on embryo implantation. Am J Obstet Gynecol 1997; 176: 1262–9. 17. Toner JP, Hassiakos DK, Muasher SJ, et al. Endometrial receptivities after leuprolide suppression and gonadotropin stimulation: histology, steroid receptor concentrations, and implantation rates. Ann NY Acad Sci 1991; 622: 220–9. 18. Toner JP, Singer GA, Jones HW Jr. Uterine receptivity after ovarian stimulation for assisted reproduction. In: Gianaroli L, Compana A, Trounson AO, eds. Implantation in Mammals. New York: Raven Press, 1993; 91: 231–8. 19. Abramowicz JS, Archer DF. Uterine endometrial peristalsis – a transvaginal ultrasound study. Fertil Steril 1990; 54: 451–4. 20. Fanchin R, Righini C, Olivennes F, et al. Uterine contractions as visualized by ultrasound alter pregnancy rates in IVF and embryo transfer. Hum Reprod 1998; 13: 1968–7. 21. Nyboe-Anderson A, Popovic-Todorivic B, Schmidt KT, et al. Progesterone supplementation during early gestations after IVF or ICSI has no effect on the delivery rates: a randomized controlled trial. Hum Reprod 2002; 17: 357–61. 22. Proctor Al, Hurst BS, Marshburn PB, et al. Effect of progesterone supplementation in early pregnancy on the pregnancy outcome after in vitro fertilization. Fertil Steril 2006; 85: 1550–2. 23. Williams SC, Oehninger S, Gibbons WE, et al. Delaying the initiation of progesterone supplementation results in decreased pregnancy rates after in vitro fertilization: a randomized, prospective study. Fertil Steril 2001; 76: 1140–3.

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24. Fanchin R, Righini C, De Ziegler D, et al. Effects of vaginal progesterone administration on uterine contractility at the time of embryo transfer. Fertil Steril 2001; 75: 1136–40. 25. Prapas Y, Prapas N, Jones EE, et al. The window for embryo transfer in oocyte donation cycles depends on the duration of progesterone therapy. Hum Reprod 1998; 13: 720–3. 26. Gelbaya TA, Kyrgiou M, Tsoumpou I, Nardo LG. The use of estradiol for luteal phase support in in vitro fertilization/intracytoplasmic sperm injection cycles: a systemic review and meta-analysis. Fertil Steril 2008; Jan 4 [Epub ahead of press]. 27. Fujimoto A, Osuga Y, Fujiwara T, et al. Human chorionic gonadotropin combined with progesterone for luteal support improves pregnancy rate in patients with low late-midluteal estradiol levels in IVF cycles. J Assist Reprod Genet 2002; 19: 550–4. 28. Farhi J, Weissman A, Steinfeld Z, et al. Estradiol supplementation during the luteal phase may improve the pregnancy rate in patients undergoing in vitro fertilization embryo transfer cycles. Fertil Steril 2000; 73: 761–6. 29. Gorkemli H, Ak D, Akyurek C, et al. Comparison of pregnancy outcomes of progesterone or progesterone + estradiol for luteal phase support in ICSI-ET cycles. Gynecol Obstet Invest 2004; 58: 140–4. 30. Levine H, Watson N. Comparison of the pharmacokinetics of Crinone 8% administered vaginally versus Prometrium administered orally in postmenopausal women. Fertil Steril 2000; 73: 516–21. 31. Nahoul K, Dehennin L, Jondet M, Roger M. Profiles of plasma estrogens, progesterone and their metabolites after oral or vaginal administration of estradiol or progesterone. Maturitas 1993; 16: 185–202. 32. Bourgain C, Devroey P, Van Waesberghe L, et al. Effects of natural progesterone on the morphology of the endometrium in patients with primary ovarian failure. Hum Reprod 1990; 5: 537–43. 33. Devroey P, Palermo G, Bourgain C, et al. Progesterone administration in patients with absent ovaries. Int J Fertil 1989; 34: 188–93. 34. Smitz J, Devroey P, Faguer B, et al. A prospective randomized comparison of intramuscular or intravaginal natural progesterone as a luteal phase and early pregnancy supplement. Hum Reprod 1992; 7: 168–75. 35. Miles R, Paulson R, Lobo R, et al. Pharmacokinetics and endometrial tissue levels of progesterone after administration by intramuscular and vaginal routes: a comparative study. Fertil Steril 1994; 62: 485–90. 36. Bulletti C, De Ziegler D, Flamigni C, et al. Targeted drug delivery in gynaecology: the first uterine pass effect. Hum Reprod 1997; 12: 1073–9. 37. Cicinelli E, De Ziegler D, Bulletti C, et al. Direct transport of progesterone from vagina to uterus. Obstet Gynecol 2000; 95: 403–6. 38. De Ziegler D, Fanchin R, Bergeron C, et al. Transvaginal administration of progesterone. Obstet Gynecol 1997; 90: 396–401. 39. Damario MA, Goudas VT, Session DR, et al. Crinone 8% vaginal progesterone gel results in lower embryonic implantation efficiency after in vitro fertilizationembryo transfer. Fertil Steril 1999; 72: 830–6.

40. Propst AM, Hill JA, Ginsburg ES, et al. A randomized study comparing Crinone 8% and intramuscular progesterone supplementation in vitro fertilization embryo transfer cycles. Fertil Steril 2001; 76: 1144–9. 41. Gibbons WE, Toner JP, Hamacher P. Experience with a novel vaginal progesterone preparation in a donor oocyte program. Fertil Steril 1998; 69: 96–101. 42. Jobanputra K, Toner JP, Denoncourt R, Gibbons WE. Crinone 8% (90 mg) given once daily for progesterone replacement therapy in donor egg cycles. Fertil Steril 1999; 72: 980–4. 43. Ludwig M, Schwartz P, Babahan B, et al. Luteal phase support using either Crinone 8% or Utrogest: results of a prospective, randomized study. Eur J Obstet Gynecol Reprod Biol 2002; 103: 48–52. 44. Kleinstein J; Luteal Phase Study Group. Efficacy and tolerability of vaginal progesterone capsules (Utrogest 200) compared with progesterone gel (Crinone 8%) for luteal phase support during assisted reproduction. Fertil Steril 2005; 83: 1641–9. 45. Geber S, Moreira AC, de Paulo SO, et al. Comparison between two forms of vaginally administered progesterone for luteal phase support in assisted reproduction cycles. Reprod Biomed Online 2007; 14: 155–8. 46. Lewin A, Pisov G, Turgeman R, et al. Simplified artificial endometrial preparation, using oral estradiol and novel vaginal progesterone tablets: a prospective randomized study. Gynecol Endocrinol 2002; 16: 131–6. 47. Doody K, Shamma FN, Paulson RJ, et al. Endometrin for luteal phase support in a randomized, controlled, open label, prospective IVF clinical trial using a combination of Menopur and Bravelle. Fertil Steril 2007; 87: S24. 48. Blake EJ, Norris PM, Yankov VI. A randomized, openlabel, single and multidose (single day and multipleday) pharmacokinetic study of a vaginal micronized progesterone tablet (Endometrin) compared to Crinone 8% vaginal gel in healthy reproductive-age female patients. Abstract presented at the 54th annual scientific meeting of the Society for Gynecologic Investigation, March 16, 2007, Reno, NV. 49. Zegers-Hochschild F, Balmaceda JP, Fabres C, et al. Prospective randomized trial to evaluate the efficacy of a vaginal ring releasing progesterone for IVF and oocyte donation. Hum Reprod 2000; 15: 2093–7. 50. Costabile L, Gerli S, Manna C, et al. A prospective randomized study comparing intramuscular progesterone and 17-alpha-hydroxyprogesterone caproate in patients undergoing in vitro fertilization-embryo transfer cycles. Fertil Steril 2001; 76: 394–6. 51. Unfer V, Casini ML, Costabile L, et al. 17 alphahydroxyprogesterone caproate versus intravaginal progesterone in IVF-embryo transfer cycles: a prospective randomized study. Reprod Biomed Online 2004; 9: 17–21. 52. Yanushpolsky E, Hurwitz S, Greenberg L, Racowsky C, Hornstein MD. Comparison of Crinone 8% intravaginal gel and intramuscular progesterone supplementation for in vitro fertilization/embryo transfer in women under age 40: interim analysis of a prospective randomized trial. Fertil Steril 2008; 89: 485–7. 53. Levine H. Luteal support from the vaginal progesterone (P) gel Crinone 8%: preliminary results of multicenter trial show higher pregnancy rates than

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The luteal phase: luteal support protocols historical controls. Poster #571, presented at the 47th annual meeting of the Society for Gynecological Investigation, 2000, Chicago, IL. 54. Gürbüz B, Yalti S, Ficicioglu C, et al. Bleeding patterns in women using intramuscular progesterone for luteal support in in-vitro fertilization cycles. J Obstet Gynaecol 2003; 23: 267–70. 55. Roman E, Aytoz A, Smitz JE, et al. Analysis of the bleeding pattern in assisted reproduction cycles with luteal phase supplementation using vaginal micronized progesterone. Hum Reprod 2000; 15: 1435–9.

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56. Yaron Y, Amit A, Mani A, et al. Uterine preparation with estrogen for oocyte donation: assessing the effect of treatment duration on pregnancy rates. Fertil Steril 1995; 63: 1284–6. 57. Younis J, Simon A, Laufer N. Endometrial preparation: lessons from oocyte donation. Fertil Steril 1996; 66: 873–84. 58. Paulus WE, Zhang M, Strehler E, et al. Influence of acupuncture on the pregnancy rate in patients who undergo assisted reproduction therapy. Fertil Steril 2002; 77: 721–4.

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44 Treatment strategies in assisted reproduction for the low responder patient Ariel Weissman, Colin M Howles

Overview In a spontaneous menstrual cycle, only one follicle out of a cohort of 10–20 usually completes maturation and ovulates to release a mature oocyte. The aim of controlled ovarian stimulation (COS) in assisted reproductive technologies (ART) protocols is to overcome the selection of a dominant follicle and to allow the growth of a cohort of follicles. This strategy leads to an increase in the number of oocytes and hence embryos available for transfer, thereby increasing the chance of transferring viable embryos. However, the chance of pregnancy and also live birth begins to dramatically decline after the age of 35 years old, and successful treatment for these patients continues to be a major challenge in ART programs. Preimplantation genetic screening studies over the last decade have identified a dramatic increase in the rate of aneuploidy as a major contributor to the reduction in embryo viability in older patients. It has also been demonstrated that women of advanced maternal age may have oocytes that are compromised by a significant reduction in the amount of mitochondrial DNA in their cytoplasm. In this chapter we describe the current strategies aimed at augmenting follicular recruitment and improve cytoplasmic integrity either in patients who are about to start COS and were estimated to have low ovarian reserve, or have been found to be poor responders in previous COS attempts. By standard and modified COS protocols as well as by various methods of manipulating endocrinology, the prognosis for these women is expected to be improved.

Introduction The decline in female fecundity associated with increasing age is well documented: the decrease occurs slowly through the 30s, and then accelerates to reach nearly zero between the ages of 45 and 50 years old.1 This decline can be based on a variety of agerelated conditions, including an increase in gynecological disorders such as endometriosis or fibroids, an

increase in ovulatory disorders due to effects on the hypothalamic–pituitary–ovarian axis, or a compromised uterine vascular supply that may impede implantation.2 Spontaneous conception is rare in women >45 years: a study carried out in orthodox Jewish sects that are proscribed from using contraceptives showed that natural pregnancies and deliveries after the age of 45 years old constitute only 0.2% of total deliveries, and >80% of these are in grand multiparas.3 Similar findings have been described in Bedouin women as well.4 In infertile couples, in vitro fertilization (IVF) may be a reasonable option for such women of advanced maternal age (>40 years) but at the age of 45 years and more deliveries were only reported if a women had > 5 oocytes retrieved.5 The peak number of oocytes present in the human ovary occurs during fetal gestation, and follicles are continually lost thereafter through the mechanism of apoptosis, a process known as atresia.6 A cohort of growing follicles is recruited each month, and the cohort enters the final stages of follicle maturation during the first half of the menstrual cycle. This maturation phase is gonadotropin dependent. Painstaking histological and in vitro studies carried out by Gougeon7 suggest that follicles require a period of approximately 70 days from the time they enter the preantral stage (0.15 mm) to reach a size of 2 mm. These 2-mm follicles have very low steroidogenic activity, and they are impervious to cyclic follicle-stimulating hormone (FSH) and luteinizing hormone (LH) changes in terms of granulosa cell (GC) proliferation. Over a 4–5 day period during the late luteal phase, follicles that are 2–5 mm in diameter enter a recruitment stage, and cyclic changes in FSH drive the development of the follicle and proliferation of GC; GC aromatase activity is not affected during this stage. Thus, as the follicle develops, it becomes increasingly responsive to gonadotropins. In the perspective of treatment management, this means that in order to influence the size of the recruitable pool of follicles, it would be necessary to ‘boost’ continued healthy follicle development over a protracted period of time (≥70 days). However, gonadotropins play a role only during the phases of recruitment and final follicular maturation,

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Percent

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22

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32 34 36 38 Age (years) Live birth date

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44

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Fig 44.1 ART success rates in USA 2004: CDC report 2006. Over the age of 35 there is a further acceleration in the decline in pregnancy rate following ART procedures.10

which occur over the last 20 days or so of this 70-day period. Therefore, extrapolating from knowledge about basic physiology, different agents would be required at different times in order to successfully reverse the agerelated decline in follicle numbers. It has also been suggested that the rate of ovarian oocyte depletion further accelerates after the age of 37 years old,8 so that older women have a decreased reserve of healthy oocytes in their ovarian pool. Women who postpone childbearing until their late 30s or early 40s are therefore frequently faced with the distressing realization that their chance of achieving a pregnancy is significantly reduced, and that they may require the help of assisted reproductive techniques, with further complex difficulties that can jeopardize their quest for successful conception. In Europe for the year 2004, women undergoing IVF or intracytoplasmic sperm injection (ICSI) procedures in the age group >40 years old now represent approximately 15% and 12.5%, respectively, of those attending IVF clinics.9 A number of different variables can affect success rates in ART, and the negative impact of increasing age is one feature that is well recognized. Not only does the response to stimulation steadily deteriorate, requiring larger amounts of gonadotropins, but also the cancellation rate is higher, and there is a significant increase in the rate of miscarriage. Data from the USA (Center for Disease Control 2004 report on ART success rates)10 clearly shows that the potential for embryo implantation and successful delivery of a live birth decreases rapidly in women >35 years old (Fig 44.1). This same report also documents the increased incidence of pregnancy loss that is related to increased maternal age, going from less than 12% in women <35 years old, then increasing from the mid 30s to reach 28% at 40 years old and 59% in women >43 years old. These data suggest that the lower age limit to define women of advanced

maternal age (AMA) should be considered as ≥35 years old. The most effective treatment option for infertile women of AMA is oocyte donation from young donors, but this is not an option in some parts of the world. Dal Prato et al11 reported a single case of a woman who successfully delivered a child after IVF treatment at the age of 46 years old, but emphasized that this is an extraordinary event, and is not a costeffective option compared with oocyte donation. Any strategy that might enhance the efficacy of treatment for these women would be of great benefit, and different areas of research have recently been explored, such as the use of pharmacogenomics to assess response to gonadotropin stimulation, manipulating the endocrinology of the treatment cycle, and screening of embryos for aneuploidy.

Ovarian response to stimulation An initial challenge in treatment of the older woman or women with low ovarian reserve is the fact that the process of biological aging often renders the ovaries increasingly resistant to gonadotropin stimulation, with the result that the number of oocytes harvested may be very low. The ability to accurately assess and predict ovarian response would reduce the burdens imposed by failure because of inadequate response to stimulation. Unfortunately, the response to stimulation cannot be reliably predicted, even for young patients with no evidence of endocrine disorders. Parameters that have been identified as exerting an influence include age,12–14 cause of infertility (Centers for Disease Control, 2004),10 body weight,15,16 and body mass index (BMI).17 Ovarian characteristics have also been assessed by ultrasound, such as the number and size of antral follicles, ovarian volume, and ovarian vascular resistance measured by Doppler ultrasound. There is a clear correlation between the number of antral follicles (defined as ≥2 mm to ≥11 mm) seen at the beginning of the follicular phase during a natural cycle and subsequent ovarian response to stimulation. However, there is as yet no consensus of agreement regarding the minimum number of antral follicles below which an influence can be seen;18–22 a minimum of less than 5 follicles of 2–5 mm diameter has been suggested as a predictive parameter.23 One of the major reasons for this is that there is not a standardized definition for the measurement of antral follicle count (AFC), whose accuracy of measurement is highly operator dependent. Small ovaries and high resistance to vascular flow have also been shown to correlate with poor ovarian response to gonadotropins.20,23–25 Klinkert et al23 suggest that patients with an AFC of <5 follicles of 2–5 mm diameter are expected to have a poor response, and in a randomized controlled trial they recently demonstrated that doubling the starting doses of gonadotropins does not lead to an improvement in

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response for these patients during IVF treatment. In this study of 52 patients, more than half were aged >40 years old, and 13 had basal FSH levels >15 IU/l. A recent meta-analysis26 has indicated that the predictive performance of ovarian volume toward poor response is clearly inferior compared with that of AFC. Basal hormone assessment at the beginning of the follicular phase has been used to predict ovarian response, including FSH,13,27–33 estradiol (E2),33,34 inhibin B,33–38 and more recently anti-Müllerian hormone (AMH).39–43 There have been attempts to develop models for ovarian response based upon algorithms made up of multiple predictive factors. For instance, PopovicTodorovic and colleagues developed a scoring system for calculating the FSH starting dose, based on four predictors: the total number of antral follicles, total Doppler score, serum testosterone levels, and smoking habit.20 This model was tested prospectively in a two-site clinical study, in which an ongoing pregnancy rate of 36.6% was reported using the algorithm to assign starting FSH doses between 100 and 250 IU, compared with an ongoing pregnancy rate of 24.4% with a standard protocol using 150 IU FSH.44 Another predictive algorithm to predict the r-hFSH (follitropin alfa) starting dose has been described but is only applicable to young (<35 years old), normogonadotropic women.45 The four factors identified as significantly predictive of ovarian response were baseline serum FSH levels, BMI, age, and AFC. The basal and dynamic tests for prediction of ovarian reserve and response to stimulation are described in detail in Chapter 54. Different clinics and investigators have their own preferences and protocols for evaluation of ovarian reserve. From a practical point of view, it is interesting to note that a recent comprehensive systematic review of tests predicting ovarian reserve and IVF outcome46 has concluded that since poor ovarian response to stimulation will provide some information on ovarian reserve status, especially if the stimulation is maximal, entering the first cycle of IVF without any prior testing seems to be the preferable strategy. Therefore, this chapter will focus on classic and specialized protocols designed for low responder patients, as well as on hormonal and pharmacologic manipulations which are expected to improve ovarian response. A large variety of strategies have been developed to improve outcome in patients with low ovarian reserve. Indeed, all of the currently available COS protocols have been used, with or without modifications, for the treatment of low responders. Unfortunately, all of these approaches have met with only limited success.47–51

High-dose gonadotropins It is generally believed that in an attempt to overcome the age-related decline in ovarian response after FSH

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stimulation, the dose of gonadotropins should be adjusted upwards. Patients who responded poorly to conventional doses (150–225 IU of FSH) may produce more follicles when given 300–450 IU or even 600 IU per day. It is expected that an enhanced response would lead to increases in the number of oocytes retrieved, the number of embryos available, and, ultimately, an increase in pregnancies and live births.52–55a These expectations, however, are not always met, and this strategy is often of limited effectiveness. Although higher circulating levels may be achieved by increasing the quantity of gonadotropins being administered, at some point saturation kinetics are attained52,56 and the ovarian response is determined more by the number of follicles available for recruitment than by circulating gonadotropin levels. This is of particular importance, since low responders generally have markedly diminished numbers of follicles available for recruitment, as reflected in low AFC. Furthermore, Ben-Rafael and colleagues53 reported poorer oocyte quality with the use of incrementally higher gonadotropin doses in their normally responding patients. It is not clear, however, whether poor oocyte quality results from exposure to high-dose gonadotropins or, more likely, is another reflection of low ovarian reserve. However, in a recent study57 of young normogonadotropic ART patients there was no indication of an adverse effect on pregnancy outcomes of higher daily doses, if FSH was given according to an algorithm based upon the four patient characteristics described by Howles et al.45 Very few studies have been conducted on the effects of increasing the dose of gonadotropins in low responders, the vast majority of the studies being small and retrospective. The available studies suffer from heterogeneity with regard to the definition of low responders, treatment protocols (use or nonuse of GnRH agonists), and main outcome measures. An interesting question is whether it is possible to rescue a cycle with initial poor response by doubling the gonadotropin dose after stimulation has already started. A prospective randomized study,58 evaluated the effect of doubling the human menopausal gonadotropin (hMG) dose in the current cycle in which the ovarian response after 5 days of ovarian stimulation with 225 IU/day was considered ‘low.’ No effect of doubling the hMG dose was noted with regards to the length of ovarian stimulation, peak E2 values, number of follicles >11 and >14 mm in diameter on the day of hCG administration, number of canceled cycles, number of oocytes retrieved, and the number of patients with ≤3 oocytes retrieved. It was concluded that doubling the hMG dose in the course of an IVF cycle is not effective in enhancing ovarian response. This is in accordance with current understanding of follicular growth dynamics, which states that follicular recruitment occurs only in the late luteal and early follicular phase of the previous menstrual cycle.

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Another relevant issue is whether high-dose stimulation should be continued throughout the stimulation phase, or the gonadotropin dose can be reduced (step down) without compromising ovarian response. In a prospective randomized study,59 a high fixed vs a step-down dose of gonadotropins on a flare-up GnRH agonist (GnRH-a) regimen were compared. Patients were pretreated for 14 days with progestin, followed by 100 µg triptorelin from day 1, reduced to 25 µg/day from day 3 of the cycle. Purified FSH at a dose of 450 IU/day was administered on days 3–5 and on cycle day 6 patients were randomized to receive either in a fixed dose of 450 IU/day or decreasing to 300 IU/day, and finally to 150 IU/day. Cancellation rates, serum E2, oocyte yield, fertilization, and pregnancy rates were similar for both groups. The duration of stimulation and gonadotropin requirements were both significantly reduced with the step-down regimen. It was concluded that in poor responders undergoing COS on a short protocol with high-dose gonadotropins, the dose of gonadotropins can be safely reduced during the second part of the follicular phase, when follicles have attained high sensitivity to gonadotropins, without compromising cycle outcome. Thus, the step-down regimen offers a substantial economic benefit and some centers routinely use this approach in order to ensure that the available follicle cohort is exposed as early as possible to elevated FSH concentrations so as to reduce the possibility of late cycle cancellation.60 In summary, increasing the starting dose of gonadotropins in poor responders is a rational approach that is widely practiced. A common starting dose would be at least 300 IU/day. Nevertheless, further dose increments are of limited effectiveness, and clinically meaningful improvements are only rarely obtained with doses >450 IU/day.

GnRH agonists in the treatment of poor responders Long GnRH-a protocols The use of GnRH-as has gained widespread popularity, and most ART programs use this approach as the predominant method of ovarian stimulation. A metaanalysis of randomized and quasi-randomized controlled trials showed that use of GnRH-as reduced cancellation rates, increased the number of oocytes retrieved, and improved clinical pregnancy rates per cycle commenced and per embryo transfer (ET), compared with conventional stimulation regimens without the use of GnRH analogs.61 The aim of the long protocol is to achieve pituitary down-regulation with suppression of endogenous gonadotropin secretion before stimulation with exogenous gonadotropins. Once pituitary down-regulation and ovarian suppression are achieved, ovarian stimulation with exogenous gonadotropins is commenced while GnRH-a

administration is continued concomitantly until the day of hCG administration. In the general IVF population, the long protocol has been found to be superior in terms of efficacy compared with the short protocol62 and is therefore most frequently used. However, the matter of which GnRH-a protocol is preferable in low responders remains controversial. Because of its substantial medical and practical advantages, the use of the long GnRH-a protocol was extended also to the treatment of low responder patients undergoing IVF. Although early studies were optimistic and suggested that long GnRH-a protocols could be beneficial for low responders,63–67 subsequent clinical experience has yielded disappointing results. Down-regulation of the hypothalamic–pituitary–ovarian axis prior to gonadotropin therapy is often associated with prolongation of the follicular phase and a significant increase in the number of gonadotropin ampules required to achieve adequate follicular development. The extent of this increase is far greater than what could be attributed to simply delaying hCG administration to the point where a larger cohort of homogeneously well-synchronized large follicles are present. Moreover, in some relatively young patients with normal ovarian reserve, it was found difficult to induce any ovarian response in the presence of pituitary down-regulation, even with very large doses of exogenous gonadotropins.68–72 Normal ovarian function was restored in these patients after withdrawal of the GnRH-a, with subsequent normal response to hMG.70,71 These early observations indicated that GnRH-as may induce a state of ovarian hyporesponsiveness, the mechanism of which has never been clearly clarified. Several theories have been suggested in an attempt to explain the dramatic (often a two-fold) increase in exogenous gonadotropin requirements during pituitary down-regulation: 1. 2. 3. 4.

Diminished circulating endogenous gonadotropin levels.68,69 Altered biologic activity of endogenous gonadotropins.73–75 Interference with follicular recruitment.76 Direct ovarian inhibition effects by GnRH-as.77–79

Thus, in many low responder patients, ovarian suppression with GnRH-a resulted in excessive dampening of the ovarian response to COS and complete refractoriness to gonadotropin stimulation.80 Consequently, cancellation rates due to lack of ovarian response were unacceptably high, and hormonal stimulation was excessively prolonged, with increased cost and duration of treatment and only a marginal benefit in the mean E2 maximum response and the yield of mature oocytes.81–83 It has been well established that there is a dosedependent duration of ovarian suppression after single implant injections of GnRH-a, and that in a

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suppressed pituitary gland the dose needed to maintain suppression gradually decreases with the length of treatment.84 This supports the concept of stepdown GnRH-a protocols, where the dose of the agonist is decreased once the criteria for ovarian suppression have been achieved. Furthermore, the minimal effective dose for sufficient pituitary suppression with GnRH-as has not been thoroughly studied before their actual introduction to clinical practice. Regarding triptorelin, for example, Janssens et al,85 in a prospective, placebo-controlled doubleblind study, demonstrated that daily administration of 15 µg of triptorelin is sufficient to prevent a premature LH surge, and that 50 µg is equivalent to 100 µg in terms of IVF results. In an attempt to maximize ovarian response without losing the benefits of GnRH-a down-regulation, Feldberg et al, 86 introduced use of the mini-dose GnRH-a protocol in low responders with elevated basal FSH levels. They found that patients who received daily triptorelin, 100 µg subcutaneous (SC) from the midluteal phase until menstruation and 50 µg thereafter, had higher peak E 2 levels, more oocytes recovered, and more embryos transferred. They also noted a trend toward improved pregnancy and implantation rates and a lower spontaneous abortion rate. Olivennes et al87 studied 98 IVF patients with a high basal FSH concentration who were previously treated by the long protocol with a GnRH-a in a depot formulation. The same patients received SC leuprolide acetate (LA) 0.1 mg/day from cycle day 21, reducing it to 0.05 mg/day upon down-regulation. The comparison was made using the previous IVF cycle of the same patient as a control. The use of a low-dose agonist protocol resulted in significantly reduced gonadotropin requirements, a shorter duration of stimulation, a higher E2 concentration on stimulation day 8, a higher number of mature oocytes, and a higher number of good-quality embryos. The cancellation rate was lower (11 vs 24%). Kowalik et al78 have demonstrated that lowering the dose of LA resulted in a faster E2 rise and higher mean peak E2 level. The higher E2 levels were obtained with a lower total gonadotropin dose. The oocyte yield was not affected. It was concluded that lowering the dosage of LA allows higher E2 response, which suggests an inhibitory in vivo effect of LA on ovarian steroidogenesis. Davis and Rosenwaks72 reported similar results using a low-dose LA protocol. Weissman et al88 prospectively compared two stimulation protocols specifically designed for low responder patients. Sixty low responders who were recruited on the basis of response in previous cycles received either a modified flare-up protocol in which a high dose of triptorelin (500 µg) was administered for the first 4 days, followed by a standard dose (100 µg), or a mini-dose long protocol in which 100 µg triptorelin was used until pituitary down-regulation, after which the

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triptorelin dose was halved during stimulation. Twentynine cycles were performed with the modified flare-up protocol and 31 were performed with the mini-dose long protocol. Significantly more oocytes were obtained with the modified long protocol than the modified flare protocol. The number and quality of embryos available for transfer was similar in both groups. One clinical pregnancy (3.4%) was achieved with the modified flare protocol, and 7 pregnancies (22.5%) were achieved using the mini-dose long protocol. Ovarian cyst formation is a common complication of the long GnRH-a protocol. It has been suggested as being typical for low responders and as being a reliable predictor of poor stimulation and low pregnancy rates in a given cycle.89,90 Although the pathophysiology of ovarian cyst formation following GnRH-a administration has not been completely elucidated, the higher the serum progesterone level at the time of commencing GnRH-a administration, the lower the incidence of cyst formation.91 Progestagen pretreatment directly inhibits endogenous gonadotropin secretion and influences the pattern of gonadotropin and hypothalamic GnRH secretion. Three prospective, randomized studies have demonstrated the successful use of progestins to prevent ovarian cyst formation during pituitary suppression in IVF cycles.92–94 We have also included successfully progestagen pretreatment in the long mini-dose protocol (88). Summarizing the above studies and observations, it appears that if the long GnRH-a protocol is to be used in low responders, then a reduced agonist dose (‘mini-dose’) is superior to a standard agonist dose in terms of oocyte yield and cycle outcome, and this approach should be preferably employed.

GnRH-a ‘stop’ protocols Pituitary recovery and resumption of gonadotropin secretion following GnRH-a treatment may take up to several weeks, depending on the dose and route of administration of the agonist. For example, with intranasal buserelin acetate (BA), suppression of endogenous gonadotropin secretion seems to continue for at least 12 days after the discontinuation of the agonist,95 as was also reported for SC BA.96 Interestingly, using the ‘ultrashort protocol,’ suppression of endogenous LH secretion was more profound when LA administration was stopped after 5 days of administration, compared with continuous LA administration, and no premature LH peak was recorded.97 This forms the basis for a variety of discontinuous GnRH-a protocols. The above observations prompted several studies in which GnRH-as were administered in the long protocol, but agonist administration was withheld once gonadotropin stimulation had started.98–102 The majority of studies have shown favorable results in terms of both cost-effectiveness and clinical outcome, but studies showing discouraging results were also

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reported.102 Corson et al98 prospectively evaluated the effect of stopping GnRH-a (SC LA) therapy upon initiation of ovarian stimulation vs simultaneous GnRH-a and gonadotropin therapy. Both groups were found comparable in terms of the duration of stimulation and amount of exogenous gonadotropins required, as well as for any other stimulation or outcome parameter studied. Stopping LA upon initiation of ovarian stimulation did not reduce its efficacy in suppressing LH secretion, as in neither group was a premature LH surge detected. Similar results were obtained in a prospective study that compared two protocols with variable duration of BA administration in an IVF/GIFT program.99 No spontaneous premature LH surges were recorded in any of the groups, and all parameters of ovarian response to stimulation were found comparable for both groups. A trend towards a higher pregnancy rate per transfer was noted in the discontinuous BA arm. Simons et al100 compared the efficacy of two early cessation protocols of triptorelin treatment with the conventional long protocol in IVF. In a double-blind, randomized, multicenter study, 178 women were randomized to one of three treatment groups at the start of stimulation. SC triptorelin was started at the midluteal phase of the previous cycle and continued until the first day of gonadotropin treatment (group S), or up to and including the fourth day of gonadotropin treatment (group M) or the day of hCG injection (group L). One premature LH surge was

(a)

observed in group M but not in groups S and L. Both early cessation protocols (S and M) were at least as effective as the long protocol (L) with regard to the number of oocytes (11.1 and 10.3 vs 9.3), number of embryos (7.3 and 6.5 vs 5.5), and ongoing pregnancy rate (28% and 24% vs 21%). It was concluded that early cessation of triptorelin on day 1 of gonadotropin treatment is as effective as the traditional long protocol in preventing a premature LH surge and results in similar reproductive outcome. In contrast, Fujii et al102 reported on a prospective randomized study, where 900 µg/day of intranasal BA were administered from the mid-luteal phase of the previous cycle until cycle day 7, when normal responding patients were randomized to receive either gonadotropin stimulation alone (group I) or combined BA and gonadotropin therapy (group II). The duration and total dose of gonadotropins administered were significantly increased in group I, as compared with the conventional long protocol (group II). The number of fertilized oocytes and embryos transferred were significantly lower and the cancellation rate and rate of failed oocyte retrieval were significantly higher in the discontinuous long protocol. Although premature LH surges were not recorded in either group, serum progesterone and LH concentrations were significantly increased on the day of hCG administration with the discontinuous long protocol. Clinical pregnancy rates per transfer were similar for both protocols. It was concluded that early discontinuation of

Day 1 7 – 8 days after estimated ovulation or cycle day 1

Day of hCG

Day 6

FSH

Individualized Dosing of FSH/LH

OCP? GnRH agonist

Progestin?

GnRH agonist

Down-regulation

Day 1

(b) 7 – 8 days after estimated ovulation or cycle day 1 OCP? Progestin?

GnRH agonist

FSH

Day 6

Individualized dosing of FSH/LH

Reduced dose of GnRH agonist

Down-regulation

Fig 44.2

(a) The long GnRH agonist protocol. (b) The ‘mini-dose’ long agonist protocol.

Day of hCG

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the GnRH-a is not beneficial and not cost-effective because of its adverse effects on follicular development and increased exogenous gonadotropin requirements, respectively. A reason for this could be because stopping daily agonist administration combined with ovarian stimulation leads to a further reduction in circulating LH concentrations,103 which supports the concept that there is still a small release of LH following daily agonist administration. Discontinuous protocols were considered to be potentially beneficial for low responder patients undergoing IVF-ET.104 Several trials with contradictory results have been reported. Faber et al.105 conducted a single group uncontrolled study, in which low responder patients were treated with LA, 0.5 mg/day starting at the mid-luteal phase of the previous cycle. With the onset of menses, LA was discontinued and high-dose gonadotropin therapy was initiated. The cancellation rate was 12.5% (28/224 cycles), and only one case of premature LH surge was observed. Despite the uncontrolled nature of the study, a clinical pregnancy rate per transfer of 32% and ongoing pregnancy rate per transfer of 23%, which seemed highly favorable for the specific subgroup of low responder patients, were achieved. Subsequently, Wang et al106 conducted a prospective nonrandomized study to determine the efficacy of a ‘stop’ protocol in previously poor responders to a standard long protocol. Fifty patients were scheduled for 52 cycles of the modified ‘stop’ agonist protocol. All patients received GnRH-a from the midluteal phase of the previous cycle to the onset of menstruation, followed by high-dose gonadotropin stimulation. Six of the 52 cycles (11.8%) were canceled because of poor ovarian response. One premature ovulation was noted, and in the other 45 cycles, an average of 6.3 mature oocytes was retrieved. A favorable embryo implantation rate (11.5%) and an acceptable clinical pregnancy rate (20.5%) were noted. In a prospective study with historical controls and involving 36 poor responders, the use of intranasal nafarelin (600 µg/day) commenced in the midluteal phase and discontinued on day 5 of ovarian stimulation, was evaluated.107 The cancellation rate was 8.3%, and there was a trend towards increased peak E2 levels and an increase in the number of oocytes retrieved. The ongoing pregnancy rate per embryo transfer was 15%. A significant improvement in both the number and the quality of cleaving embryos was observed, and it was therefore suggested that discontinuation of the GnRH-a leads to improved oocyte quality. In another prospective study with historical controls,108 39 ‘stop’ nafarelin cycles in 30 previously poor responder patients were compared to 60 past cycles in the same individuals. A significantly higher number of oocytes were retrieved and a higher number of embryos were available for transfer. No cases of premature LH surge were recorded. Pregnancy rates per embryo transfer and per cycle were 10.4% and 7.7%, respectively.

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In contrast, Dirnfeld et al109 reported on a prospective randomized, controlled trial involving 78 cycles, in which a ‘stop agonist’ regimen was compared with a standard long luteal protocol. Intranasal BA (1 mg/day) or SC triptorelin (0.1 mg/day) was initiated on day 21 of the previous cycle and ceased once pituitary suppression was confirmed. Ovarian stimulation was induced with the use of 225–375 IU/day hMG or purified FSH, commencing on the day of down-regulation. A significantly higher cancellation rate was noted with the stop regimen compared with the controls (22.5% vs 5%, respectively). The stop and long regimens resulted in similar stimulation characteristics and clinical pregnancy rates (11% vs 10.3%, respectively). Only in patients with a basal FSH level that was not persistently high did the stop regimen result in a significantly higher number of retrieved oocytes compared with the standard long protocol (7.6 vs 4.0, respectively). It was concluded that, for most low responders, the stop regimen offers no further advantage over the standard long protocol. Similarly, Garcia-Velasco et al110 designed a prospective, randomized controlled trial in order to evaluate whether early cessation of the GnRH-a (LA) is more beneficial than just increasing the doses of gonadotropins in low responder patients. Seventy low responder patients with normal basal FSH concentrations and a previous canceled IVF cycle were randomly allocated to either a standard long protocol or a stop protocol. A significantly higher number of mature oocytes were obtained with the stop protocol compared with the standard long protocol (8.7 vs 6.2). The stop protocol significantly reduced the gonadotropin requirements. Both protocols resulted in a similar cancellation rate (2.7 vs 5.8%), pregnancy rate (14.3 vs 18.7%), and implantation rate (12.1 vs 8.8%). It was concluded that the stop protocol combined with high doses of gonadotropins permitted the retrieval of a significantly higher number of oocytes, but did not influence the reproductive outcome.

Short GnRH-a regimens In patients who fail to obtain adequate multifollicular growth with the long GnRH-a protocol, one of the options is to decrease the length of suppression by decreasing the duration of GnRH-a use (short and ultrashort regimens). The short protocol consists of early follicular phase initiation of GnRH-as, with minimal delay before commencing gonadotropin ovarian stimulation. It takes advantage of the initial agonistic stimulatory effect of GnRH-a on endogenous FSH and LH secretion, also known as the flare-up effect. In theory, it eliminates excessive ovarian suppression associated with prolonged agonist use. The duration of the endogenous gonadotropin flare has not been completely characterized, but pituitary desensitization is generally achieved within 5 days of initiating treatment,111 and therefore,

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patients are protected from premature LH surges by the end of the stimulation phase. The short protocol has been proposed by many authors as a better stimulation protocol for low responders.112–114 Although widely used in low responders, no prospective randomized controlled trials comparing the efficacy of the short protocol with standard long protocols have been published. In an early prospective study with historical controls and using an ultrashort protocol, Howles et al114 treated seven patients who had previously responded poorly to stimulation with clomiphene citrate (CC) and hMG with 0.5 mg/day BA during only the first 3 days of the cycle (ultrashort protocol). All seven patients had oocytes recovered, and embryos replaced, and three out of these seven conceived (42.9%). Similarly, Katayama et al115 reported improved cycle outcomes in seven prior poor responder patients with the short regimen. Garcia et al113 conducted a nonrandomized prospective trial comparing long luteal and short flare-up agonist initiation in 189 cycles. They noted a significant decrease in exogenous gonadotropin requirements, higher pregnancy rates, and decreased miscarriage rates in patients receiving the flare-up regimen. In a retrospective comparison, Toth et al116 also reported that pregnancy and implantation rates were significantly higher and cancellation rates lower in patients with basal serum FSH levels ≥15 mIU/ml undergoing a flare-up regimen vs a long luteal agonist regimen. In a prospective uncontrolled study, Padilla et al112 administered a flare-up protocol with high-dose gonadotropins to 53 patients who were thought to be at risk for poor response after a ‘leuprolide acetate screening test’. The cancellation rate was higher in poor flareup LA test responders (11.3%) compared with good flare-up LA responders (1.1%) and luteal phase long protocols cycles (1.8%). Despite a low number of oocytes retrieved, the ongoing pregnancy rate was 29% per retrieval and was considered favorable for this group of potentially poor responder patients. Despite these encouraging findings, other authors failed to confirm any substantial benefit of using a classic flareup protocol. In a prospective study with historical controls,117 80 poor responders were treated using a classic flare-up regimen with LA 0.5 mg/day from cycle day 2, and high-dose hMG from cycle day 3. While the number of retrieved oocytes was increased (10 ± 6.6), the cancellation rate was high (23.4%), and the ongoing pregnancy rate (PR) of 6.5% per retrieval and 7.6% per transfer were disappointing. Brzyski et al118 reported that not only did concomitant initiation of GnRH-a with purified urinary FSH result in poorer cycle outcome but also an increased number of atretic oocytes were retrieved. A significant increase in LH and progesterone levels during the follicular phase was noted. Other groups using this approach also reported failure to improve ovarian response or cycle outcome in generally similar patient populations.119–121 In a randomized prospective study, San Roman et al122 have shown that a combination of early follicular phase LA administration and hMG stimulation was associated with a significant increase in serum LH

levels, beginning with the first follicular phase agonist dose, and with significant increases in serum progesterone and testosterone levels during the follicular phase, compared with midluteal GnRH-a administration. The live birth rate/retrieval for the long protocol was 25% as compared with 3.8% in the flare-up group. All these adverse effects may result through the initial flare-up effect of GnRH-as on LH secretion, with a subsequent increase in intrafollicular androgen levels. The androgen-rich environment may interfere with the process of folliculogenesis by untimely inhibition of meiosis inhibiting factors, leading to overaging of oocytes, with a significant reduction in their fertilization and implantation capacity.123–125 Evidence of an adverse effect of high endogenous LH level during the follicular phase has led to the establishment of the ceiling theory.126 According to this theory, beyond a certain ceiling level, LH suppresses granulosa cell proliferation and initiates atresia of less mature follicles. Further support for this view comes from a study of Gelety et al,127 who performed a prospective randomized crossover study of five regularly cycling women in order to determine the short-term pituitary and ovarian effects of GnRH-a administered during differing phases of the menstrual cycle in the absence of gonadotropin stimulation. Each patient was administered LA 1 mg/day SC for 5 days beginning on cycle day 3, 8 days post-LH surge, and 13 days post-LH surge with an intervening ‘washout’ month. Significant increases in serum LH, E2, estrone, androgens, and progesterone levels were noted in the early follicular phase group, compared with the midluteal group. Early follicular initiation of the agonist resulted in a more pronounced suppression of FSH. It was suggested that relative FSH suppression and marked androgen elevations could have potential detrimental effects on oocytes of the developing cohort that are often observed with flare-up regimens. Can the ill effects of the gonadotropin flare be prevented without losing the potential benefits of the short protocol? Two possible solutions have been suggested: the first is pretreatment with an oral contraceptive pill (OCP) or a progestin. Cédrin-Durnerin et al128 noted that pretreatment with a 12–20 day course of the progestin norethisterone before initiation of a flare-up regimen effectively lowered LH and progesterone levels during the early stages of gonadotropin stimulation. Many clinicians thus regard pretreatment with an OCP or a progestin as integral in flare-up regimens, although this issue also became a matter of controversy.129 The second solution is dose reduction of the GnRH-a causing the flare, which forms the basis for ‘micro-dose flare’ regimens (Fig 44.3).

Micro-dose flare GnRH-a regimens In theory, micro-flare regimens decrease the enhanced LH, progesterone, and androgen secretion associated with standard flare-up regimens, as described above. Bstandig et al130 studied the hormonal profiles during the flare-up period using 25 µg and 100 µg of triptorelin

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OCP GnRH agonist

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GnRH agonist

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in the short protocol. No significant difference in the magnitude of FSH and E2 release was observed between the two groups, but the maximal plasma LH level was significantly reduced after injection of 25 µg of triptorelin. It was suggested that in the flare protocol, a lower dose of GnRH-a induces a hormonal flareup that is more conducive for optimal follicular recruitment. Deaton et al131 have demonstrated that an extremely low dose of LA (25 or 50 µg) is needed to cause a pituitary flare of gonadotropins. Following a flare from 25 µg of LA on cycle day 2, the pituitary is able to recover and respond with a repeat flare on cycle day 5. These observations support the rationale behind the so-called micro-dose flare protocols. Navot et al132 studied the effect of very low doses of GnRH-a in cynomolgus monkeys and humans and established that 10 mg of historelin in four divided doses (micro-doses) could induce ovarian hyperstimulation in humans. Scott et al133 reported that an increase in gonadotropin levels could be induced in baboons with LA doses as low as 0.017 mg/kg. Although the minimal and optimal effective dose of GnRH-as that can be successfully used to induce a gonadotropin flare in humans has not been thoroughly evaluated, several investigators have reported an improved outcome with doses as low as 20–40 µg of LA twice daily in low responders. In a prospective study with historical controls, Scott and Navot134 treated 34 low responder patients with an OCP followed by 20 µg LA twice daily, beginning on cycle day 3 and supplemented with exogenous

Fig 44.3 (a) The short GnRH agonist protocol. (b) The ‘mircro-dose’ flare GnRH agonist protocol.

gonadotropins, beginning on cycle day 5. Ovarian responsiveness was enhanced with the micro-dose GnRH-a stimulation cycle when compared with previous stimulation cycles. Specifically, the patients had a more rapid rise in E2 levels, much higher peak E2 levels, the development of more mature follicles, and the recovery of larger numbers of mature oocytes. None of the patients had a premature LH surge. Impressive results using the micro-dose flare protocol were also reported in a prospective study with historical controls.135 Thirty-two patients, whose prior long luteal agonist cycles had been canceled because of poor response, were now pretreated with an OCP followed by follicular phase administration of 40 µg LA twice daily beginning on cycle day 3 and high-dose FSH supplemented with growth hormone (GH) beginning on cycle day 5. Compared with the prior long luteal GnRHa cycle, there was a higher E2 response, more oocytes retrieved (10.9 per patient), fewer cycle cancellations (12.5%), and no premature LH surge or luteinization. For patients who were not canceled, a favorable ongoing pregnancy rate of 50% was achieved. In a prospective nonrandomized trial with historical controls, Surrey et al136 treated 34 patients with a prior poor response to a standard midluteal long protocol with an OCP followed by LA 40 µg twice daily and high-dose gonadotropins. Cycle cancellation rates were dramatically reduced, and the mean maximal serum E2 levels obtained were significantly higher. The ongoing pregnancy rates per ET were 33% in patients ≤39 years old and 18.2% in patients >39

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years old. Significant increases in circulating FSH levels occurred after 5 days of gonadotropin stimulation. No abnormal rises in LH, progesterone, or testosterone during the follicular phase were noted. This could result from either the lower GnRH-a dose, the OCP pretreatment, or a combination of the two. Recently, a retrospective cohort study assessed the efficacy of three different GnRH-a stimulation regimens to improve ovarian response in poor responders.104 Women diagnosed as poor responders underwent three different stimulation regimens during IVF cycles: 1.

2.

3.

Stop protocol: LA 500 µg/day administered from the midluteal phase to the start of menses, then gonadotropins from day 2 of cycle. Micro-dose flare: LA 20 µg administered twice daily with gonadotropins from day 2 to the day of hCG administration. Regular dose flare: gonadotropins beginning with LA on day 2 at 1 µg/day for 3 days, followed by 250 LA µg/day until the day of hCG administration.

Since only 61 cycles were included in the analysis, none of the comparisons reached statistical significance; however, the micro-dose flare group demonstrated a trend toward a higher delivery rate. It is noteworthy that, in a general IVF population (excluding poor responders), retrospective analysis failed to find the micro-dose flare protocol superior over the long midluteal agonist regimen.137 Significantly higher cancellation rates (22.5 vs 8.2%), lower clinical pregnancy rates (47.3 vs 60%, NS), and a decreased number of oocytes retrieved per cycle (13.3 vs 16.5, NS) were noted with the micro-dose flare-up regimen. Overall, all studies evaluating the micro-dose flare protocol were retrospective in nature. Obviously, large prospective randomized controlled trials are needed to validate the true efficacy of the micro-dose flare-up GnRH-a regimens in poor responder patients.

GnRH antagonists in the treatment of poor responders GnRH antagonists competitively block the GnRH receptor in the pituitary gland, producing an immediate doserelated suppression of gonadotropin release. Within 6 hours of GnRH antagonist administration, LH levels are significantly reduced. On the principle of maximizing potential endogenous pituitary stimulation, a GnRH antagonist can be administered later in the follicular phase to suppress the LH surge,138,139 thus avoiding suppression during the phase of early follicular recruitment. In the general IVF population, the GnRH antagonists offer comparable therapeutic efficacy to agonists and have a number of potential advantages over agonists for use in ovarian stimulation protocols, such as avoiding the initial ‘flare-up’ of LH, shortening the overall treatment period, reducing the risk of ovarian hyperstimulation syndrome (OHSS), and menopausal side effects.138,139

The GnRH antagonists are administered in the late follicular phase, either according to the fixed or according to the flexible protocol (see Chapter 40). Thus, at the beginning of COS, the pituitary is fully susceptible to GnRH pulses. This may allow us to obtain a more natural follicular recruitment without any inhibitory effect possibly induced by the GnRHas. It has been therefore suggested by several authors as a suitable protocol for poor responders. GnRH antagonists also permit the revival of stimulation protocols of the pre-agonist era using for example, CC.140 The combination of CC treatment in the early follicular phase and subsequent, overlapping, gonadotropin stimulation has been a standard therapy in the past.140,141 Owing to the synergistic effect of these compounds, the amount of gonadotropins required is lower and so are the costs.142,143 In addition, the gonadotropins counteract the detrimental effects of CC on the endometrium.142 As a result of the high rate of premature LH surges, and therefore the high cancellation rate, this stimulation regimen was abandoned when GnRH-as were introduced in IVF. Craft et al144 were first to suggest the use of GnRH antagonists for COS in low responders. In a small retrospective series, 18 previously poor responders were stimulated with a combination of gonadotropins and CC, and started on a GnRH antagonist according to the flexible protocol. Compared to their poor response in a previous GnRH-a cycle, modest improvements in cycle cancellation rates (29% vs 57%), oocyte yield (6.4 vs 4.7), and gonadotropin requirements (4506 IU vs 5468 IU) were noted with the GnRH antagonist. Two live births resulted (11.8%). Several studies were subsequently undertaken in order to examine the efficacy of GnRH antagonists in COS regimens designed for low responders. The majority of these studies were of small scale and retrospective. Retrospective studies will be presented first, followed by more recently reported randomized controlled trials.

Retrospective studies Nikolettos et al145 compared 21 poor responders who underwent IVF–ICSI and were treated with a GnRH antagonist protocol with 21 matched poor responders treated according to the long GnRH-a protocol. Fifteen patients of the GnRH antagonist group were treated with the combination of CC plus gonadotropins, while six patients were treated with gonadotropins alone. The use of the GnRH antagonist protocol resulted in a significantly shorter treatment duration and lower gonadotropin consumption as compared with the use of the long GnRH-a protocol. Three pregnancies (14.3%) were achieved with the antagonist and two (9.5%) with the long agonist protocol (NS). Several retrospective studies have compared the GnRH antagonist protocol with GnRH-agonist flareup and micro-dose flare regimens. In a retrospective cohort study, Posada et al146 compared the clinical outcome of COS in unselected patients undergoing

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Day 1

± OCP or oral E2

FSH/LH

Day 6

Day of hCG

Individualized Dosing of FSH/LH 250 µg per day Antagonist

Day 2 or 3 of menses

Fig 44.4

GnRH antagonist protocol.

IVF with a GnRH antagonist (133 cycles) vs a 4-day ultrashort GnRH agonist regimen (236 cycles). The GnRH antagonist protocol was shown to reduce treatment duration and amount of gonadotropin used. In younger women, the antagonist protocol was associated with significantly better pregnancy and implantation rates, but no difference was observed in pregnancy rates in patients >38 years old. Mohamed et al147 retrospectively compared the agonist flare-up and antagonist protocols in the management of poor responders to the standard long protocol. One hundred and thirty-four patients undergoing IVF– ICSI treatment, who responded poorly to the standard long protocol in their first treatment cycle, were studied. In the second cycle, 77 patients received a short GnRH-a flare-up regimen and 57 patients received an antagonist protocol, based solely on physician preference. There were no cycle cancelations in the flare-up protocol and a 7% cancellation rate in the antagonist protocol due to lack of response. A significantly higher number of patients had embryo transfer in the flare-up protocol. Similar numbers of oocytes (5.4 vs 5.2) and similar implantation and pregnancy rates per cycle (12.8% and 17.5% vs 12.8% and 24.7%) were reported in the antagonist and flare-up groups, respectively. It was concluded that both the flare-up and the antagonist protocols significantly improved the ovarian response of previously poor responders. However, a significantly higher cycle cancellation rate and less patients having embryo transfer in the antagonist group suggested a higher efficacy for the flare-up regimen. Conflicting results were reported by Fasouliotis et al148 who also conducted a retrospective analysis between the flare-up and antagonist regimens in poor responders. Of 56 poor responders treated with flare-up protocol, 53 who failed to conceive were subsequently treated in the next cycle with a GnRH antagonist regimen. While ovarian response did not differ between the two protocols, the number of embryos transferred was significantly higher in the GnRH-antagonist group (2.5 ± 1.6 vs. 2.0 ± 1.4, respectively). The clinical pregnancy and implantation rates per transfer in the GnRH-antagonist group tended to be higher than in the flare-up group, but did not reach significance (26.1 and 10.7 compared with 12.2 and 5.9%, respectively). However, the ongoing pregnancy rate per transfer was significantly higher in the GnRH antagonist than in the GnRH agonist flare-up group (23.9 vs 7.3%, respectively).

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Copperman149 conducted a retrospective analysis with historic controls comparing cycle outcomes in poor responders who had stimulation protocols that included an antagonist with those with the micro-dose flare protocol. Patients were placed in the antagonist or micro-dose flare treatment group usually after failing in an LA down-regulation cycle, and often according to physician preference. The results of this retrospective analysis indicated that, for poor responders, the inclusion of a GnRH antagonist in the treatment regimen significantly increased clinical pregnancy rates and significantly lowered cancellation rates compared with patients treated with the microdose flare protocol. The use of OCP pretreatment in antagonist cycles for poor responder patients is also of clinical relevance, as their ovarian reserve may be especially sensitive to suppression of endogenous gonadotropins by the pill. Copperman149 reported a retrospective study of 1343 patients, where poor responders were given a starting dose of 450 IU of gonadotropin. In the OCP pretreatment group, patients were administered OCP for 18–24 days, beginning on cycle day 3. Patients were first administered a combination of recombinant FSH and hMG on cycle day 3, and were administered GnRH antagonist when their lead follicle reached 14 mm. An additional 75 IU of hMG was administered beginning the first day of antagonist. Patients whose antagonist stimulation cycle included OCP pretreatment had a significantly higher pregnancy rate and a significantly lower cancellation rate. In addition, a higher proportion of patients obtained more than eight oocytes following OCP pretreatment. In contrast, Shapiro et al150 reported significantly increased cancellation rates (23%) in a group of poor responder patients pretreated with an OCP compared with patients not receiving OCP pretreatment (9%). The two studies, however, differed both in inclusion criteria and in the use of LH in the stimulation protocol.

Prospective studies Akaman et al151 compared a GnRH antagonist protocol to a protocol using gonadotropins alone in low responders. In total, 20 women were randomized to each group. Women assigned to the antagonist received 0.25 mg of cetrorelix according to the flexible protocol, and all women were initially stimulated with 600 IU of urinary-derived gonadotropin. There was no statistically significant difference between the groups for cancellation rates, gonadotropin requirements, number of mature oocytes retrieved, E2 concentrations on the day of hCG administration, fertilization rates, and number of embryos transferred. The clinical pregnancy and implantation rates in the antagonist group appeared higher, but were not significantly different (20 and 13.33% compared with 6.25 and 3.44%, respectively) because of the small numbers involved. There are very few prospective studies that actually compare the agonist flare-up and the antagonist protocols. In a prospective randomized trial, Akman et al152

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compared clinical outcomes of 48 poor responder patients who were treated with either a micro-dose flare (LA, 40 µg SC per day) protocol or the antagonist (cetrorelix 0.25 mg daily) protocol. All patients received 300 IU of highly purified FSH and 300 IU of hMG for 4 days, followed by individual adjustments in the dose of highly purified FSH. Patients in the microdose flare group also received OCP pretreatment. There was no difference in the median total treatment doses of gonadotropins between the two groups. Serum E2 levels on the day of hCG administration and the number of oocytes retrieved were significantly lower in the antagonist group. No differences were observed between the two groups for fertilization rates, number of embryos transferred, and most importantly, implantation rates and ongoing pregnancy rates per transfer. It was concluded that the efficacy of these stimulation protocols in poor responder patients was comparable, but larger studies were needed. Schmidt et al153 randomized 48 previously poor responder patients to either a GnRH antagonist protocol (ganirelix 0.25 mg daily in a flexible protocol) or a micro-dose flare regimen (LA, 40 µg bid, after OCP pretreatment). Ovarian stimulation consisted of 300 IU of recombinant FSH every morning and 150 IU of hMG every evening. Cancellation rates due to an inadequate response were equally high, close to 50% in both groups. While only 13 women in the antagonist group, and 11 women who received a micro-dose flare completed their cycle, no significant differences in oocyte yield (8.9 vs 9), fertilization rate (69.1% vs 63.5%), or clinical pregnancy rate (38.5% vs 36.4%) were detected. It was concluded that the antagonist protocol appears to be as effective as the micro-dose flare protocol for COS in poor responders, but could be a superior choice in terms of cost and convenience for the patient. Malmusi et al154 compared the efficacy of the flare-up GnRH agonist protocol to the flexible GnRH antagonist in poor responders. Fifty-five poor-responder patients undergoing IVF–ICSI were randomized to receive either triptorelin (100 µg daily) from the first day of menstruation, followed by exogenous gonadotropins from the second day of menstruation (30 cycles), or exogenous gonadotropins from the first day of menstrual cycle, and later ganirelix (0.25 mg daily) once the leading follicle reached 14 mm in diameter (25 cycles). Gonadotropin requirements were significantly reduced with the flareup protocol. The number of mature oocytes retrieved, fertilization rate, and top-quality embryos transferred were significantly increased in the flare-up compared to the GnRH antagonist group. The implantation and pregnancy rates were similar in both groups. Two randomized prospective trials that compared the long agonist and the antagonist protocol in low responders have been published. D’Amato et al155 randomized (according to the week day) 145 infertile women, to receive either a combination of CC and high-dose recombinant FSH plus a GnRH antagonist (cetrorelix 0.25 mg daily) once the leading follicle reached 16 mm in

diameter (85 patients), or a standard long protocol with a depot LA preparation (60 patients). Patients who received an antagonist protocol obtained significantly lower cancelation rates (4.7% vs 34%) and higher E2 levels (945.9 ± 173.2 pg/ml vs 169.6 ± 45.1 pg/ml), more oocytes and mature oocytes retrieved (5.6 ± 1.1 vs 3.4 ± 1.3), better-quality embryos, and a trend for higher pregnancy rates (22.2% vs 15.3%) and implantation rates (13.5% vs 7.6%) compared with those receiving the long protocol. Thus, the antagonist protocol was found to be of higher efficacy compared to the long agonist protocol. It should be stated, however, that patients were enrolled in the study based on an already prior poor response to the long agonist protocol, so that their poor performance in the current study was not surprising, especially since a depot preparation of the GnRH-a was used. In the second study by Cheung et al,156 66 poor responders were randomized into two groups: the study group received 0.25 µg of cetrorelix daily starting on stimulation day 6 (fixed protocol); the control group received 600 µg of BA daily starting in the midluteal phase of the preceding cycle. Both groups were pretreated with an OCP and stimulated with a fixed dose of recombinant FSH (300 IU daily). There were no significant differences in the cycle cancelation rates, duration of stimulation, consumption of gonadotropins, and mean numbers of mature follicles, oocytes, and embryos obtained. The implantation rates were similar, but the number of embryos transferred was significantly higher for the antagonist group (2.3 ± 0.6 vs 1.5 ± 0.8). The pregnancy rates were also higher in the antagonist group, but the difference was not statistically significant. It was concluded that, despite the fact that the study failed to demonstrate an overall improvement in ovarian responsiveness, a fixed GnRH antagonist protocol is feasible for patients who are poor responders.

Alternative approaches and treatment protocols using GnRH antagonists GnRH antagonists are recently gaining much popularity in COS protocols for normal, high, and low responder patients. Much work, however, remains to be done in optimizing the antagonist protocols and individualizing them to different patient and cycle characteristics. Several of such recent attempts in low responder patients are outlined below. The baseline FSH level remains the most convenient and reliable hormonal indicator of ovarian reserve and predictor of IVF performance. It is well known that wide intercycle fluctuation of baseline hormones occurs in low responder patients. Because GnRH antagonist protocols do not require pituitary desensitization, an unaltered, up-to-date baseline hormonal assessment of ovarian reserve may be obtained during the actual cycle of stimulation. A recent retrospective analysis by Jurema et al157 suggests that restricting the initiation of COS with GnRH antagonists in poor-prognosis patients to cycles in which the day 3 FSH was <8 IU/l would

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have halved the cycle cancellation rate, and almost tripled the clinical PR in this patient population. One of the problems often seen in poor responding patients is a shortened follicular phase, which limits the ability to recruit a sizable cohort of follicles. Frankfurter et al158 recently described a novel use of a GnRH antagonist before ovarian stimulation in an attempt to lengthen the follicular phase, aiming to lengthen the recruitment phase of the cycle to allow for the rescue of more follicles once gonadotropin stimulation was initiated. Twelve patients who previously exhibited a poor response to a standard (long, short or antagonist) protocol were included. According to the new regimen, patients received two doses of 3 mg of cetrorelix, the first on cycle days 5–8, and the second 4 days later. With cetrorelix start, medroxyprogesterone acetate (MPA, 10 mg daily) was given and was continued until ovarian suppression was confirmed. Then, a combination of recombinant FSH (225 IU SC, twice a day) and recombinant hCG (2.5 mg SC, four times a day) was initiated, and MPA was discontinued to allow for vaginal bleeding. When a lead follicle size of 13 mm was observed, daily cetrorelix (0.25 mg SC) was started and continued until the triggering hCG injection. By using a GnRH antagonist in the follicular phase before ovarian stimulation, a significant improvement in oocyte, zygote, and embryo yield was achieved. A trend toward improved implantation (21%), clinical pregnancy (41.7%), and ongoing pregnancy (25%) rates in the follicular GnRH antagonist cycle was also noted. Larger prospective studies are needed in order to examine the efficacy of this novel therapeutic approach. A recent publication by Orvieto et al159 describes the combination of the micro-dose GnRH-a flare protocol and a GnRH antagonist protocol in poor responders. This protocol combines the benefits of the stimulatory effect of the micro-dose flare on endogenous FSH release with the immediate LH suppression induced by the GnRH antagonist, and was therefore suggested as a valuable new tool for treating poor responders.159,160 The stimulation characteristics of 21 consecutive ultrashort GnRH agonist/GnRH antagonist cycles in 21 patients were compared with their previous failed cycles.159 Triptorelin (100 µg SC) was started on the first day of menses and continued for 3 consecutive days, followed by high-dose gonadotropins, which were initiated 2 days later. Once the lead follicle had reached a size of 14 mm or/and E2 levels exceeded 400 pg/ml, cetrorelix (0.25 mg/day) was introduced and continued up to and including the day of hCG administration. The number of follicles >14 mm on the day of hCG administration, number of oocytes retrieved, and number of embryos transferred were all significantly higher in the study protocol as compared with the historic control cycles. A reasonable clinical pregnancy rate (14.3%) was achieved. Another innovative protocol using GnRH-a/antagonist conversion with estrogen priming (AACEP) in low responders has been recently reported by Fisch

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et al161 and is described later in this chapter (section on luteal phase manipulations).

Natural and modified natural cycle The natural cycle The yield of lengthy, high-dose, and cost-stimulation regimens used in poor responders to increase the number of oocytes retrieved is often disappointing. It was therefore suggested to perform natural cycle IVF in such cases, an approach which is less invasive and less costly for the patient. In a prospective study with historical controls, Bassil et al162 analyzed 11 patients who underwent 16 natural cycles for IVF. These were compared with 25 previous failed cycles with poor response in the same patients. The cancellation rate in natural cycles was 18.8%, as compared with 48% in stimulated cycles. Three ongoing pregnancies were obtained in natural cycles (18.8% per started cycle) compared to none in stimulated cycles. In another prospective study with historical controls, Feldman et al163 compared 44 unstimulated IVF cycles in 22 poor responder patients with those of 55 stimulated cycles of the same patients during the 12 months prior to the study. Eighteen (82%) patients had at least one oocyte retrieved, while nine (41%) had at least one cycle with embryo transfer. Two (9%) patients each gave birth to a healthy term baby. These results were comparable with those of the stimulated cycles. In a small retrospective study,164 30 patients who had previously been canceled because of poor ovarian response underwent 35 natural cycles, achieving an ongoing pregnancy rate of 16.6% per oocyte retrieval and an implantation rate of 33%. All patients, however, were <40 years old and had a mean day 3 FSH of 11.1 IU/l. Similar results were found in an observational study with no controls, in which patients aged 44–47 years old were included.165 These patients were recruited based on age only, without prior demonstration of poor response. Out of 48 treatment cycles conducted in 20 women, oocyte retrieval was successful in 22 cycles (46%). Fertilization and cleavage rates of 48% and 100%, respectively, were obtained. One biochemical and one ongoing pregnancy were achieved. Thus, the ongoing pregnancy rate was 5% per patient and 2.08% per cycle. In addition, Check et al166 reported on 259 retrieval cycles and 72 transfers in poor responders using minimal or no gonadotropin stimulation and without GnRHas or antagonists. These patients were divided into four age groups (<35, 36–39, 40–42, and >43 years old) and their mean serum day 3 FSH levels were 19.7, 20.6, 18.8, and 21.9 mIU/ml, respectively. In total, 12 deliveries were achieved after 259 IVF cycles (4.6%). Eliminating the oldest age group, the delivery rate for 47 embryo transfers in women ≤42 years of age was 25.5%. Approximately 50% of retrievals resulted in an embryo (about half were transferred fresh and half frozen). The median number of embryos transferred was one. The

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implantation rate was 21.6% for the three groups and was 33.3% for patients aged <35 years old and 28.6% for women aged 36–39 years old. It was concluded that the pregnancies and live births can be achieved in poorprognosis/poor responder patients with elevated basal FSH levels, and age was found to be a more adverse infertility factor than elevated serum FSH. The only prospective, randomized, controlled trial on this topic,167 compared the efficacy of natural cycle IVF with the micro-dose GnRH-a flare protocol in poor responders. A total of 129 patients who were poor responders in a previous IVF cycle were included: 59 women underwent 114 attempts of natural cycle IVF and 70 women underwent 101 attempts of IVF with COS by micro-dose agonist flare. In the natural cycle patients, the oocyte retrieval procedure was performed in 114 cycles, and oocytes were found in 88 of these (77.2%). The poor responders treated with naturalcycle IVF and those treated with micro-dose GnRH-a flare showed similar pregnancy rates per cycle and per transfer (6.1% and 14.9% vs 6.9% and 10.1%, respectively). The women treated with natural cycle IVF showed a statistically significant higher implantation rate (14.9%) compared with controls (5.5%). When subdivided into three groups according to age (≤35 years old, 36–39 years old, and >40 years old), younger patients had a better pregnancy rate than the other two groups. It was concluded that in poor responders, natural cycle IVF is at least as effective as COS, especially in younger patients, with a higher implantation rate. Finally, Papaleo et al168 recently reported on a series of poor-prognosis patients, all of them with advanced maternal age, elevated serum FSH, and reduced antral follicle counts, who underwent natural cycle IVF. A total of 26 natural cycles in 18 patients were analyzed. Pregnancy was achieved in three patients, of which two patients were ongoing (11.5% per cycle, 20.0% per ET). It was suggested that since the overall pregnancy rates achieved were comparable with those of conventional IVF–ET in poor responders, considering the lower costs and risks and the patient-friendly nature of such protocols, natural cycle IVF can provide an acceptable alternative option for persistent poor responders.

Modified natural cycle The efficacy of natural cycle IVF is hampered by high cancellation rates because of premature LH rise and premature ovulations.169 The possibility of enhancing the efficacy of unstimulated IVF cycles by the concomitant addition of a GnRH antagonist and exogenous gonadotropins in the late follicular phase was introduced by Paulson et al as early as 1994.170 This protocol, later known as the ‘modified natural cycle’ (MNC), is expected to reduce the rate of premature ovulation and to improve control of gonadotropin delivery to the developing follicle. In a preliminary report on 44 cycles in 33 young normal responder patients,171 the cancellation rate was 9%, and in 25% of retrievals no oocyte was obtained.

Embryo transfer was performed in 50% of the started cycles, leading to a clinical pregnancy rate of 32.0% per transfer and 17.5% per retrieval, of which five (22.7% per transfer) were ongoing. It was suggested that the MNC could represent a first-choice IVF treatment with none of the complications and risks of current COS protocols, a considerably lower cost, and an acceptable success rate. Large experience with the MNC protocol in the general IVF population has been accumulated by the Dutch group in Groningen.172–174 In a preliminary report, the cumulative ongoing pregnancy rate after three cycles with this protocol was 34% and the live birth rate per patient was 32%.173 Summarizing a much larger experience, the same group174 later reported on a total of 336 patients who completed 844 cycles (2.5 per patient). The overall ongoing pregnancy rate per started cycle was 8.3% and the cumulative ongoing pregnancy rate after up to 3 cycles was 20.8% per patient. In a recent report of further follow-up of up to 9 cycles,172 a total of 256 patients completed 1048 cycles (4.1 per patient). The embryo transfer rate was 36.5% per started cycle. The ongoing pregnancy rate was 7.9% per started cycle and 20.7% per embryo transfer. Including treatment-independent pregnancies, the observed clinical pregnancy rate after up to 9 cycles was 44.4% (95% confidence interval [CI] 38.3–50.5) per patient. Pregnancy rates per started cycle did not decline in higher cycle numbers (overall 9.9%), but drop-out rates were high (overall 47.8%). Several studies have been reported on the use of the MNC protocol in poor responders. Kolibianakis et al175 evaluated the use of the MNC for IVF in poor responders with an extremely poor prognosis, as a last resort prior to oocyte donation. Thirty-two patients with regular menstrual cycles, basal FSH levels >12 IU/l and one or more failed IVF cycles with ≤5 oocytes retrieved were included. Recombinant FSH 100 IU and ganirelix 0.25 mg/day were started concomitantly when a follicle with a mean diameter of 14 mm was identified. hCG was administered as soon as the mean follicular diameter was ≥16 mm. Twenty-five out of 78 cycles performed (32.1%) did not result in oocyte retrieval. In nine out of 53 cycles (16.9%), in which oocyte retrieval was performed, no oocytes were retrieved. Embryo transfer was performed in 19 out of 44 cycles in which oocytes were retrieved (43.2%), but no ongoing pregnancy was achieved in 78 MNC cycles. It was concluded that the MNC does not offer a realistic chance of live birth in poor-prognosis/poor responder patients, when offered as a last resort prior to oocyte donation. Studies with somewhat more encouraging outcome were also reported. Elizur et al176 retrospectively evaluated 540 cycles in 433 poor responders who were divided by treatment protocol into MNC, GnRH antagonist, and long agonist groups: there were 52 MNC cycles, 200 GnRH antagonist cycles, and 288 long GnRH-a cycles. In the MNC protocol, a GnRH antagonist 0.25 mg/day and 2–3 ampules of hMG were administered daily once the lead follicle reached a diameter of 13 mm.

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The mean number of oocytes retrieved in the MNC group was significantly lower than in the stimulated antagonist and long agonist groups (1.4 ± 0.5 vs 2.3 ± 1.1 and 2.5 ± 1.1, respectively, p <0.05). The respective implantation and pregnancy rates were comparable, 10% and 14.3%, 6.75% and 10.2%, and 7.4% and 10.6%. The number of canceled cycles was significantly higher in the MNC group. Cancellations due to premature luteinization or failure to respond to stimulation were significantly more common in patients >40 years old. Since pregnancy rates were comparable for all groups, it was concluded that the MNC is a reasonable alternative to COS in poor responders. In summary, the natural and/or modified natural cycle certainly has a role in poor responders, especially in those who are refractory to COS and decline the option of oocyte donation. Its exact role has yet to be determined, as are also several key issues that have not yet been subjected to testing such as: 1. 2.

3. 4. 5. 6. 7. 8.

Is the MNC protocol superior to the simple natural cycle protocol? What is the best timing for hCG administration and what is the ideal time interval between hCG administration and egg retrieval? Are oocyte and embryo quality improved in natural cycles? How many attempts should be made? What is the role of follicle flushing? Should cleavage- or blastocyst-stage transfers be performed? What is the dose of gonadotropins that should be administered in the MNC protocol? Should LH be included in the gonadotropin regimen?

More research is needed before these questions can be effectively answered.

Manipulating endocrinology The role of follicle-stimulating hormone Inherent biological mechanisms such as follicle sensitivity to FSH and pharmacodynamics of drug metabolism or receptor interaction177 may have a bearing on the ovarian response to stimulation. Recent research in the fields of genetics and pharmacogenomics has revealed genetic factors that may facilitate improved cycle management. FSH secreted from the pituitary is a heterodimer glycoprotein hormone with two covalently linked subunits, α and β. The molecule is glycosylated by posttranslational modification, and the presence and composition of the carbohydrate glycan moieties determine its in vivo biological activity (Fig 44.5).178,179 In vivo, the native FSH hormone consists of a family of at least 20 different isohormones that differ in their pattern of glycosylation. For follitropin-α, isoelectric focusing has identified seven major bands of FSH isoforms between pI 4.2 and 5.05, five minor bands between pI 5.25 and

Fig 44.5 Follicle-stimulating hormone is a complex glycoprotein with two noncovalently associated α and β protein subunits. Two oligosaccharides are linked to each protein subunit. (Molecular model created by Merck Serono Reproductive Biology Unit, USA.)

6.30, and one minor band at pI 4.20. These have been demonstrated to be consistent between different manufactured batches (Fig 44.6).180 The ovarian response to stimulation by FSH relies on an interaction of the hormone with membrane receptors (FSHR) on granulosa cells, and a normal response is dependent on correct molecular structure of the hormone, the receptor, and factors associated with their interaction. Any defect in the genes encoding FSH or its receptor may result in ovarian resistance, and therefore genotype may play a fundamental role in determining the physiological response to FSH stimulation. The FSH receptor is a member of the family of G-protein receptors linked to adenyl cyclase signaling, with extensive extracellular ligand-binding domains. The gene encoding the FSH receptor is located on the short arm of chromosome 2, and is made up of 2085 nucleotides that translate into a polypeptide with 695 amino acids. This molecule has four potential N-linked glycosylation sites located at amino acids 191, 199, 293, and 318. Mutations in the receptor gene can result in amino acid changes that affect function, and mutations that result in complete FSH resistance181 as well as partial loss of FSHR function have been identified.182 Screening different populations for mutations of the FSHR gene have shown that single nucleotide polymorphisms (SNPs) can be identified, and two discrete polymorphisms have been studied: (1) position 307 (Ala or Thr) in the extracellular domain, and (2) position 680 (Asn or Ser) in the intracellular domain. Both polymorphic

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Fig 44.6 IEF gel showing the FSH isoform pattern of nine batches of follitropin-α filled by mass (Gonal-F). First lane pI markers, lanes 2–10, nine batches of follitropin-α. (Reproduced from Bassett and Driebergen,186 with permission.)

sites give rise to two discrete allelic variants of the FSHR, i.e. Thre307/Asn680 and Ala307/Ser680. Recent studies suggest that there is an association between these polymorphisms and ovarian response in patients undergoing assisted reproduction treatments,183,184 and that their frequency may vary among different ethnic groups: a lower frequency of Ser/Ser polymorphism at position 680 has been found in Asian women than in Caucasians (Table 44.1). In a Korean IVF patient population, Jun et al184 grouped 263 young patients according to their FSHR genotype, and found that basal FSH levels differed between the groups. The Ser/Ser (p.N680S) homozygous group required higher total doses of gonadotropins to achieve multiple follicular development compared to the other two groups (Asn/Asn and Asn/Ser at position 680). Additionally, significantly fewer oocytes were recovered in patients with the Ser/Ser FSHR genotype. Perez Mayorga et al183 also suggest that the FSHR genotype plays a fundamental role in determining the physiological response to FSH stimulation, and that subtle differences in FSHR might fine-tune the action of FSH in the ovary. In a study conducted in 161 ovulatory young (<40 years old) women who underwent IVF treatment, a wide variation in the number of ampules of FSH required to achieve an adequate response was observed. They confirmed that this observation could be correlated with the patient’s FSHR genotype, i.e. type of polymorphism. Behre et al185 also carried out a prospective, randomized controlled study to further investigate this observation, and found that the Ser/Ser (p.N680S) homozygous group results in lower E2 levels following FSH stimulation. This lower FSH receptor sensitivity could be overcome by higher FSH doses in the trial patients. An analysis was undertaken with the objective of determining whether specific factors could optimally predict a response to stimulation in ART, and then to develop a corresponding treatment algorithm that could be used to calculate the optimal starting dose of recombinant human follicle-stimulating hormone (rhFSH;

follitropin-α) for selected patients.45 Backwards stepwise regression modeling indicated that in ART patients <35 years old (N = 1378) who were treated with r-hFSH monotherapy, predictive factors for ovarian response included basal FSH, BMI, age, and number of follicles <11 mm at baseline screening. The concordance probability index was 59.5% for this model. Using these four predictive factors, a follitropin-α starting dose calculator was developed that can be used to select the FSH starting dose required for an optimal response. A prospective cohort study has been completed using this r-hFSH starting dose calculator and demonstrated a similar number of oocytes and pregnancy rates across the doses used.57 Taken together, these studies suggest that, in the future, it might be possible to tailor FSH therapy to the patient’s genetic background, and thereby adjust the doses and the timing of stimulation. This would be of particular benefit in the treatment of older women, who cannot afford any delay in their race against the biological clock.

The role of luteinizing hormone Ovulation induction studies in hypogonadotropic women using r-hFSH have demonstrated that FSH can induce follicular growth to the preovulatory stage, but E2 and androstenedione concentrations remain extremely low.186,187 This suggests that follicular maturation depends on the action of LH to stimulate androstenedione biosynthesis as a substrate for aromatase activity. Below a minimal level of LH, follicular development will plateau – this has been observed in patients with profound pituitary downregulation after GnRH-a depot.188,189 In women with hypogonadotropic hypogonadism, E2 concentrations may be inadequate for cytoplasmic maturation of the follicle, endometrial proliferation, and corpus luteum function.186,187 Adequate folliculogenesis and steroidogenesis required for successful fertilization and implantation therefore depend upon a certain threshold level of LH. Although the amount of LH necessary for normal follicle and oocyte development is not known, it is likely to be very low, since a maximal steroidogenic response can be elicited when <1% of follicular LH receptors are occupied.190 On this basis, resting levels of LH (1–10 IU/l) should be sufficient to provide maximal stimulation of thecal cells.191 There is also evidence that excessive levels of LH can have an adverse effect on follicular development,192 associated with impaired fertilization and pregnancy rates, as well as higher miscarriage rates, the so-called ‘ceiling’ effect (Fig 44.7). LH levels must be below this ceiling in order for the LH-dependent phase of development to proceed normally. It seems that there is a clinical therapeutic window:193,194 ‘low-dose’ treatment with LH generally enhances steroidogenesis, but ‘high-dose’ treatment can enhance progesterone synthesis, suppress aromatase activity, and inhibit cell growth. Huirne et al195 administered different GnRH antagonist doses to five groups of patients and measured the

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The frequency of the FSH receptor polymorphism at p.N680S in published reports SNP680

Author

Ethnic origin

Patient number (diagnosis)

Asn/Asn

Asn/Ser

Ser/Ser

Perez Mayorga et al183 Sudo et al295 Laven et al296 Laven et al296 De Castro et al297 Daelemans et al298 Daelemans et al298 Daelemans et al298 Choi et al299 Schweickhardt 2004 – unpublished thesis

Caucasian Japanese Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Korean Not stated (USA)

161 (male/tubal) 522 (mixed) 148 (anovulatory) 30 (ovulatory) 102 (male/tubal/both) 99 (non-IVF control) 130 (mixed?) 37 (mixed–OHSS) 172 (mixed, non-PCO) 663 (mixed)

29% 41% 16% 23% 31.4% 38% 24% 16% 41.9% 30.6%

45% 46.9% 44% 61% 50% 45% 51% 54% 47.7% 48.7%

26% 12.1% 40% 16% 18.6% 17% 25% 32% 10.5% 20.7%

The Ser/Ser (p.N680S) homozygous group is generally lower in Asian populations than in Caucasian populations.

subsequent change in LH levels between the groups. The aim of this study was to deliberately induce different LH levels, and assess the effect of an LH range on IVF outcome in order to estimate what the optimal level might be. No pregnancies were observed in relation to either very high or very low LH, suggesting an optimal window. However, their data led them to conclude that not the absolute levels, but instead excessive changes in LH – either increases or decreases – were the more significant parameter. They suggest that the correct sequence of stages in oocyte maturation, together with synchrony between nuclear and cytoplasmic maturation, is dependent upon an appropriate endocrine milieu. Excessive fluctuations in LH levels might disrupt this balance, as well as affect maturation of the endometrium – i.e. stable and appropriate LH levels are needed during IVF cycles. It is possible that specific patient groups, such as those with polycystic ovaries (PCOS) or diminished ovarian reserve may be prone to larger changes in LH levels and sensitive to high fluctuations. In addition, serum LH levels

assayed by immunoassay do not necessarily reflect circulating LH bioactivity, particularly in these specific patient groups. The initial availability of a pure recombinant human LH (r-hLH) (Luveris, Merck Serono International, Geneva, Switzerland) preparation has provided a new tool that allows the endocrinology of ovarian stimulation to be examined more accurately. This has been followed by the recent commercial availability of a combination r-hFSH/r-hLH (2:1 ratio) preparation (Pergoveris, Merck Serono International, Geneva, Switzerland) indicated for use in women with severe gonadotropin deficiency. The use of r-hLH in controlled ovarian stimulation protocols has been recently reviewed.196 A clear relationship between the dose of r-hLH and serum E2 has been found in hypogonadotropic patients.197 The optimal LH levels required to provide the best results in IVF are still a matter of debate, and a number of studies have tried to assess the role of LH supplementation in GnRH-a and GnRH antagonist cycles.

Atresia of follicles LH over exposure

Poor oocyte/embryo quality Disturbed endometrial maturation

Ceiling

LH deficiency

Poor cytoplasmic maturation and post-fertilization development

• Gonadotropin-deficient (WHO I) anovulation • Patients treated with GnRH agonist depot (~ 15%) • Poor responders/older patients

Fig 44.7

The LH therapeutic window concept.

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LH supplementation may have an effect via intraovarian mechanisms that affect steroid biosynthesis, and therefore oocyte maturation. Foong et al198 recently conducted a study that included patients who showed an inadequate response to r-hFSH-only stimulation, and reported that although peak E2 levels were similar to those found in normal responders, intrafollicular E2 levels were significantly lower, and progesterone was significantly higher in poor responders to FSH. E2 plays an important role in human oocyte cytoplasmic maturation in vitro,199 as manifested by improved fertilization and cleavage rates. Growth hormone has also been shown to stimulate E2 production by follicular cells.200,201 High intrafollicular E2 concentrations in the pre-ovulatory follicle predict an increased chance of pregnancy.202 On the other hand, androstenedione can irreversibly block the effect of E2,203 and it is clear that maintaining an appropriate steroid balance within the follicle is very important. In the ovine, E2 is associated with an up-regulation of oocyte DNA repair enzymes.204 In the rhesus monkey, adding an aromatase inhibitor during the late stages of follicular development, just prior to the period of ovulation, resulted in a reduced capacity of the oocyte to mature, and a reduced rate of fertilization in vitro.205 Overall, it seems that LH may have a beneficial effect through a mechanism that improves oocyte cytoplasmic maturation (increasing mitochondrial function and/or up-regulating DNA repair enzymes), either through E2 or some other intraovarian factor. However, an additional effect on the endometrium itself cannot be excluded. A number of further studies have examined the effect of LH supplementation in poor responders206 or patients who respond inadequately to FSH stimulation.188,189,207 LH supplementation in unselected ART patients has also been investigated.14,208 Following stratification of the data, a subsegment of patients aged ≥35 years old has been identified, who seem to benefit from LH supplementation in terms of an increased number of mature oocytes retrieved and improved implantation and pregnancy rates. This benefit was maintained even when LH supplementation was initiated from stimulation day 6 or 8. This seems logical in terms of physiology, as the granulosa cells, through FSH stimulation, acquire LH receptors only after the follicle reaches a diameter of at least 11 mm.209 In hyporesponsive women, the need for higher FSH doses might be an individual biological index of LH deficiency, with an effect on oocyte competence. A requirement for LH supplementation in order to achieve good ovarian response and follicular maturation in AMA patients could be based on a number of theoretical explanations. With age and the onset of the menopause, endogenous LH as well as FSH levels increase and testosterone levels decrease.210,211 The number of functional LH receptors also decreases with age.212 Kim et al213 found that the best predictor of ovarian reserve (reproductive age) in normally cycling women was the combination of the FSH and LH levels on menstrual cycle day 1. There is also evidence that

endogenous LH may be less biologically active or potent than it should be, or the immunologic LH many not be comparable to the biologically active LH.214,215 Overall, this could result in an increasing ovarian resistance to LH-mediated events. It has been suggested that follicular recruitment in women >38 years old can be improved by supplementing r-hFSH stimulation with LH-containing preparations.216,217 Since hMG contains hCG and a number of unknown contaminating proteins in addition to FSH and LH, Gomez-Palomares et al218 conducted a prospective randomized cohort study comparing the effects of hMG with r-hLH supplementation in a group of women 38–40 years old in order to determine whether LH is the hMG component that favors early follicular recruitment. The patients were randomly assigned by computer to two groups: 58 patients received r-hFSH 225 +hMG one ampule, and 36 were treated with r-hFSH 225 +r-hLH 75 until day 6. Follicular recruitment was evaluated on day 6, and stimulation was continued with r-hFSH alone, without further hMG or r-hLH. Both groups recruited a similar number of follicles after 5 days of stimulation, but the r-hLH group showed a significant increase in the number of metaphase II oocytes retrieved, and higher clinical pregnancy rate (47% vs 26%; NS). In another study, Ferraretti et al207 demonstrated that supplementation from the mid to late stimulation phase with r-hLH but not hMG was associated with significantly improved implantation and clinical pregnancy rates, in patients who were responding inadequately to FSH-only stimulation. This is an interesting category of ART patients and it seems that such a response may be more common in a GnRH-a depot regimen.188,189 Here, de Placido and colleagues described the beneficial use of r-hLH supplementation administered following the occurrence of a plateau in E2 secretion and a lack of continued follicle growth around day 7 of FSH stimulation. Several studies have also supported the need for additional LH in poor responders when short and long protocols of GnRH agonists are used.219–221 From such studies, it has been theorized that ovarian stimulation in patients with diminished ovarian reserve may be enhanced by the LH-induced production of E2 precursors such as androstenedione. Because of the sudden and often dramatic inhibition of LH secretion associated with the use of GnRH antagonists, there has been concern about the need for supplementation with exogenous LH. There is, however, paucity of data regarding the significance of LH supplementation in poor responders or patients with advanced maternal age undergoing COS using a GnRH antagonist protocol. In a retrospective cohort study,222 240 GnRH antagonist cycles in poor responders were evaluated. Of 153 that reached the stage of oocyte retrieval, 75 patients received r-hFSH for ovarian stimulation, and 66 received hMG in combination with r-hFSH. In patients aged <40 years old, there were no significant differences between treatment

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groups in the amount and duration of treatment, number of oocytes retrieved, and number of embryos. In patients aged ≥40 years old, significantly fewer oocytes were retrieved in patients who received exogenous LH in their stimulation, resulting in significantly fewer fertilized embryos. Implantation and clinical pregnancy rates did not differ by treatment group. It was concluded that in poor responders undergoing IVF with GnRH antagonists, outcomes are comparable whether stimulation is achieved in the presence or absence of supplemental LH. Similar results have been reported recently by Barrenetxea et al.223 In a prospective randomized trial, 84 patients who had a basal FSH level of >10 mIU/ml, were >40 years old, and were undergoing their first IVF cycle, were randomly allocated into two study groups: group A, in which ovarian stimulation included GnRH-a flare-up and r-hFSH and r-hLH; and group B, in which patients received the flare-up protocol by r-hFSH without further LH addition. The overall pregnancy rate was 22.6%. The pregnancy wastage rate was 30.0% in group A and 22.2% in group B. There were no differences in the ongoing pregnancy rate per retrieval and implantation rate per embryo transferred. The number of days of gonadotropin treatment, E2 level on hCG administration day, number of developed follicles, number of retrieved oocytes, number of normally fertilized zygotes, cumulative embryo score, and number of transferred embryos were all comparable for the two groups. It was concluded that the addition of r-hLH at a given time of follicular development produces no further benefit in poor responder patients stimulated with the short protocol, and a reduced ovarian response cannot be overcome by changes in the stimulation protocol. Finally a last word on this issue comes from a recent Cochrane Systematic Review.224 Overall, there was no evidence of a statistical difference in clinical pregnancy rates or in ongoing pregnancy rates in ART patients administered r-hFSH alone or r-hFSH combined with r-hLH. However, in three reviewed trials, including only poor responders, there was a significant increase in the pregnancy rate, in favor of co-administrating r-hLH. In summary, the addition of LH to the COS regimen of poor responders remains controversial probably because of the heterogeneity of the patient population so far studied. Future large-scale studies with a prospective randomized design in well-defined patient subgroups are needed in order to define the circumstances under which LH supplementation would be beneficial.

The role of androgens The human follicle has internal (granulosa) and external (theca) cell layers, and folliculogenesis is regulated by endocrine and paracrine factors that interact within a microenvironment; steroidogenesis is coordinated by

595

Fig 44.8 Schematic diagram of the ‘two cells–two gonadotropins theory’ which interacts to produce estradiol (E2). Luteinizing hormone (LH) acts on the theca cells, which produce androgens. Through follicle-stimulating hormone (FSH) stimulation, granulosa cell aromatase converts androgens into E2.

the two different cell types (Fig 44.8). Pituitary LH acts on theca cells through surface receptors to promote androgen synthesis in the early follicular phase, and FSH acts through membrane-associated granulosa cell receptors to promote their proliferation and differentiation. Granulosa cells then express an aromatase enzyme system that catalyzes the conversion of androgens to estrogen.225 In order for normal folliculogenesis to continue, adequate levels of bioavailable estrogen are needed, and paracrine signaling activated by FSH and LH (steroids, cytokines, and other growth factors) sustains growth and estrogen secretion as the follicle develops. Theca cell enzyme activity is increased to enhance androgen production, thus contributing further to estrogen synthesis within the follicle. FSH also stimulates granulosa cell LH receptor expression in the late follicular phase, so that they become receptive to LH stimulation. Larger follicles with granulosa cell LH receptors continue to grow, and LH can then also stimulate the GC aromatase system. FSH and LH together stimulate granulosa cells to produce inhibin, which has a synergistic effect on theca cells to further promote androgen synthesis. Androgens are synthesized in thecal cells through cell-specific expression of P450C17α (CYP17), which is under LH control, and granulosa cells express androgen receptors (AR) throughout antral development. During the late stages of follicle development prior to ovulation, transcription of the granulosa AR gene and AR protein levels decline, so that granulosa cell responsiveness to gonadotropins is diminished.

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This mechanism could delay terminal differentiation until the LH surge signals the onset of ovulation, when the cells begin to switch their steroid synthesis to progesterone for the luteal phase. The rate-limiting step in androgen synthesis, conversion of cholesterol to pregnenolone, occurs within theca cells, and close cooperation between the two types of somatic cells ensures that sufficient E2 is produced during oocyte maturation.198 In the primate ovary, androgens stimulate early stages of follicular growth,226 and primate experiments indicate that androgens may influence the responsiveness of ovaries to gonadotropins. In rhesus monkeys, treatment with dehydrotestosterone (DHT) or testosterone (T) augments follicular FSH receptor expression in granulosa cells, promotes initiation of primordial follicle growth, and increases the number of growing preantral and small antral follicles. These studies strongly suggest that androgen treatment may amplify the effects of FSH on the ovary. Hillier and Tetsuka123 confirmed that T enhances FSH-induced granulosa cell gene expression, and that androgens also exert a paracrine effect in the early follicular phase.227 Hugues and Cédrin-Durnerin228 have recently reviewed the role of androgens in fertility treatment. They suggest that androgen status should be more carefully assessed prior to treatment. Thus, androgens might have two separate roles: 1.

2.

During early follicle growth before the follicle becomes sensitive to gonadotropins (reducing apoptosis?) Enhancing FSH action during the early gonadotropin-sensitive phase of follicular growth.

In premenopausal cycling women, circulating T is derived from direct secretion by the ovary and adrenal, and from conversion of precursors such as androstenedione (Fig 44.9). Testosterone circulates in 3 forms: free, bound to albumin, and bound to sex hormone-binding globulin (SHBG). The free and albumin-bound fractions are believed to be bioavailable, and the fraction bound to SHBG is thought to be unavailable for action in the periphery. With increasing age, androgen levels in women decline significantly.229,230 Frattarelli et al231 evaluated androgen levels in 43 normo-ovulatory women before IVF treatment, and observed that patients who had a low level of T after down-regulation (<20 ng/dl) required a higher FSH dose, a longer duration of stimulation, and were less likely to achieve a pregnancy than patients with higher baseline testosterone levels. Barbieri et al232 investigated the association of BMI, age, and cigarette smoking with serum T levels in women undergoing IVF. They observed that T levels decreased significantly with advancing age, and suggest that this may be because LH stimulation of ovarian androgen secretion begins to decline during the decade of the 30s. This effect occurs before a decline in ovarian

estrogen secretion, possibly due to the fact that a compensatory increase in FSH with ovarian aging at 35–45 years old maintains ovarian estrogen secretion but does not maintain ovarian androgen secretion. They also found a positive correlation between serum T and the number of oocytes retrieved; advancing age and years of cigarette smoking were associated with a decreased number of oocytes. GnRH-a administration during ART treatment cycles reduces the level of circulating LH, and therefore the amount or bioactivity of aromatizable androgen substrate available for FSH-induced E2 synthesis is reduced. We suggest that in women with diminished ovarian reserve who undergo ART treatment, boosting intraovarian androgens might increase the number of follicles available to enter the recruitment stage, as well as the process of follicle recruitment itself. Three different strategies have been proposed: 1. 2. 3.

Stimulating theca cells with r-hLH prior to r-hFSH stimulation in the long agonist protocol. Testosterone or dehydroepiandrosterone (DHEA) supplementation prior to gonadotropin stimulation. Blocking intraovarian androgen conversion with the use of an aromatase enzyme inhibitor.

Pretreatment with r-hLH Normogonadotrophic women (n = 146) were treated in a long depot GnRH-a protocol and were randomized to receive r-hLH (Luveris, 300 IU/day) for a fixed 7 days, or no r-hLH treatment. This was followed by a standard r-hFSH stimulation regimen (Gonal-F, 150 IU/day). Pretreatment with r-hLH was associated with an increase in small antral follicles prior to FSH stimulation (p = 0.007), and an increased yield of normally fertilized (2 PN) embryos (p = 0.03).232a

Androgen supplementation Massin et al233 treated low responders with transdermal T application for 15 days prior to FSH stimulation. Testosterone gel application resulted in a significant increase in plasma T levels but did not significantly improve the antral follicle count. Furthermore, after gel application, the main parameters of ovarian response (numbers of preovulatory follicles, total and mature oocytes and embryos) did not significantly differ between T and placebo-treated patients. It was concluded that no significant beneficial effects of androgen administration on ovarian response to FSH can be demonstrated. However, further trials are needed to determine whether an optimal dose and/or a longer duration of T administration may be helpful. Balasch et al60 investigated the usefulness of T pretreatment in poor responders. In a prospective, therapeutic, self-controlled clinical trial, 25 consecutive infertile patients who had a background of the first and second IVF treatment cycle cancellations due to

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Cytochrome P450 C17 α

597

LH

Cholesterol

17 α-Hydroxylase Pregnenolone 3 β OH steroid Deshydrogenase Progesterone

C 17-20 Desmolase ∆5 Pathway

17. OH P

Androstenedione 17 § OH Deshydrogenase

Estrogens Aromatase

Testosterone FSH

poor follicular response, in spite of vigorous gonadotropin ovarian stimulation and having normal basal FSH levels, were included. In their third IVF attempt, all patients received transdermal T treatment (20 µg/kg per day) during the 5 days preceding gonadotropin treatment. Twenty patients (80%) showed an increase of over five-fold in the number of recruited follicles, produced 5.8 oocytes, received two or three embryos, and achieved a clinical pregnancy rate of 30% per oocyte retrieval. There were 20% canceled cycles. It was concluded that pretreatment with transdermal T may be a useful approach for women known to be low responders but having normal basal FSH concentrations. Casson et al234 postulated that DHEA administration to poor responders might augment the effect of gonadotropin stimulation, via a paracrine effect mediated by insulin-like growth factor I (IGF-I). In a preliminary small series of five patients (<41 years old) with documented poor response to high doses of gonadotropins, DHEA was administered orally (80 mg/day) for 2 months, and continued during ovarian stimulation prior to intrauterine insemination (IUI). After 2 months of treatment, all patients showed increased serum androgen and E2 levels during the stimulation cycle, and all had an improved response to stimulation by approximately two-fold, even after controlling for gonadotropin dose. One of the patients delivered twins after IUI. This preliminary report was recently confirmed by several publications reported by Barad and Gleicher.235–237 The effect of treatment with DHEA on fertility outcomes among women with diminished ovarian reserve was evaluated in a case-control study.236 Twenty-five women with significantly diminished ovarian reserve had one IVF cycle before and after DHEA treatment, with otherwise identical hormonal stimulation. Women received 75 mg of DHEA daily for an average of 17.6 ± 2.1 weeks. Paired analysis of IVF cycle outcomes in 25 patients, who

Fig 44.9 Steroid biosynthesis in the human ovary; the ∆5 pathway predominates in the follicle for estrogen synthesis.

underwent cycles both before and after DHEA supplementation, demonstrated significant increases in fertilized oocytes (p <0.001), normal day 3 embryos (p = 0.001), embryos transferred (p = 0.005), and average embryo scores per oocyte (p <0.001) after DHEA treatment. This study supported the previously reported beneficial effects of DHEA supplementation on ovarian function in women with diminished ovarian reserve. A recent case-control study of 190 women with diminished ovarian function further assessed the role of DHEA supplementation on pregnancy rates.235 The study group included 89 patients who used supplementation with 75 mg/day of oral, micronized DHEA for up to 4 months prior to entry into IVF. The control group was composed of 101 couples who received infertility treatment but did not use DHEA. Cumulative clinical pregnancy rates were significantly higher in the study group (25 pregnancies; [28.4%] vs 11 pregnancies [11.9%]). These data support a beneficial effect of DHEA supplementation among women with diminished ovarian function.

Blocking intraovarian androgen conversion Aromatase inhibitors were initially approved to suppress estrogen levels in postmenopausal women with breast cancer. They inhibit the enzyme by competitive binding to the heme of the cytochrome P450 subunit, blocking androgen conversion into estrogens so that there is a temporary accumulation of intraovarian androgens. Mitwally and Casper238 showed that aromatase inhibition improves ovarian response to FSH in poor responder patients undergoing ovulation induction and IUI. Garcia-Velasco et al239 undertook a study to assess whether the use of an aromatase inhibitor improves ovarian response and IVF cycle outcome in patients with diminished ovarian reserve, using an OCP/GnRH antagonist protocol. A cohort of 147 patients with diminished ovarian reserve, on the basis of at least one previously canceled IVF attempt with ≤4 × 16 mm follicles,

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was divided into two groups: 71 patients were treated with a high-dose gonadotropin regimen supplemented with 2.5 mg letrozole during the first 5 days of stimulation, and 76 patients were treated with the high-dose gonadotropin regimen alone. The GnRH antagonist was administered daily after the leading follicles reached a diameter of 14 mm. In this study, aromatase inhibitortreated patients showed significantly higher levels of follicular fluid T and androstenedione, and these patients had a higher number of oocytes retrieved (6.1 vs 4.3) as well as a higher implantation rate (25% vs 9.4%) despite similar doses of gonadotropins.

The role of human growth hormone The use of human growth hormone (hGH) in the management of female and male subfertility was reported in the early 1990s,240,241 and there has been a great deal of controversy about its use in patient management since that time. There are substantial data that demonstrate the critical importance of the IGF–IGFBP family (the growth factors IGF-I, IGF-II, and their binding proteins) to follicular development. In particular, IGF-I is GH-dependent and is involved in potentiating the effect of FSH.242,243 Early trials were promising, showing improvements in follicular responsiveness and pregnancy rates.244–246 Additional clinical trials utilizing hGH supplementation in normogonadotropic patients or in poor responders to gonadotropin stimulation did not demonstrate any therapeutic advantage.247–252 This is not completely surprising, as the early work of Blumenfeld and Lunenfeld253 demonstrated that hGH was only effective in hGH-deficient individuals, who failed to mount a normal GH response to clonidine challenge. Subsequent studies using hGH or growth hormone releasing factor in IVF poor responders demonstrated no improvement in stimulation characteristics, clinical pregnancy, or live birth rates, and the interest and use of adjuvant GH has subsided. This was further confirmed by a Cochrane Review, which reported a meta-analysis of the trials assessing the effectiveness of adjuvant hGH therapy in women undergoing ovulation induction.254 In previous poor responders treated with hGH, the common odds ratio for pregnancy per cycle instituted was 2.55 (95% CI 0.64–10.12). No significant difference was noted in either the number of follicles and oocytes, or gonadotropin usage. It was concluded that a trend towards improved outcome with hGH treatment deserved further study. In a recent randomized, placebo-controlled study, Tesarik et al reported the use of hGH in women >40 years old undergoing ART.255 They used a high-dose FSH stimulation regimen (600 IU FSH) supplemented with 8 IU/day hGH from FSH stimulation day 7 until day +1 hCG. Although no improvement in the number of oocytes retrieved was observed, significantly higher plasma and intrafollicular E2 levels were found in the hGH group, and the clinical pregnancy and live

birth rates were also significantly higher in the hGHtreated group. They concluded that hGH may improve the potential for oocyte development. The action of GH in vitro in stimulating E2 production by follicular cells has been previously reported.200,201 It has recently been demonstrated that hGH receptor mRNA is expressed in human oocytes and throughout preimplantation embryonic development. Mendoza et al256 reported that the low hGH concentrations in follicular fluid were associated with cleavage failure and poor embryo morphology, whereas the addition of hGH to culture medium improves in vitro maturation of immature human oocytes. Izadyar et al257 suggested that hGH stimulates cytoplasmic maturation and may have a positive role in increasing total cell number in the embryo and in decreasing apoptosis, as described in the bovine species.258 It may also stimulate the mechanisms of DNA repair, as described in the liver.259 Further studies are eagerly awaited to elucidate the mechanisms through which hGH may exert a positive effect on embryonic development (Fig 44.10). In summary, considering the extra cost and limited data available, there is not a well-established clinical role for adjuvant hGH in the treatment of low responders at the current time. Further studies should be directed at defining the dose of hGH, and determining if select populations may benefit from hGH co-treatment.

Oral contraceptive pill pretreatment It has been suggested that the use of an OCP in the previous cycle may increase pregnancy rates in IVF.260 Because OCPs have a putative role in enhancement of estrogen receptor sensitization due to their estrogen content, in addition to exerting pituitary suppression, they have been used in combination with GnRH agonists. Biljan et al261 reported that pituitary suppression with OC and a GnRH-a was superior to GnRH-a alone regarding the time required to achieve pituitary suppression, as well as pregnancy and implantation rates. Because of these promising effects, OCPs have also been used in poor responders. However, there are only very few retrospective studies evaluating the actual contribution of OCPs in this group of patients. Lindheim et al262 found higher pregnancy rates with OCP alone compared with GnRH-a–treated cycles (both long and flare protocols). They concluded that the good outcome associated with OCP pretreatment might reflect production or alterations of local ovarian growth factors and/or changes at the endometrial level. In contrast with the above observations, Kovacs et al263 also compared retrospectively the use of OCPs with GnRH agonists for hypothalamic–pituitary suppression in poor responder IVF patients. Hypothalamic-pituitary suppression was performed with either an OCP or a GnRH-a followed by stimulation with gonadotropins. Cycle outcomes, including cancellation rates, gonadotropin requirements,

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599

• In vitro GH stimulates E2 production200,201 • Oocytes from follicles with high antral fluid GH levels have better developmental potential than oocytes from low GH follicles256 • GH receptor mRNA in human oocyte and preimplantation embryos (Menezo et al256a) • Nuclear and cytoplasmic maturation enhanced by GH (bovine – Izadyar et al257) • Role for GH in stimulation of DNA repair (as in liver, Thompson et al256b) • Improvement in normal fertilization and embryo development E 2 GH/IGF-I

FSH

Androgens

LH

number of oocytes retrieved, number of embryos transferred, and embryo quality, were similar. Patients in the OCP group required fewer days of stimulation to reach oocyte retrieval. Pregnancy rates were similar in the two groups. Overall, there was no improvement in IVF cycle outcome in poor responders who received OCPs to achieve pituitary suppression instead of a GnRH-a. In summary, although there is a general feeling that OCP pretreatment might be of assistance in the ovarian response of poor responders, especially in flareup regimens, only a minimal amount of published data exist to support this approach.

Luteal phase manipulations During the early follicular phase of the menstrual cycle, antral follicle sizes are often markedly heterogeneous. These follicle size discrepancies may, at least in part, result from the early exposure of FSH-sensitive follicles to gradient FSH concentrations during the preceding luteal phase. This phenomenon, which often occurs in women in low ovarian reserve, may potentially affect the results of ovarian stimulation. Pre-existing follicle size discrepancies may encumber coordinated follicular growth during ovarian stimulation, thereby reducing the number of follicles that reach maturation at once. Interventions aimed at coordinating follicular growth by manipulations at the midluteal phase of the preceding cycle are largely based on the innovative work of Fanchin et al.264 To investigate this issue, three clinical studies were conducted to test the hypothesis that luteal FSH suppression could coordinate follicular growth. First, luteal FSH concentrations were artificially lowered by administering physiological E2 doses and measured

Fig 44.10 E2 and GH/IGF-I may enhance oocyte quality by enhancing and coordinating cytoplasmic and nuclear maturation.

follicular characteristics on the subsequent day 3 in healthy volunteers.265 In this study, luteal E2 administration was found to reduce the size and to improve the homogeneity of early antral follicles on day 3. Secondly, it was verified whether luteal E2 administration could promote the coordination of follicular growth during ovarian stimulation and improve its results.266 Ninety IVF patients were randomly pretreated with 17β-estradiol (4 mg/day) from cycle day 20 until next cycle day 2 (n = 47) or controls (n = 43). On cycle day 3, all women started r-FSH treatment followed by a GnRH antagonist in the flexible protocol. The authors focused on the dynamics of follicular development, including magnitude of size discrepancy of growing follicles on day 8 of r-FSH treatment and number of follicles >16 mm in diameter on the day of hCG administration. On day 8, follicles were significantly smaller (9.9 ± 2.5 vs 10.9 ± 3.4 mm) and their size discrepancies attenuated in the treatment group compared with controls. This was associated with more >16 mm follicles, and more mature oocytes and embryos in the E2-treated group. It was concluded that luteal E2 administration reduces the pace of growth, improves size homogeneity of antral follicles on day 8 of r-FSH treatment, and increases the number of follicles reaching maturation at once. Thirdly, the effects of premenstrual GnRH antagonist administration on follicular characteristics were assessed during the early follicular phase.267 Twentyfive women underwent measurements of early antral follicles by ultrasound and serum FSH and ovarian hormones on cycle day 2 (control/day 2). On day 25, they received a single 3 mg cetrorelix acetate administration. On the subsequent day 2 (premenstrual GnRH

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antagonist/day 2), participants were re-evaluated as on control/day 2. The main outcome measure was the magnitude of follicular size discrepancies. Follicular diameters (4.1 ± 0.9 vs 5.5 ± 1.0 mm) and follicle-to-follicle size differences decreased on premenstrual GnRH antagonist/day 2 as compared with control/day 2. Consistently, FSH (4.5 ± 1.9 vs 6.7 ± 2.4 mIU/ml), E2 (23 ± 13 vs 46 ± 26 pg/ml), and inhibin B (52 ± 30 vs 76 ± 33 pg/ml) were lower on GnRH antagonist/day 2 than on control/day 2. It was concluded that premenstrual GnRH antagonist administration reduces diameters and size disparities of early antral follicles on day 2, probably through the prevention of luteal FSH elevation and early follicular development. Taken together, the results of the above studies suggest that luteal FSH suppression by either E2 or GnRH antagonist administration improves the size homogeneity of early antral follicles during the early follicular phase, an effect that persists during ovarian stimulation. Coordination of follicular development has the potential to optimize ovarian response to COS protocols, and constitutes an attractive approach for improving their outcome. An opposite approach of enhancing follicular recruitment by initiating FSH therapy during the late luteal as opposed to the early follicular phase has been attempted in prior poor responders but without success. In a prospective randomized trial, Rombauts et al268 failed to demonstrate any benefit of this regimen, with the exception that follicular maturation was achieved sooner after the onset of menses. Several studies evaluated the effects of combining pretreatment with E2 and/or GnRH antagonist during the luteal phase of the preceding cycle on the outcome of COS in poor responders. Dragisic et al269 reported lower cancellation rates and improved IVF outcome via a combination of estrogen patch therapy and GnRH antagonist started in the midluteal phase of the preceding menstrual cycle. Recently, Frattarelli et al270 reported a retrospective paired cohort analysis where they compared embryo and oocyte data between a standard protocol and a luteal phase E2 protocol. The results of 60 poor responder patients who underwent IVF with a luteal phase E2 protocol (17βestradiol 4 mg/day from cycle day 21 through the first 3 days of gonadotropin stimulation) were compared to 60 cycles in the same patients without E2 pretreatment. The luteal phase E2 protocol showed significant increases in the number of embryos with >7 cells, number of oocytes retrieved, number of mature oocytes, and number of embryos generated than did the standard protocol. There was no difference between the two protocols with respect to basal antral follicle count, days of stimulation, number of follicles >14 mm on day of hCG administration, or endometrial thickness. A trend toward improved pregnancy outcomes was found with the luteal E2 protocol. Using a slightly different approach, Fisch et al161 recently described their experience with a protocol

using GnRH-a/antagonist conversion with estrogen priming (AACEP) in low responders with prior IVF failures. The AACEP protocol focuses on promoting estrogenic dominance in the stimulated ovary, and opposing the potential ill effects of the LH flare and overproduction of androgens, which are commonly seen in GnRH-a flare, and in antagonist protocols. Patients received an OCP and a GnRH-a overlapping the last 5–7 days of the pill until the onset of menses. From cycle day 2, low-dose GnRH antagonist (0.125 mg/day), and estradiol valerate (2 mg) was given IM every 3 days for two doses, followed by estrogen suppositories until a dominant follicle was detected. Ovarian stimulation consisted of high-dose FSH/hMG. Although women aged <38 years old and those on 600 IU/day produced more mature eggs and fertilized embryos than women aged 38–42 years old, there were no differences in peak serum E2, endometrial thickness, or embryos transferred. Outcomes were similar for all patients, regardless of age or FSH dosage. Ongoing pregnancy rates were 27% for all patients, 25% for patients <38 years old, and 28% for patients 38–42 years old. It was concluded that the AACEP protocol may improve the prognosis and outcome for low responders with prior IVF failures. In summary, manipulating the luteal phase preceding the IVF treatment cycle may improve the coordination of follicular development and increase the number and quality of embryos achieved in poor responder patients. Ultimately, this may translate into improved cycle and pregnancy outcomes in these patients. It remains to be seen whether this approach is superior to pretreatment with an OCP, which is commonly practiced in various protocols designed for low responders. Properly designed randomized controlled trials (RCTs) are needed to test this innovative therapeutic approach.

Additional interventions Low-dose aspirin Low-dose aspirin therapy has been demonstrated to enhance blood perfusion in multiple different organ systems. This may be accomplished by proportionally greater inhibition of vascoconstricting prostaglandins (thromboxane A2) than the vasodilating prostaglandins (prostacyclin). Early reports on the use of aspirin in ART were encouraging. Rubinstein et al,271 reported a prospective, randomized, controlled evaluation of the impact of daily 100 mg aspirin on multiple outcome parameters, including ovarian responsiveness, oocyte number, implantation rates, and pregnancy rates in a general IVF population. Dramatic improvements in gonadotropin responsiveness, pregnancy rates, and implantation rates were reported. Low-dose aspirin therapy was also found to improve implantation rates in oocyte donation recipients with a thin endometrium,272 as well as in IUI cycles.273

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Following these early encouraging reports, multiple studies were conducted on general IVF populations, yielding conflicting results. Finally, two meta-analyses published in 2007 have reached a similar conclusion that currently available evidence does not support the use of aspirin in IVF or ICSI treatment.274,275 Interestingly, Ruopp et al276 reanalyzed the effects of low-dose aspirin in IVF and raised methodological questions regarding the analysis by Gelbaya et al.274 They conclude that aspirin may increase clinical pregnancy rates and that more data are needed to resolve the issue. In their opinion there is no reason to change clinical management and discontinue the use of aspirin, reflecting the ongoing controversy regarding the use of low-dose aspirin in IVF. The use of low-dose aspirin was specifically studied in low responder patients as well. Lok at el277 conducted a prospective, randomized, double-blind, placebo-controlled study evaluating the effect of adjuvant low-dose aspirin on utero-ovarian blood flow and ovarian responsiveness in poor responders undergoing IVF. Sixty patients received 80 mg aspirin daily or placebo during long down-regulation protocol. Doppler measurements of intraovarian and uterine pulsatility index were performed before (baseline) and after ovarian stimulation. Duration of use and dose of gonadotropins, cycle cancellation rate, number of mature follicles recruited, and oocytes retrieved were also compared. High cancellation rates were found in both groups (33.3% vs 26.7%, placebo vs treatment). There were no significant differences in gonadotropin requirements, median number of mature follicles recruited (3.5 vs 3.0), or median number of oocytes retrieved (4 vs 3). No significant differences were found in either intraovarian or uterine artery pulsatility index measured at baseline or on the day of hCG administration. It was concluded that supplementation with low-dose aspirin failed to improve either ovarian and uterine blood flow or ovarian responsiveness in poor responders undergoing IVF. Scoccia et al278 reported a retrospective cohort study, where 133 poor responder cycles were reviewed. There were 108 patients who received 81 mg aspirin daily during ovarian stimulation and 25 who did not. There was no improvement with aspirin use in pregnancy or live birth rates. However, there was a trend (p = 0.05) towards an improved implantation rate in cycles without aspirin. Recently, another retrospective cohort analysis was reported by Frattarelli et al.279 A total of 1250 poor-responder patients undergoing IVF were studied, of which 417 patients used 81 mg of aspirin before and during the IVF cycle, and 833 did not. Patients taking 81 mg of aspirin had significantly higher basal AFCs, required more days of stimulation, needed more ampules of gonadotropins, achieved higher peak E2 levels, and had more follicles that were ≥14 mm in diameter on the day of hCG administration. There was a decrease in the overall fertilization rate for the patients taking

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aspirin. There was no difference in IVF outcome (implantation, pregnancy, loss, or live birth). Overall, no improvement in IVF outcome secondary to 81-mg aspirin intake was found. Currently available evidence does not support the use of aspirin in poor responder patients undergoing IVF. However, the paucity of prospective randomized studies highlights the need for additional trials.

Aneuploidy screening Oocytes produced in women of AMA may have inadequate reserves of energy, due to age-related accumulated effects on their mitochondrial DNA.280 Energy produced by mitochondria is necessary for correct function throughout the processes of oocyte maturation and the final stages of meiosis, and mitochondrial dysfunction can lead to an increase in the frequency of nondysjunction, abnormalities in chromatid separation, and increased rates of apoptosis. As a result, the oocytes retrieved during ART procedures may be biochemically or chromosomally defective. Tzeng et al281 transferred a homologous mitochondrial enriched fraction of cytoplasm obtained from granulosa cells to compromised oocytes, and reported an increase in fertilization, embryo development, and pregnancies. It has long been known that women over the age of 35 years old are at increased risk of having a fetus with a chromosome abnormality, and this is probably due to an age-related increase in oocyte aneuploidy. Chromosome abnormalities are the primary cause of embryo wastage in patients >35 years old and up to 80% of embryos in women over 40 years old may be aneuploid (Table 44.2). Therefore, screening oocytes or embryos for aneuploidy so that only chromosomally normal embryos are transferred should increase implantation rates, reduce spontaneous abortion, and increase live-birth rates. Munné et al282 conducted a controlled clinical study to assess the incidence of chromosomal aneuploidy in women with a history of recurrent miscarriage presenting for assisted reproduction treatment. This group was compared with a group undergoing preimplantation genetic screening (PGS) because of advanced maternal age. Each embryo was biopsied on day 3, and blastomeres were analyzed by using fluorescent probes for chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22. Their findings confirmed that for women aged ≥35 years old, PGS significantly reduced pregnancy losses and increased the number of viable pregnancies. Taranissi et al283 also assessed the influence of maternal age on the outcome of PGS in IVF cycles carried out in patients with recurrent implantation failure. Their prospective study included 160 couples with a history of three or more failed fresh IVF attempts; the study population was divided into two groups: 78 patients aged ≤40 years old, and 38 patients aged ≥41 years old. Their results also confirmed the

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Table 44.2

The majority of embryos are chromosomally abnormal in older patients Age (years old)

Abnormality Aneuploidy (9 chromosomes)a Other aneuploidy (by CGH) Postmeiotic abnormalitiesb Total abnormal

20–34

35–39

24% 5% 35% 64%

27% 6% 36% 69%

40–47 39% (p<0.001) 8% 35% (NS) 82% (p<0.001)

From Munné et al,300 Marquez,301 and Gutierrez-Mateo et al.302 CGH comparative genomic hybridization. a Aneuploidy for chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22. b Mosaics, polyploid, haploid.

detrimental effects of increasing age: younger patients have a significantly higher proportion of euploid oocytes/embryos, cycles reaching embryo transfer, clinical pregnancy (36.1% vs 16.6%) and ongoing delivery (32% vs 12.5%) per embryo transfer. Similarly, Rubio et al284 performed 341 PGS cycles in women ≥38 years old (mean age = 40.5), and compared the results with a control group of women <37 years old. Their study confirmed a significant increase in the percentage of abnormal embryos in women of AMA (70.3% vs 33.1% in the control group; p <0.05). They were able to achieve acceptable ongoing pregnancy rates with PGS until the age of 42 years old (28.8%), but women >42 years old had a poorer prognosis. Conflicting results, however, were recently reported by Mastenbroek el al,285 who conducted a multicenter, randomized, double-blind, controlled trial comparing three cycles of IVF with and without PGS in women 35–41 years old: 408 women (206 assigned to PGS and 202 assigned to the control group) underwent 836 cycles of IVF (434 cycles with and 402 cycles without PGS). The ongoing pregnancy rate was significantly lower in the women assigned to PGS (25%) than in those not assigned to PGS (37%): (rate ratio = 0.69; 95% CI 0.51–0.93). The women assigned to PGS also had a significantly lower live birth rate (24%) vs (35%) (rate ratio = 0.68; 95% CI 0.50–0.92). It was concluded that PGS did not increase but instead significantly reduced the rates of ongoing pregnancies and live births after IVF in women of AMA.

Assisted hatching Assisted hatching involves the artificial thinning or breaching of the zona pellucida (ZP) and has been proposed as one technique to improve implantation and pregnancy rates following IVF. The assisted hatching procedure is generally performed on day 3 after fertilization using various methods. These include the creation of an opening in the zona either by drilling with acidified Tyrode’s solution, PZD with a glass microneedle, laser photoablation, or use of a piezomicromanipulator. The ZP can be artificially

thinned without breaching its integrity with proteolytic enzymes, acidified Tyrode’s solution, or laser. For a comprehensive review on assisted hatching the reader is referred to Chapter 13. A randomized, prospective trial on assisted hatching suggested an improvement in implantation rates when the procedure was selectively applied to embryos with a poor prognosis (based on zona thickness, blastomere number, fragmentation rates, maternal age, etc.).286 Since its introduction, many ART programs have incorporated the use of assisted hatching in efforts to improve clinical outcomes. Success rates following the use of assisted hatching have varied considerably. However, differences in patient populations, operator experience, hatching technique, and study design make it difficult to compare directly reports from different centers. Assisted hatching has been suggested for increasing the chance for implantation and pregnancy in poorprognosis women, such as those with multiple IVF failures or those >38 years old.286,287 The role of assisted hatching in the treatment of poor responders has never been directly assessed, but poor responders have been often included as poor-prognosis patients either because of advanced maternal age or because of multiple previous failed IVF attempts. Schoolcraft et al288 compared prospectively 33 poor-prognosis IVF patients (elevated day 3 FSH level, age ≥39 years old, and multiple prior IVF failures) who were treated with assisted hatching, with 43 control subjects without assisted hatching. The implantation and ongoing pregnancy rate in the assisted hatching group were 33% and 64%, compared with 6.5% and 19% in the control group, respectively. It was concluded that assisted hatching, when applied to poor-prognosis patients, improves embryonic implantation and pregnancy rates. Similarly, Stein et al287 have demonstrated that, in a selected group of patients (aged >38 years old, who have failed to conceive in >3 previous IVF attempts), assisted hatching significantly increases the clinical pregnancy rate (23.9% in the study group compared with only 7% of the controls). Focusing on maternal age, Meldrum et al289 demonstrated that following

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assisted hatching, the implantation rate was increased in women aged 35–39 years old and markedly increased in women aged 40–42 years old, but not in women over 43 years old. Magli et al290 conducted a prospective randomized study on the efficacy of the assisted hatching in poorprognosis patients with (1) maternal age ≥38 years old (45 cycles); (2) ≥3 failed IVF attempts (70 cycles), and (3) patients possessing both inclusion criteria (20 cycles). The control groups included patients with similar characteristics who did not undergo assisted hatching. The clinical pregnancy rate per cycle following assisted hatching was significantly higher than in controls for the first (31% vs 10%) and second groups (36% vs 17%), but not for the third group. Similarly, higher implantation rates were obtained (11.5%, 15% and 11%) compared with the respective controls (4%, 6.3%, and 1.5%). Finally, in a prospective randomized study, Mansour et al291 demonstrated that transfer of zona-free embryos significantly increased pregnancy rates in poor-prognosis patients (age ≥40 years old, and/or ≥2 previous failed IVF–ICSI attempts) compared with controls (23% vs 7.3%), but not in good-prognosis patients (age <40 years old undergoing the first ICSI attempt). Other prospective randomized prospective studies have failed to find a benefit for assisted hatching in women with advanced maternal age. Bider et al292 focused on patients >38 years old undergoing IVF. A total of 839 embryos from 211 patients underwent assisted hatching during 312 cycles of therapy and compared to 540 nonhatched embryos transferred to 174 patients during 274 cycles of therapy. The pregnancy rate was not statistically different between the groups (8.9% in the assisted hatching group vs 5.1% in the controls) as were implantation rates (3.75% and 3.55%, respectively), and delivery rates (3.8% and 3.4%, respectively). It was concluded that assisted hatching in patients aged >38 years old does not increase the takehome baby rate after IVF. Lanzendorf et al293 reached similar conclusions in a randomized controlled study in which patients ≥36 years old were treated with (n = 41) or without (n = 48) assisted hatching. No significant differences were observed in the rates of implantation (11.1% vs 11.3%), clinical pregnancy (39.0% vs 41.7%), and ongoing pregnancy (29.3% vs 35.4%) between the hatched and control groups, respectively. These results suggest that assisted hatching may have no significant impact on IVF success rates in the patient population studied. It should be stated that while all of the above studies focused on patients with AMA, in some of them a mean of 8–10 oocytes were retrieved,291,293 which automatically excludes them from the poor-responders category. A recent comprehensive review and meta-analysis294 identified 23 RCTs involving 2668 women undergoing assisted hatching during ART. Only six of the studies included in the analysis (involving 516 women) reported live birth rates with and without assisted

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hatching. Women undergoing assisted hatching were significantly more likely to achieve clinical pregnancy (23 RCTs, OR = 1.33, 95% CI 1.12–1.57). There was no significant difference in the odds of live births in the assisted hatching compared with control groups (OR = 1.19 95% CI 0.81–1.73). Subgroups of patients who demonstrated the greatest improvement in clinical pregnancy rates were those with prior failed ART cycles and older women. Due to lack of power, the numbers of live births reported in studies do not allow a confident conclusion regarding the clinical efficacy of assisted hatching procedures. The authors concluded that, despite significantly improved odds of clinical pregnancy, there is insufficient evidence to determine any effect of assisted hatching on live birth rates. Currently, there is insufficient evidence to recommend assisted hatching. With regard to low responder patients, large prospective studies involving documented poor responders should be conducted in order to assess the efficacy of this intervention in this subgroup of patients.

Summary: practical considerations There are several key issues which make the challenge of developing treatment strategies for low responder patients difficult and frustrating: 1.

2.

3.

There is no universally accepted definition of low responders. While many papers are referenced in this text, all use a large variety of inclusion criteria and are therefore not readily comparable. Similarly, there is no universally accepted definition of diminished ovarian reserve during infertility investigation prior to ART treatment. The lack of any large-scale, prospective RCTs of the different management strategies does not allow any definitive conclusion to be drawn.

It is therefore suggested that a real effort to achieve a consensus by a panel of experts on the definitions and categories of poor response and diminished ovarian reserve should be made, in a similar manner to the consensus that was reached on the definition of polycystic ovarian syndrome (PCOS). This would allow both the scientific community and our patients to better profit from the many studies that are constantly being conducted on these issues. In the mean time, the following practical considerations represent a combination of the evidence presented above with long-standing clinical experience.

High-dose gonadotropins Patients with either diminished ovarian reserve (by testing prior to treatment) or poor ovarian response in previous cycles may benefit from high-dose gonadotropin therapy (300–450 IU of FSH daily) in order to maximize oocyte yield.

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Long GnRH-a protocol The long GnRH-a protocol might be the protocol of choice if ovarian reserve is estimated to be low but not critically diminished. If the long protocol is to be used, progestagen pretreatment may reduce the incidence of cyst formation. Reducing the dose of the GnRH-a once pituitary down-regulation has been achieved (minidose agonist), might help to augment ovarian response. Depot GnRH-a preparations should not be used.

Short or micro-dose flare GnRH-a protocol Oral contraceptive pretreatment is extremely important in short GnRH-a regimens, as it may prevent the ill effects of LH and androgen secretion caused by the endogenous gonadotropin flare.

Failure to respond to a GnRH-a regimen Failure to respond to a long or short GnRH-a regimen does not necessarily mean cycle cancellation. Agonist administration can be withdrawn, but gonadotropin stimulation continued. The cycle can be converted either to an antagonist or a modified natural cycle regimen, once ovarian response is observed.

The poor response that is commonly observed in women of advanced maternal age is directly related to a diminished ovarian reserve. The associated reduction in oocyte quality as manifested by the increase in aneuploidy embryos is most likely due to suboptimal cytoplasmic maturation (including reduced capacity of oocyte mitochondria to generate sufficient quantities of energy required for fertilization and cell division). The strategies outlined above, utilizing pharmacogenomics and manipulating endocrinology, may provide a means of augmenting follicular recruitment and cytoplasmic integrity, and thus improve the prognosis for these women. Recent studies indicate that androgen supplementation may be one area to explore further. The availability of r-hLH has made it possible to investigate the role of LH in the endocrinology of follicular recruitment: it appears that a defect in the balance of LH/FSH might be involved in the subtle age-related decline in follicular recruitment, and patients of older reproductive age undergoing ART might benefit from the addition of LH and/or hGH. Further studies are required to investigate the physiological mechanisms behind this observation, and to assess the possible effect of r-hLH and/or hGH supplementation on the age-related decline in pregnancy rate.

Addition of LH activity Since (1) there is evidence suggesting that patients with AMA and/or low responders may benefit from the addition of LH activity to the stimulation regimen, and (2) it is not currently possible to identify those patients who are LH deficient following GnRH-a or antagonist administration, it is recommended to add LH (a minimum of 150IU daily) during the final stages of follicle and oocyte maturation (from stimulation day 6 onwards).

Conclusions Women who have entered the declining years of fecundity and then require assisted reproduction have always been a major challenge in ART treatment. In addition to the obstacles of diminished ovarian reserve, resistance to ovarian stimulation, and higher frequency of potential gynecological disorders, they are also at higher risk of producing aneuploid oocytes and embryos. Uterine factors, as well as the possibility of aneuploid embryos, result in an increased miscarriage rate. Their situation is further compounded by the psychological stress of knowing that the ‘biological clock’ is ticking, and time is against them. Although the use of donor oocytes has proved to be a very successful alternative treatment, this is not an option in many parts of the world, and efforts must be made to maximize each patient’s potential to use her own oocytes. If a sufficient number of oocytes and embryos can be obtained, aneuploidy screening by PGS may allow abnormal embryos to be eliminated, thus increasing the chance of implantation and reducing miscarriage rates.

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Treatment strategies in ART for low responder patients 180. Bassett RM, Driebergen R. Continued improvements in the quality and consistency of follitropin alfa, recombinant human FSH. Reprod Biomed Online 2005; 10: 169–77. 181. Aittomaki K, Herva R, Stenman UH, et al. Clinical features of primary ovarian failure caused by a point mutation in the follicle-stimulating hormone receptor gene. J Clin Endocrinol Metab 1996; 81: 3722–6. 182. Touraine P, Beau I, Gougeon A, et al. New natural inactivating mutations of the follicle-stimulating hormone receptor: correlations between receptor function and phenotype. Mol Endocrinol 1999; 13: 1844–54. 183. Perez Mayorga M, Gromoll J, Behre HM, et al. Ovarian response to follicle-stimulating hormone (FSH) stimulation depends on the FSH receptor genotype. J Clin Endocrinol Metab 2000; 85: 3365–9. 184. Jun JK, Yoon JS, Ku SY, et al. Follicle-stimulating hormone receptor gene polymorphism and ovarian responses to controlled ovarian hyperstimulation for IVF-ET. J Hum Genet 2006; 51: 665–70. 185. Behre HM, Greb RR, Mempel A, et al. Significance of a common single nucleotide polymorphism in exon 10 of the follicle-stimulating hormone (FSH) receptor gene for the ovarian response to FSH: a pharmacogenetic approach to controlled ovarian hyperstimulation. Pharmacogenet Genomics 2005; 15: 451–6. 186. Balasch J, Miro F, Burzaco I, et al. The role of luteinizing hormone in human follicle development and oocyte fertility: evidence from in-vitro fertilization in a woman with long-standing hypogonadotrophic hypogonadism and using recombinant human follicle stimulating hormone. Hum Reprod 1995; 10: 1678–83. 187. Hull M, Corrigan E, Piazzi A, Loumaye E. Recombinant human luteinising hormone: an effective new gonadotropin preparation. Lancet 1994; 344: 334–5. 188. De Placido G, Alviggi C, Mollo A, et al. Effects of recombinant LH (rLH) supplementation during controlled ovarian hyperstimulation (COH) in normogonadotrophic women with an initial inadequate response to recombinant FSH (rFSH) after pituitary downregulation. Clin Endocrinol (Oxf) 2004; 60: 637–43. 189. De Placido G, Alviggi C, Perino A, et al. Recombinant human LH supplementation versus recombinant human FSH (rFSH) step-up protocol during controlled ovarian stimulation in normogonadotrophic women with initial inadequate ovarian response to rFSH. A multicentre, prospective, randomized controlled trial. Hum Reprod 2005; 20: 390–6. 190. Howles CM. Role of LH and FSH in ovarian function. Mol Cell Endocrinol 2000; 161: 25–30. 191. Chappel SC, and Howles C. Reevaluation of the roles of luteinizing hormone and follicle-stimulating hormone in the ovulatory process. Hum Reprod 1991; 6: 1206–12. 192. Hillier SG. Current concepts of the roles of follicle stimulating hormone and luteinizing hormone in folliculogenesis. Hum Reprod 1994; 9: 188–91. 193. Balasch J, Fábregues F. Is luteinizing hormone needed for optimal ovulation induction? Curr Opin Obstet Gynecol 2002; 14: 265–74.

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poor-responder women: a randomized controlled trial on the effect of luteinizing hormone supplementation on in vitro fertilization cycles. Fertil Steril 2008; 89: 546–53. 224. Mochtar MH, Van der Veen, Ziech M, van Wely M. Recombinant luteinizing hormone (rLH) for controlled ovarian hyperstimulation in assisted reproductive cycles. Cochrane Database Syst Rev: 2007; (2): CD005070. 225. Adashi EY. The ovarian follicular apparatus. In: Adashi EY, Rock J, Rosenwaks Z, eds. Reproductive Endocrinology, Surgery and Technology. Philadelphia: Lippincott-Raven 1996: 7–40. 226. Weil SJ, Vendola K, Zhou J, et al. Androgen receptor gene expression in the primate ovary: cellular localization, regulation, and functional correlations. J Clin Endocrinol Metab 1998; 83: 2479–85. 227. Hillier SG. Gonadotropic control of ovarian follicular growth and development. Mol Cell Endocrinol 2001; 179: 39–46. 228. Hugues JN, Durnerin IC. Impact of androgens on fertility – physiological, clinical and therapeutic aspects. Reprod Biomed Online 2005; 11: 570–80. 229. Piltonen T, Koivunen R, Perheentupa A, et al. Ovarian age-related responsiveness to human chorionic gonadotropin in women with polycystic ovary syndrome. J Clin Endocrinol Metab 2004; 89: 3769–75. 230. Guay A, Munarriz R, Jacobson J, et al. Serum androgen levels in healthy premenopausal women with and without sexual dysfunction: Part A. Serum androgen levels in women aged 20–49 years with no complaints of sexual dysfunction. Int J Impot Res 2004; 16: 112–20. 231. Frattarelli JL, Peterson EH. Effect of androgen levels on in vitro fertilization cycles. Fertil Steril 2004; 81: 1713–14. 232. Barbieri RL, Sluss PM, Powers RD, et al. Association of body mass index, age, and cigarette smoking with serum testosterone levels in cycling women undergoing in vitro fertilization. Fertil Steril 2005; 83: 302–8. 232a.Durnerin CI, Erb K, Fleming R, et al. Effects of recombinant LH treatment on folliculogenesis and responsiveness to FSH stimulation. Hum Reprod 2008; 23: 421–6. 233. Massin N, Cédrin-Durnerin I, Coussieu C, et al. Effects of transdermal testosterone application on the ovarian response to FSH in poor responders undergoing assisted reproduction technique – a prospective, randomized, double-blind study. Hum Reprod 2006; 21: 1204–11. 234. Casson PR, Lindsay MS, Pisarska MD, Carson SA, Buster JE. Dehydroepiandrosterone supplementation augments ovarian stimulation in poor responders: a case series. Hum Reprod 2000; 15: 2129–32. 235. Barad D, Brill H, Gleicher N. Update on the use of dehydroepiandrosterone supplementation among women with diminished ovarian function. J Assist Reprod Genet 2007; 24: 629–34. 236. Barad D, Gleicher N. Effect of dehydroepiandrosterone on oocyte and embryo yields, embryo grade and cell number in IVF. Hum Reprod 2006; 21: 2845–9.

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Treatment strategies in ART for low responder patients 237. Barad DH, Gleicher N. Increased oocyte production after treatment with dehydroepiandrosterone. Fertil Steril 2005; 84: 756. 238. Mitwally MF, Casper RF. Aromatase inhibition improves ovarian response to follicle-stimulating hormone in poor responders. Fertil Steril 2002; 77: 776–80. 239. Garcia-Velasco JA, Moreno L, Pacheco A, et al. The aromatase inhibitor letrozole increases the concentration of intraovarian androgens and improves in vitro fertilization outcome in low responder patients: a pilot study. Fertil Steril 2005; 84: 82–7. 240. Shoham Z, Homburg R, Owen EJ, et al. The role of treatment with growth hormone in infertile patients. Baillières Clin Obstet Gynaecol 1992; 6: 267–81. 241. Owen EJ, Shoham Z, Mason BA, Ostergaard H, Jacobs HS. Cotreatment with growth hormone, after pituitary suppression, for ovarian stimulation in in vitro fertilization: a randomized, double-blind, placebo-control trial. Fertil Steril 1991; 56: 1104–10. 242. Adashi EY, Resnick CE, Hurwitz A, et al. Insulinlike growth factors: the ovarian connection. Hum Reprod 1991; 6: 1213–19. 243. Adashi EY, Resnick CE, Hernandez ER, et al. Insulin-like growth factor I as an intraovarian regulator: basic and clinical implications. Ann NY Acad Sci 1991; 626: 161–8. 244. Ibrahim ZH, Matson PL, Buck P, Lieberman BA. The use of biosynthetic human growth hormone to augment ovulation induction with buserelin acetate/ human menopausal gonadotropin in women with a poor ovarian response. Fertil Steril 1991; 55: 202–4. 245. Wu MY, Chen HF, Ho HN, et al. The value of human growth hormone as an adjuvant for ovarian stimulation in a human in vitro fertilization program. J Obstet Gynaecol Res 1996; 22: 443–50. 246. Busacca M, Fusi FM, Brigante C, et al. Use of growth hormone-releasing factor in ovulation induction in poor responders. J Reprod Med 1996; 41: 699–703. 247. Howles CM, Loumaye E, Germond M, et al. Does growth hormone-releasing factor assist follicular development in poor responder patients undergoing ovarian stimulation for in-vitro fertilization? Hum Reprod 1999; 14: 1939–43. 248. Suikkari A, MacLachlan V, Koistinen R, Seppala M, Healy D. Double-blind placebo controlled study: human biosynthetic growth hormone for assisted reproductive technology. Fertil Steril 1996; 65: 800–5. 249. Shaker AG, Fleming R, Jamieson ME, Yates RW, Coutts JR. Absence of effect of adjuvant growth hormone therapy on follicular responses to exogenous gonadotropins in women: normal and poor responders. Fertil Steril 1992; 58: 919–23. 250. Hughes SM, Huang ZH, Morris ID, et al. A doubleblind cross-over controlled study to evaluate the effect of human biosynthetic growth hormone on ovarian stimulation in previous poor responders to in-vitro fertilization. Hum Reprod 1994; 9: 13–18. 251. Dor J, Seidman DS, Amudai E, et al. Adjuvant growth hormone therapy in poor responders to invitro fertilization: a prospective randomized placebo-controlled double-blind study. Hum Reprod 1995; 10: 40–3.

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252. Younis JS, Simon A, Koren R, et al. The effect of growth hormone supplementation on in vitro fertilization outcome: a prospective randomized placebocontrolled double-blind study. Fertil Steril 1992; 58: 575–80. 253. Blumenfeld Z, Lunenfeld B. The potentiating effect of growth hormone on follicle stimulation with human menopausal gonadotropin in a panhypopituitary patient. Fertil Steril 1989; 52: 328–31. 254. Kotarba D, Kotarba J, Hughes E. Growth hormone for in vitro fertilization. Cochrane Database Syst Rev 2000; (2): CD000099. 255. Tesarik J, Hazout A, Mendoza C. Improvement of delivery and live birth rates after ICSI in women aged >40 years by ovarian co-stimulation with growth hormone. Hum Reprod 2005; 20: 2536–41. 256. Mendoza C, Cremades N, Ruiz-Requena E, et al. Relationship between fertilization results after intracytoplasmic sperm injection, and intrafollicular steroid, pituitary hormone and cytokine concentrations. Hum Reprod 1999; 14: 628–35. 256a Menezo YJ, el Mouatassim S, Chavrier M, et al. Human oocytes and preimplantation embryos express mRNA for growth hormone receptor. Zygote 2003; 11: 293–7. 256b Thompson BJ, Shang CA, Waters MJ. Identification of genes induced by growth hormone in rat liver using cDNA arrays. Endocrinology 2000; 141: 4321–4. 257. Izadyar F, Van Tol HT, Hage WG, Bevers MM. Preimplantation bovine embryos express mRNA of growth hormone receptor and respond to growth hormone addition during in vitro development. Mol Reprod Dev 2000; 57: 247–55. 258. Kolle S, Stojkovic M, Boie G, Wolf E, Sinowatz F. Growth hormone inhibits apoptosis in in vitro produced bovine embryos. Mol Reprod Dev 2002; 61: 180–6. 259. Thompson BJ, Shang CA, Waters MJ. Identification of genes induced by growth hormone in rat liver using cDNA arrays. Endocrinology 2000; 141: 4321–4. 260. Gonen Y, Jacobson W, Casper RF. Gonadotropin suppression with oral contraceptives before in vitro fertilization. Fertil Steril 1990; 53: 282–7. 261. Biljan MM, Mahutte NG, Dean N, et al. Effects of pretreatment with an oral contraceptive on the time required to achieve pituitary suppression with gonadotropin-releasing hormone analogues and on subsequent implantation and pregnancy rates. Fertil Steril 1998; 70: 1063–9. 262. Lindheim SR, Barad DH, Witt B, Ditkoff E, Sauer MV. Short-term gonadotropin suppression with oral contraceptives benefits poor responders prior to controlled ovarian hyperstimulation. J Assist Reprod Genet 1996; 13: 745–7. 263 Kovacs P, Barg PE, Witt BR. Hypothalamic–pituitary suppression with oral contraceptive pills does not improve outcome in poor responder patients undergoing in vitro fertilization–embryo transfer cycles. J Assist Reprod Genet 2001; 18: 391–4. 264. Fanchin R, Mendez Lozano DH, Schonauer LM, Cunha-Filho JS, Frydman R. Hormonal manipulations in the luteal phase to coordinate subsequent antral follicle growth during ovarian stimulation. Reprod Biomed Online 2005; 10: 721–8.

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265. Fanchin R, Salomon L, Castelo-Branco A, et al. Luteal estradiol pre-treatment coordinates follicular growth during controlled ovarian hyperstimulation with GnRH antagonists. Hum Reprod 2003; 18: 2698–703. 266. Fanchin R, Cunha-Filho JS, Schonauer LM, et al. Coordination of early antral follicles by luteal estradiol administration provides a basis for alternative controlled ovarian hyperstimulation regimens. Fertil Steril 2003; 79: 316–21. 267. Fanchin R, Castelo Branco A, Kadoch IJ, et al. Premenstrual administration of gonadotropinreleasing hormone antagonist coordinates early antral follicle sizes and sets up the basis for an innovative concept of controlled ovarian hyperstimulation. Fertil Steril 2004; 81: 1554–9. 268. Rombauts L, Suikkari AM, MacLachlan V, Trounson AO, Healy DL: Recruitment of follicles by recombinant human follicle-stimulating hormone commencing in the luteal phase of the ovarian cycle. Fertil Steril 1998; 69: 665–9. 269. Dragisic KG, Davis OK, Fasouliotis SJ, Rosenwaks Z. Use of a luteal estradiol patch and a gonadotropinreleasing hormone antagonist suppression protocol before gonadotropin stimulation for in vitro fertilization in poor responders. Fertil Steril 2005; 84: 1023–6. 270. Frattarelli JL, Hill MJ, McWilliams GD, et al. A luteal estradiol protocol for expected poor-responders improves embryo number and quality. Fertil Steril 2007 Jul 19 [Epub ahead of print] 271. Rubinstein M, Marazzi A, Polak de Fried E. Lowdose aspirin treatment improves ovarian responsiveness, uterine and ovarian blood flow velocity, implantation, and pregnancy rates in patients undergoing in vitro fertilization: a prospective, randomized, double-blind placebo-controlled assay. Fertil Steril 1999; 71: 825–9. 272. Weckstein LN, Jacobson A, Galen D, Hampton K, Hammel J. Low-dose aspirin for oocyte donation recipients with a thin endometrium: prospective, randomized study. Fertil Steril 1997; 68: 927–30. 273. Hsieh YY, Tsai HD, Chang CC, Lo HY, Chen CL. Low-dose aspirin for infertile women with thin endometrium receiving intrauterine insemination: a prospective, randomized study. J Assist Reprod Genet 2000; 17: 174–7. 274. Gelbaya TA, Kyrgiou M, Li TC, Stern C, Nardo LG. Low-dose aspirin for in vitro fertilization: a systematic review and meta-analysis. Hum Reprod Update 2007; 13: 357–64. 275. Khairy M, Banerjee K, El-Toukhy T, Coomarasamy A, Khalaf Y. Aspirin in women undergoing in vitro fertilization treatment: a systematic review and metaanalysis. Fertil Steril 2007; 88: 822–31. 276. Ruopp MD, Collins TC, Whitcomb BW Schisterman EF: Evidence of absence or absence of evidence? A reanalysis of the effects of low-dose aspirin in in vitro fertilization. Fertil Steril 2008; 90: 71–6. 277. Lok IH, Yip SK, Cheung LP, Yin Leung PH, Haines CJ. Adjuvant low-dose aspirin therapy in poor responders undergoing in vitro fertilization: a prospective, randomized, double-blind, placebocontrolled trial. Fertil Steril 2004; 81: 556–61.

278. Scoccia H, Puccini M, Horlick N, Winston N. Advancing maternal age and low-dose aspirin effect in poor responders undergoing in vitro fertilization. Fertil Steril 2005; 84: S248. 279. Frattarelli JL, McWilliams GD, Hill MJ, Miller KA, Scott RT Jr. Low-dose aspirin use does not improve in vitro fertilization outcomes in poor responders. Fertil Steril 2008; 89: 1113–17. 280. May-Panloup P, Chretien MF, Jacques C, et al. Low oocyte mitochondrial DNA content in ovarian insufficiency. Hum Reprod 2005; 20: 593–7. 281. Tzeng CR, Hsieh RH, Au HK, et al. Mitochondria transfer (MIT) into oocyte from autologous cumulus granulosa cells (cGCs). Fertil Steril 2004; 82: S53. 282. Munne S, Chen S, Fischer J, et al. Preimplantation genetic diagnosis reduces pregnancy loss in women aged 35 years and older with a history of recurrent miscarriages. Fertil Steril 2005; 84: 331–5. 283. Taranissi M, El-Toukhy T, Gorgy A, Verlinsky Y. Influence of maternal age on the outcome of PGD for aneuploidy screening in patients with recurrent implantation failure. Reprod Biomed Online 2005; 10: 628–32. 284. Rubio C, Rodrigo L, Perez-Cano I, et al. FISH screening of aneuploidies in preimplantation embryos to improve IVF outcome. Reprod Biomed Online 2005; 11: 497–506. 285. Mastenbroek S, Twisk M, van Echten-Arends J, et al. In vitro fertilization with preimplantation genetic screening. N Engl J Med 2007; 357: 9–17. 286. Cohen J, Alikani M, Trowbridge J, Rosenwaks Z. Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Hum Reprod 1992; 7: 685–91. 287. Stein A, Rufas O, Amit S, et al. Assisted hatching by partial zona dissection of human pre-embryos in patients with recurrent implantation failure after in vitro fertilization. Fertil Steril 1995; 63: 838–41. 288. Schoolcraft WB, Schlenker T, Gee M, Jones GS, Jones HW Jr. Assisted hatching in the treatment of poor prognosis in vitro fertilization candidates. Fertil Steril 1994; 62: 551–4. 289. Meldrum DR, Wisot A, Yee B, et al. Assisted hatching reduces the age-related decline in IVF outcome in women younger than age 43 without increasing miscarriage or monozygotic twinning. J Assist Reprod Genet 1998; 15: 418–21. 290. Magli MC, Gianaroli L, Ferraretti AP, et al. Rescue of implantation potential in embryos with poor prognosis by assisted zona hatching. Hum Reprod 1998; 13: 1331–5. 291. Mansour RT, Rhodes CA, Aboulghar MA, Serour GI, Kamal A. Transfer of zona-free embryos improves outcome in poor prognosis patients: a prospective randomized controlled study. Hum Reprod 2000; 15: 1061–4. 292. Bider D, Livshits A, Yonish M, et al. Assisted hatching by zona drilling of human embryos in women of advanced age. Hum Reprod 1997; 12: 317–20. 293. Lanzendorf SE, Nehchiri F, Mayer JF, Oehninger S,Muasher SJ. A prospective, randomized, doubleblind study for the evaluation of assisted hatching

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in patients with advanced maternal age. Hum Reprod 1998; 13: 409–13. Seif MM, Edi-Osagie EC, Farquhar C, et al. Assisted hatching on assisted conception (IVF & ICSI). Cochrane Database Syst Rev 2006; (1): CD001894. Sudo S, Kudo M, Wada S, et al. Genetic and functional analyses of polymorphisms in the human FSH receptor gene. Mol Hum Reprod 2002; 8: 89–90. Laven JS, Mulders AG, Suryandari DA, et al. Follicle- stimulating hormone receptor polymorphisms in women with normogonadotropic anovulatory infertility. Fertil Steril 2003; 80: 986–92. de Castro F, Moron FJ, Montoro L, et al. Pharmacogenetics of controlled ovarian hyperstimulation. Pharmacogenomics 2005; 6: 629–37. Daelemans C, Smits G, de Maertelaer V, et al. Prediction of severity of symptoms in iatrogenic ovarian hyperstimulation syndrome by follicle-stimulating

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hormone receptor Ser680Asn polymorphism. J Clin Endocrinol Metab 2004 89: 6310–15. Choi D, Lee EY, Yoon S, et al. Clinical correlation of cycline D2 mRNA expression in human luteinized granulosa cells. J Assist Reprod Genet 2000; 17: 574–9. Munne S, Alikani M, Tomkin G, et al. Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities. Fertil Steril 1995; 64: 382–91. Marquez, C. Sandalinas M, Bahce M, et al. Chromosome abnormalities in 1255 cleavage-stage human embryos. Reprod Biomed Online 2000; 1: 17–26. Gutierrez-Mateo C, Wells D, Benet J, et al. Reliability of comparative genomic hybridization to detect chromosome abnormalities in first polar bodies and metaphase II oocytes. Hum Reprod 2004; 19: 2118–25.

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45 Repeated implantation failure: the preferred therapeutic approach Mark A Damario, Zev Rosenwaks

Overview The treatment of human infertility through the assisted reproductive technologies (ART) continues to be comparatively inefficient. Despite the common practice of multiple embryo transfers, all in vitro fertilization-embryo transfer (IVF-ET) procedures performed in the United States in 2001 resulted in a mean 31.6% delivery rate per oocyte retrieval.1 Although this IVF-ET delivery rate is actually an improvement over the preceding years, it is obvious that the majority of IVF-ET cycles still fail. While a clearly attributable cause for cycle failure may occasionally be present, in most circumstances there is no apparent explanation other than failure of the implantation process. Although both subclinical and clinical pregnancy losses occur, the largest percentage of failed IVF-ET cycles simply exhibit lack of implantation. In some patients, implantation failure occurs repeatedly. These latter patients continue to present unique challenges for the infertility specialist. Age is perhaps the most important single variable influencing outcome in assisted reproduction. The effect of advancing female age on clinical IVF-ET is manifested not only in the pattern of ovarian response to gonadotropin stimulation but also in reduced implantation efficiency and an increased spontaneous abortion rate.2 Determination of diminished ovarian reserve by timed hormonal evaluation provides useful prognostic information regarding assisted reproductive treatment.3,4 Ovarian reserve testing, however, still does not provide 100% sensitivity in the detection of women with reduced IVF-ET treatment potential. Embryonic loss which occurs repeatedly after assisted reproduction may be attributable to many factors. These include embryonic genetic abnormalities, a lack of endometrial receptivity, and suboptimal laboratory culture conditions. Genetic abnormalities may perhaps account for at least as many as 30–40% of implantation failures.5 It is likely that, even in the best of circumstances, some embryonic loss occurs due to the artificial laboratory environment. This is supported by the differences in morphology and

cleavage rates of in vivo human embryos and human embryos that have been supported in vitro.6 This chapter summarizes several of the contemporary strategies used to enhance IVF-ET outcome in cases of repeated implantation failure. Included in these strategies will be our preferred treatment approaches, which will be outlined in further detail.

Methods Prophylactic salpingectomy It has recently become apparent that patients with severe tubal damage have a poor prognosis with IVFET. In many retrospective reports, patients with hydrosalpinges have been identified as having lower implantation and pregnancy rates than patients suffering other types of tubal damage.7–9 Similar adverse effects on embryonic implantation specifically attributable to hydrosalpinges were noted in two metaanalyses of published comparative studies.10,11 Different theories have evolved to explain the mechanism behind the association of hydrosalpinges with poorer pregnancy outcome. Reflux of hydrosalpinx fluid into the uterine cavity may simply result in mechanical factors diminishing embryonic endometrial apposition.12 Hydrosalpinx fluid is commonly slightly alkaline and may also contain cytokines, prostaglandins, or other inflammatory compounds.13 These inflammatory mediators may result in either direct embryotoxicity or adverse effects on the endometrium.14,15 One group has demonstrated an association of hydrosalpinges with altered endometrial histology and a lack of expression of endometrial adhesion molecules (integrins) which may play important roles in the implantation process.16 Clinical evidence has shown that improved clinical outcomes are seen after prophylactic bilateral salpingectomy. Several retrospective studies have demonstrated that bilateral salpingectomy results in improved implantation as well as pregnancy rates compared with controls who harbor hydrosalpinges.17,18

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Assisted hatching It has been found that only a relatively small percentage of human embryos cleave beyond the 8-cell stage in vitro to form expanded blastocysts. In addition, fewer than 25% of such expanded blastocysts have been shown to hatch in vitro, presumably secondary to abnormal zona hardening.19 It was also noted that cleavage-stage embryos with a reduced zona thickness seemed to have a good prognosis for implantation.20 Furthermore, microsurgically fertilized embryos with artificial gaps in their zonae (from partial zona dissection) seemed to have higher rates of implantation.21 From these observations, techniques were developed to promote improved embryo implantation efficiency. Assisted hatching was first tested experimentally by introducing small incisions in the zonae of 4-cell embryos by a mechanical method.22 However, the observation of embryonic entrapment in the narrow zona openings during hatching23 and the potential for embryo damage due to micromanipulation prior to the formation of blastomere structural junctions led to the development of an alternative zona drilling procedure which is performed with acidified Tyrode’s solution on 3-day-old embryos.24 Assisted hatching has also been accomplished utilizing a piezo-micromanipulator25 and a laser.26 Early randomized, prospective trials examining the efficacy of assisted hatching were undertaken at the Center for Reproductive Medicine and Infertility at the Weill Cornell Medical College and New YorkPresbyterian Hospital.27 The initial trials included patients with normal basal follicle-stimulating hormone (FSH) concentrations. Assisted hatching appeared to benefit patients with thick zonae (>15 µm). Further trials employed zona biometric criteria as the indication for zona drilling. These latter selective assisted hatching trials indicated that women aged >38 years old appeared to derive the most benefit from the procedure.

Preimplantation genetic diagnosis (aneuploidy screening) There is significant evidence that implantation failure in women of advanced maternal age is closely linked to embryonic aneuploidy. This is based on data from spontaneous abortions28 as well as recent data on oocytes and embryos.29,30 Utilizing blastomere biopsy and fluorescent in situ hybridization (FISH) to diagnose X, Y, 18, 13, and 21 aneuploidy, Munné et al noted that even in embryos judged to be of good quality, aneuploidy rates were 4.0%, 9.4%, and 37.2%, in women aged 20–34, 35–39, and 40–47 years old, respectively.30 A relationship between maternal age and aneuploidy for chromosome 16 was also identified.31 Therefore, considering only these data, rates of aneuploidy in women aged 40 years old would be expected to exceed 40%. There remains a possibility

that the rate of embryonic aneuploidy may be even higher in these women when future assessment of additional chromosomes is included. Methods have been developed for the detection of aneuploidy in older women undergoing IVF-ET. These methods were developed in order to improve the implantation efficiency, reduce the spontaneous abortion rate, as well as decrease the incidence of chromosomal abnormalities at term. The first method involves embryo biopsy on day 3, in which one to two blastomeres are removed from an 8- to 10-cell embryo. Early work suggested that removing single blastomeres from 8-cell embryos did not affect their viability or ability to progress to the blastocyst stage.32 While analyses of two blastomeres may be preferable in order to reduce misdiagnoses, there is presently less clinical experience with two-blastomere biopsies. Following blastomere biopsy, the cells are fixed on a slide and analyzed by FISH. Most studies to date have been carried out with a multiple probe technique in a time frame compatible with clinical IVF (embryo transfer on day 4 or 5).33 Up to five chromosomes can be detected by FISH at the single-cell level. Further recent investigations suggest that even more chromosomes may be investigated incorporating techniques of FISH–FISH cell recycling in which two or more rounds of hybridization are employed.34 Recent work has included the use of comparative genomic hybridization (CGH) on single blastomeres for the purpose of aneuploidy screening. CGH is a molecular cytogenetic technique that allows for the simultaneous assessment of every chromosome in single interphase cells.35 Recently, investigators have applied CGH to single human blastomeres from disaggregated human embryos.36 The clinical application of CGH for preimplantation detection of embryonic aneuploidy has also been reported.37,38 Chief limitations of single-cell CGH are that it is complex and requires 4 days to complete, thereby requiring initial freezing of biopsied embryos and later thawing prior to transfer. The use of first polar body analysis for aneuploidy detection has also been proposed as an alternative to blastomere analysis since most aneuploidies originate from maternal meiosis I nondisjunction.39,40 In order to further uncover aneuploidies deriving from errors in the second meiotic division, the sequential analysis of the first and second polar bodies using multiprobe FISH has also been reported.41 Polar body analyses, however, are hampered by the inability to diagnose paternally derived chromosome abnormalities as well as those resulting from post-fertilization events.

Blastocyst culture and transfer In attempts to improve the overall efficiency of human IVF-ET, investigators have strived to identify embryos with higher implantation potential. One method that has been repeatedly explored is the culture of human embryos to the blastocyst stage (day 5 of culture). In the human embryo, activation of the embryonic genome

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occurs at the 8–10-cell stage (day 3 of culture). Embryos which cleave after day 3 in culture therefore are no longer dependent on maternal RNA transcripts and have made the successful transition from maternal to embryonic genomic control. Embryos which progress to the blastocyst stage may thereby represent embryos with higher implantation potential. Blastocyst culture and transfer approaches may potentially provide certain patients with a history of repeated implantation failures an improved chance for pregnancy. Early attempts to culture human embryos to the blastocyst stage, however, were discouraging as it was clear that the culture medium in use was primitive and would not support long-term growth of human embryos. In 1992, significant improvements were obtained when human embryos were cocultured in vitro along with Vero cell monolayers.42 The availability of new culture media has recently furthered interest in the culture of human embryos to the blastocyst stage.43 New sequential culture media have been designed specifically for the first 2 days after fertilization (early cleavage) and the third to fifth days of embryo growth (morula and blastocyst). These new sequential culture media systems clearly have resulted in improved blastocyst culture and transfer results over those seen with conventional culture media.44 A few investigators have suggested that blastocyst culture and transfer may potentially improve clinical outcome in women with repeated implantation failure.45,46 Since cleaving embryos do not normally reside in the uterine cavity, it is felt that there is a possibility that some embryos may experience nutritional or homeostatic stress when introduced during day 2–3 transfers. Of perhaps more significance is the fact that blastocyst culture and transfer may allow for better embryo selection. A higher incidence of aneuploidy has been detected in embryos which fail to progress to the blastocyst stage in vitro.47 On the other hand, simply progression to blastocyst stage certainly would not be expected to guarantee chromosomal normality. Blastocyst culture and transfer, however, may be used as an additional tool in older women with a history of repeated implantation failure by providing an indirect screening tool for aneuploidy. Owing to the continued limited efficiency of blastocyst culture and transfer techniques, this would be expected to work favorably only if satisfactory ovarian responses can be achieved.

Coculture methods The quality of in vitro culture conditions is one of the most crucial aspects of successful IVF-ET. Studies in many lower mammalian species have suggested that growth, biochemical synthetic activity, and survival after transfer are inferior in in vitro derived embryos when compared with in vivo derived embryos.48–50 Embryonic developmental blocks are frequently encountered during the transition from maternal to embryonic genomic activation.

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Various attempts at improving in vitro culture conditions by modifying the medium electrolyte and energy sources have met with limited success.51 An alternative approach has been the development of coculture systems in which a variety of ‘helper cells’ have provided a more efficient means to maintain human embryos in vitro. Various cell types have been used, including tubal or endometrial epithelium (from humans or animals), autologous cumulus or granulosa cells, or an established cell line (monkey kidney epithelial cells [Vero cells]).52–56 Use of coculture methods has produced somewhat variable results, although most investigators have noted at least improvements in embryonic developmental rates.54,55 The variability in success rates associated with coculture systems probably is attributable to differences in cell lines, maintenance of the cells, and various environmental factors within each laboratory. Although the beneficial effects of coculture systems have been demonstrated by a number of researchers, the mechanism of action of these helper cells remains uncertain. Coculture cells have been demonstrated to both produce embryotrophic factors57 as well as serve to detoxify the culture medium.58 There is a fair consensus that coculture improves embryo morphology, blastocyst development, and hatching.59 In addition, a better synchrony between embryo development and the uterine environment may occur. At the Center for Reproductive Medicine and Infertility at the Weill Cornell Medical College and New York-Presbyterian Hospital we have developed a unique coculture system that uses the patient’s own endometrial cells and successfully applied this system to our clinical IVF-ET program.60,61 In this system, patients undergo an endometrial biopsy performed in the mid to late luteal phase of a cycle preceding their actual IVF-ET treatment cycle. Endometrial glandular epithelial and stromal cells are then separated by differential sedimentation and plated until monolayers are obtained. The cells are then cryopreserved and later thawed at a precise time in synchrony with the patient’s treatment. An equal mixture of glandular epithelial and stromal cells is seeded into a four-well tissue plate containing Ham’s F-10 medium supplemented with 15% patient’s serum. In general, approximately 75% confluence is achieved by the time embryos are placed into the system (Fig 45.1). Embryos are introduced into the coculture system after fertilization checks (Fig 45.2) and maintained with the autologous endometrial cells until day 3 (Fig 45.3) when embryo transfer is performed.

Results Prophylactic salpingectomy In women with tubal factor infertility, the presence of hydrosalpinges has been related to poorer IVF outcomes in comparison to women without hydrosalpinges in

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Fig 45.1 Autologous endometrial cell cultures at confluence consisting of a mixture of glandular epithelial cells and stromal cells.

Fig 45.2 Pronuclear oocytes placed on autologous endometrial coculture.

Fig 45.3 Day 3 embryos developed on autologous endometrial coculture.

numerous retrospective studies.7–9 It seems that there may be a relation between the size of the hydrosalpinx and reduced implantation, as one group of investigators has noted that only when hydrosalpinges were large enough to be visualized by ultrasonography were clinical outcomes diminished.62 Two meta-analyses estimated that hydrosalpinges diminished implantation rates by 35–50%.10,11 In addition, both meta-analyses

also reported an increased risk of early pregnancy loss in patients with hydrosalpinges. Two prospective randomized trials involving prophylactic salpingectomy in patients with severe tubal factor infertility and hydrosalpinges have explored whether implantation rates and clinical outcomes can be improved in these patients.63,64 In the first limited monocentric study, Dechaud et al63 reported an improved implantation rate (10.4%) in the group with salpingectomy in comparison to the group without salpingectomy (4.6%) during the first IVF attempt. For all IVF-ET attempts, the respective implantation rates in the two groups were 13.4% and 8.6%, respectively. In addition, the ongoing pregnancy rate per transfer was 34.2% in the group with salpingectomy compared with 18.7% in the group without salpingectomy. A prospective randomized multicenter trial of salpingectomy prior to IVF was conducted in Scandinavia.64 Inclusion criteria included the presence of unilateral or bilateral hydrosalpinges as determined by either hysterosalpingography, or laparoscopy and age <39 years old. A total of 204 patients were available for an intention to treat analysis and 192 actually started IVF. Clinical pregnancy rates per included patient were 36.6% in the salpingectomy group and 23.9% in the nonintervention group (not significant, p = 0.067). Subgroup analyses, however, revealed significant differences in favor of salpingectomy in patients with bilateral hydrosalpinges (implantation rates of 25.6% vs 12.3%, p = 0.038) and in patients with ultrasound visible hydrosalpinges (clinical pregnancy rates of 45.7% vs 22.5%, p = 0.029; delivery rates of 40.0% vs 17.5%, p = 0.038). In addition, the delivery rate was increased 3.5-fold in patients who had exhibited bilateral hydrosalpinges on ultrasound (p = 0.019). Further studies have only strengthened the evidence that IVF clinical outcomes in women with severe tubal disease are improved following bilateral salpingectomy. Strandell et al65 reported on the effect of cumulative cycles after reviewing further data from the Scandinavian randomized, prospective trial. After taking into account the number of cycles per patient and the presence of salpingectomy after a previous transfer, salpingectomy resulted in a significant increase in birth rate (hazard ratio = 2.1, 95% confidence interval [CI] 1.6–3.6, p = 0.014). Similar findings were included in a recent Cochrane review.66 In this meta-analysis of published randomized, controlled trials, the odds ratios (ORs) for both pregnancy and live birth were statistically increased with laparoscopic salpingectomy prior to IVF. One retrospective study has reported improved pregnancy rates in patients with severe tubal factor infertility who underwent laparoscopic salpingectomy after experiencing repeated implantation failure.67 One can therefore conclude that patients with severe tubal factor infertility have improved clinical outcomes following prophylactic salpingectomy,

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particularly if they have either bilateral hydrosalpinges or hydrosalpinges large enough to be visualized by ultrasound. Laparoscopic salpingectomy appears to improve clinical outcomes in patients who have hydrosalpinges and repeated implantation failures. On the other hand, the clinical efficacy of prophylactic salpingectomy in the presence of either unilateral hydrosalpinges or hydrosalpinges that are not visible on ultrasound requires further study.

Assisted hatching The clinical results after assisted hatching in poor prognosis patients undergoing IVF-ET have been mixed. In the initial randomized clinical trials from our institution, it appeared that breaches in the zona pellucida impaired the clinical pregnancy rate after transfers of drilled embryos with a thin zona pellucida (<13 µm), while facilitating the implantation of embryos in the setting of a thick zona pellucida (>15 µm), compared to controls.27 In a later trial, selective assisted hatching was performed only on those embryos with a thick zona pellucida or poor morphology (<5 cells or >20% fragmentation on day 3). The overall implantation rates per transferred embryos were improved in the selectively zona drilled group (25%) when compared with controls (18%) (p <0.05). Selective assisted hatching, however, seemed to have the largest impact in improving the implantation rate of women >38 years old (16% vs 3%, p <0.05). Women with elevated basal FSH concentrations greater than 15 mIU/ml also seemed to benefit, particularly, although this has not been subsequently corroborated. In nonrandomized studies using historical controls, several investigators reported improved implantation efficiency following assisted hatching in poor prognosis patients (>40 years old or several IVF-ET failures).68–70 Most of these centers attempted to use assisted hatching globally rather than selectively use assisted hatching according to zona characteristics. One investigator challenged the zona thickness theory by reporting no differences in mean zona thickness in subsequently pregnant (18.5 µm) as opposed to nonpregnant (18.7 µm) patients.71 Other investigators also failed to demonstrate clinical benefits from assisted hatching in patients selected for advanced age, zona thickness, or previous failed attempts.72 There have been relatively few prospective, randomized controlled trials examining the efficacy of assisted hatching in poor prognosis patients. Magli et al73 reported the clinical efficacy of assisted hatching in 135 cycles with a poor prognosis for pregnancy: (1) maternal age = 38 years old (45 cycles); (2) three or more previous failed IVF-ET attempts (70 cycles); and (3) patients possessing both criteria (20 cycles). The control group (113 cycles) included patients possessing the same characteristics (42, 53, and 18 cycles, respectively) who did not undergo the assisted hatching procedure. The percentage of clinical pregnancies per

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cycle was significantly higher for the first (31% vs 10%, p <0.05) and second groups (36% vs 17%, p <0.05). No significant difference in pregnancy rates was noted in the third group, although the numbers were limited. Chao et al.74 also reported a prospective randomized study of assisted hatching exclusively in patients with a history of repeatedly failed IVF-ET and noted significantly improved pregnancy and implantation rates in the assisted hatching group following transcervical, but not transtubal, embryo transfers. Lanzendorf et al,75 however, were unable to ascertain any statistically significant benefits of assisted hatching in a prospective randomized study that comprised unselected patients of ≥36 years old. In addition, a recent Cochrane systematic review of randomized trials reported that, despite the significantly improved odds of clinical pregnancy, there was insufficient evidence to demonstrate an improvement in live birth rates with assisted hatching in unselected patients.76 To account for some of the variable clinical results seen with assisted hatching, investigators have examined some of the different available techniques of assisted hatching. Hsieh et al26 reported that the 1.48 µm noncontact diode laser was more effective for assisted hatching than the chemical method in older patients. In a multicenter prospective randomized trial, Primi et al77 showed that following recurrent implantation failure, implantation and pregnancy rates trended higher (although not statistically significant) with inclusion of assisted hatching with the diode laser. In contrast, Balaban et al78 did not find any appreciable differences when selective assisted hatching was performed by either the partial zona dissection, acid Tyrode’s, diode laser, or pronase thinning methods. Other researchers have reported on a modification of the laser-assisted hatching technique in which the zona is partially thinned without a total breach.79 In fact, Mantoudis et al80 reported that the highest pregnancy rates occurred in patients treated with laser partial thinning extended to a quarter segment of the zona. A further prospective randomized study by Petersen et al81 demonstrated significantly higher implantation rates (p = 0.02) in patients with recurrent implantation failure who received quarterlaser zona thinning. Meldrum et al70 have suggested that results of chemical-assisted hatching are highly technique dependent. They noted a time-dependent improvement in clinical results associated with the technique which they attributed to increasing technical experience. With experience, embryologists can perform the procedure rapidly, thus limiting temperature and pH changes around the embryo. In addition, the size and shape of the gap in the zona may be fashioned in a more consistent manner. Although hard to assess, perhaps operator-related variables have contributed to some of the inconsistent reported clinical results seen with assisted hatching.

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Preimplantation genetic diagnosis (aneuploidy screening) The extensive use of polar body testing for aneuploidy in order to improve IVF-ET outcome has been reported by Verlinsky et al.82 These investigators reported the application of first and second polar body testing with multi-probe FISH in 659 cycles of women of advanced maternal age (>35 years old). Specific probes for chromosomes 13, 18, and 21 were used. Fluorescent in situ hybridization results were available for 3217 (81.6%) of 3943 oocytes studied, of which 1388 (43.1%) had aneuploidies; 35.7% of aneuploidies were of first meiotic division origin, and 26.1% of second meiotic division origin. The transfer of embryos derived from 1558 aneuploidy-free oocytes in 614 treatment cycles resulted in 131 clinical pregnancies (21.3%) and 88 healthy children born (with an additional 18 pregnancies ongoing). As this was a nonrandomized study, the precise impact of the preselection of aneuploidy-free oocytes on the overall IVF-ET efficiency is hard to determine. A larger body of literature is available on the use of blastomere biopsy approaches in the screening for aneuploidy in patients with a poor prognosis (older women and those with repeated IVF-ET failure).83–86 In an initial nonrandomized trial, Gianaroli et al83 reported on preimplantation genetic diagnosis (PGD) for aneuploidy of chromosomes X, Y, 13, 18, and 21 on 196 embryos from 36 infertile patients classified with a poor prognosis due to: (1) maternal age = 38 years old (n = 11); (2) repeated IVF failure (n = 22); and (3) altered karyotype (46XX/45XO mosaics) (n = 3). The percentage of abnormal embryos was comparable in the three groups: maternal age (63%); repeated IVF failure (57%); and mosaic karyotype (62%). They noted an increase in the percentage of chromosomally abnormal embryos that was directly proportional to the number of IVF failures. This led these investigators to propose that the high rate of chromosomally abnormal embryos may have been the cause of implantation failure. Subsequently, these investigators performed a prospective randomized controlled trial using a similar PGD scheme in women with either maternal age ≥38 years old or ≥3 previous IVF failures.84 Assisted hatching was performed on day 3 embryos in the control group. In the study group, a total of 61 embryos were analyzed, with 55% detected to be chromosomally abnormal. Embryo transfer with at least one normal embryo was carried out in 10 cycles, resulting in four clinical pregnancies and a 28.0% implantation rate. In the control group, 41 embryos were transferred in 17 cycles, resulting in four clinical pregnancies and a statistically lower implantation rate (11.9%). A later multicenter PGD for aneuploidy study was performed in women aged ≥35 years old.85 Initially, this study was intended to be randomized. Owing to lack of available data at the time supporting a beneficial

clinical effect of aneuploidy screening, however, few patients agreed to the study, and those who committed to it rejected randomization. Therefore, PGD cases were matched retrospectively with controls based on maternal age, number of previous IVF cycles, duration of stimulation, estradiol concentration, and number of mature follicles. One or two cells per embryo were biopsied on day 3 and analyzed by FISH. In most cases, embryos classified as normal after PGD were transferred on the same day of analysis. During the beginning of the trial, probes for the simultaneous detection of chromosomes X, Y, 18, and the shared alpha satellite region of chromosomes 13 and 21 were used (n = 14). Later, specific probes for X, Y, 13, 18, and 21 (n = 22) were used. Even later, a probe for chromosome 16 was added to the previous mixture and used in an additional proportion of cases (n = 50). Finally, a small fraction of cases (n = 31) benefited from having the biopsied cells analyzed with the X, Y, 13, 16, 18, and 21 probe mixture and then reanalyzed with a second probe mixture specific for chromosomes 14, 15, and 22. Only embryos classified as normal were transferred after PGD. The rates of fetal heart beat (FHB)/embryo transferred were similar between the test and control groups. However, spontaneous abortions, measured as FHB aborted/FHB detected, decreased after PGD (24.2% vs 9.6%, p <0.05) and ongoing fetuses or delivered babies per embryo transferred increased after PGD (15.9% vs 10.6%, p <0.05). From this trial, the authors concluded that while increased implantation efficiency was not proven, PGD for aneuploidy reduced the rate of embryo loss after implantation. Later trials have further expanded the testing capabilities of FISH on biopsied blastomeres.33,86,87 In one trial, Gianaroli et al86 reported on the outcomes of patients of maternal age ≥36 years old, ≥3 previous IVF failures, or abnormal karyotypes who underwent PGD testing, including two rounds of FISH (initial analysis of chromosomes X, Y, 13, 16, 18, and 21 followed by reanalysis for chromosomes 14, 15, and 22). In many of these cases, embryo transfer was carried out on day 4 to allow time for the two rounds of FISH analysis. The investigators reported in a randomized controlled trial that an increase in the ongoing implantation rate (22.5% vs 10.2%, p <0.001) was achieved in the PGD patients compared with controls. The clinical benefits of PGD were most notable in women ≥38 years old and in carriers of an altered karyotype. In a further large randomized controlled aneuploidy screening (AS) trial, Staessen et al87 compared blastocyst transfer combined with PGD-AS using FISH for chromosomes X, Y, 13, 16, 18, 21, and 22, with a control group without PGD-AS, in couples with advanced maternal age (mean = 39 years old in control and 40 years old in study groups, respectively). The implantation rates were similar (17.1% and 11.5%, respectively), but a significantly higher number of embryos were replaced in the control group. In PGD-AS cycles with genetically normal embryos, no morula or blastocyst formation

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occurred in 11 of 148 cycles. The authors reported that in patients from which an expanded blastocyst on day 5 results, the chance of selecting a genetically normal embryo is 65% vs 41.3% if an 8-cell stage embryo is selected on day 3. Mastenbroek et al88 reported on a recent multicenter randomized controlled trial comparing three cycles of IVF with and without preimplantation genetic screening (PGS) in women 35–41 years old. The PGS method used in this trial included analysis for chromosomes 1, 16, and 17, followed by analysis for chromosomes 13, 18, 21, X, and Y. In this report, 206 women were assigned to PGS and 202 women were assigned to the control group. Ultimately, 434 cycles with PGS and 402 cycles without were performed. Interestingly, in this report of consecutive PGS cycles, the ongoing pregnancy rate was actually significantly lower in the women assigned to PGS (25%) than in those not assigned to PGS (37%). The women receiving PGS also demonstrated a lower live birth rate (24% vs 35%; rate ratio = 0.68; 95% CI 0.50–0.92). Further trials have looked specifically at the outcomes of PGD for aneuploidy screening in recurrent implantation failure patients.89–91 Pehlivan et al89 reported using FISH on one or two blastomeres from 49 implantation failure patients (defined as three or more failed IVF attempts) and compared them with nine fertile controls. In each case, three rounds of FISH were utilized (assessing chromosomes 13, 16, 18, 21, 22, X, and Y) and transfer was undertaken on day 5. There was a significantly higher rate of chromosomal abnormalities in the implantation failure patients (67.4%) than in controls (36.3%). Following PGD-AS, the implantation failure patients demonstrated a pregnancy rate of 34.0% and implantation rate of 19.8%, which was comparable to controls (33.1% and 24.1%, respectively). On the other hand, Munné et al90 reported that the major clinical effect seen with PGD-AS was in women of advanced maternal age with eight or more 2-pronuclear zygotes. In this report, an increase in implantation rate was not observed in patients with two or more previous IVF attempts or in patients with fewer than eight zygotes. Finally, Taranissi et al91 reported on the use of FISH of the first and second polar bodies using probes specific for chromosomes 13, 16, 18, 21, and 22 in couples with recurrent implantation failure. Subsequent diploid oocytes that cleaved were further tested using the same probes on a single blastomere from day 3 embryos and subsequent chromosomally normal embryos were replaced on day 5. In this study, patients aged ≤40 years old had a significantly higher proportion of euploid oocytes and embryos, cycles reaching embryo transfer, and ongoing delivery rates per transfer (32 vs 12.5%) than patients aged ≥41 years old. Voullaire et al38 reported on the use of CGH in single blastomeres from 20 women with repeated implantation failure. Biopsied embryos were initially

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cryopreserved. Individual blastomeres underwent alkaline lysis followed by whole genome amplification and CGH. Abnormalities detected include aneuploidy for one or two chromosomes (25%) and complex chromosomal abnormalities (29%). Approximately 40% of the embryos were considered suitable for transfer, although the investigators did not report on clinical outcomes. In comparison to CGH, multicolor FISH utilizing a five probe set would have detected 77% of the abnormalities and incorrectly diagnosed 38% of abnormal embryos. In comparison to CGH, repetitive rounds of FISH utilizing a nine probe set would have detected 85% of the abnormalities but still incorrectly diagnosed 25% of abnormal embryos as normal.

Blastocyst culture and transfer As stated earlier, it has been hypothesized that in some patients cleavage-stage embryos prematurely transferred into the uterine environment may undergo nutritive and homeostatic stress.92 On the other hand, uterine hostility to cleavage-stage embryos seems doubtful in lieu of the excellent clinical results achieved in some clinics. In addition, perhaps blastocyst culture and transfer may serve as an indirect method of screening out aneuploidic embryos as it is known that the rate of aneuploidy is increased in embryos which arrest in culture.47 Since the proportion of aneuploidic embryos appears to increase directly with the number of failed previous IVF-ET cycles,83 blastocyst culture and transfer may be of clinical benefit in patients with a history of repeated implantation failure. Cruz et al93 reported the use of blastocyst culture and transfer in patients who had previously failed three or more IVF cycles and who had at least three 8–12-cell embryos on day 3. In this nonrandomized small trial using a selected ‘poor prognosis’ patient group, a statistically significant increase in clinical pregnancy and implantation rates was seen in the blastocyst group compared with controls. Guerif et al94 similarly reported the use of blastocyst transfer in a nonrandomized analysis in patients who previously failed at least two cleavage-stage embryo transfers with at least two good-quality embryos. These investigators reported that live birth rate and implantation rates were subsequently higher when day 5 or day 6 transfers were used. Barrenetxea et al95 performed a retrospective analysis of day 5 and day 6 transfers in patients with repeated implantation failure. In this analysis, day 5 transfers had nearly five-fold the implantation rate as day 6 transfers (23% vs 5%, respectively). Finally, Levitas et al96 performed a prospective randomized study utilizing patients with recurrent cleavage-stage implantation failure. In this trial, the clinical pregnancy rates per oocyte retrieval were 21.7% and 12.9% per blastocyst and day 2–3 embryo transfers, respectively.

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The main disadvantage of blastocyst culture and transfer is that the rate of blastocyst development is still limited. Even with newer sequential culture media, blastocyst formation occurs in only <55% of embryos. While a higher proportion of embryos which fail to develop to the blastocyst stage in culture are apparently chromosomally abnormal, it is still uncertain whether some embryos may fail to progress simply because of presently suboptimal culture conditions. In addition, it remains difficult to fully evaluate the clinical benefits of blastocyst culture and transfer since many trials have used selected patient groups. Patients who are high responders to gonadotropin stimulation seem to be excellent candidates for blastocyst culture approaches since they are not only likely to have many embryos available for transfer but also the ability to select among available blastocysts in many instances probably also enhances the implantation rate.97 The clinical efficacy of blastocyst culture and transfer in unselected patient groups is less certain. In particular, patients with multiple failed previous IVF-ET cycles will frequently exhibit poor responses to gonadotropin therapy and have few embryos available. Therefore, blastocyst culture and transfer may not be the best approach for all patients with repeated implantation failure.

Coculture methods Favorable clinical results have been achieved utilizing various cellular preparations (both human and animal) in coculture systems. Vero cells (monkey kidney epithelial cells) have been documented to be beneficial in long-term cocultures of embryos, resulting in an increase in the total number and quality of blastocysts when compared to embryos that were not cultured in the presence of Vero cells.98 Use of bovine oviductal epithelial cells in combination with the use of assisted hatching and day 3 embryo replacements were noted to yield relatively high pregnancy rates in poor prognosis patients.99,100 Coculture of human embryos with buffalo rat liver cells seemed to exhibit a favorable trend towards improving pregnancy rates in patients with previous in vitro fertilization failure (34% coculture vs 28% control).101 Because of the potential infectious risks associated with the use of animal cells, recent investigators have focused on utilizing human cells (both autologous and homologous) in coculture systems. To mimic the in vivo environment of the fallopian tube, tubal cells from the ampullary portion of the fallopian tube have been used.102,103 In these reports, the cells were harvested during postpartum tubal ligations or hysterectomies and passaged several times in order to achieve adequate numbers of cells for multiple patients. Embryonic viability, morphological appearance, and the number of blastocysts were reported to be enhanced with the tubal epithelial coculture system.102 Further clinical benefits of tubal epithelial

coculture have included a higher pregnancy rate, a higher implantation rate, lower spontaneous abortion rate, and an increased number of spare embryos available for cryopreservation.103 Autologous systems for coculture have also been developed. One of the simplest involves the use of granulosa or cumulus cells derived from the cells collected during the patient’s retrieval. The use of autologous cells in coculture is relatively safe and ethical for the patient, although it can be time consuming as each coculture is individualized. Furthermore, since the granulosa or cumulus cells are plated after retrieval, any coculture benefit provided to either the gametes or early embryo is probably limited. Nevertheless, Plachot et al104 noted convincing evidence of the benefits of granulosa cell coculture. Using each patient as her own control, one-half of the zygotes was cultured using either standard methods or autologous granulosa cell coculture. Eighty-three percent of granulosa cell coculture embryos were available for transfer compared with only 3% of controls. Other investigators have also noted beneficial morphological effects with cumulus cells utilized in the coculture of supernumerary embryos.105,106 More recently, Carrell et al107 reported that the use of autologous cumulus coculture improved embryo morphology, implantation rates, and clinical pregnancy rates following IVF. At The Center for Reproductive Medicine and Infertility at the Weill Medical College and New YorkPresbyterian Hospital we have developed a unique coculture system using autologous cryopreserved endometrial cells. Advantages of this system include use of a readily available source of autologous cells, avoidance of the infectious and ethical risks when using either animal or homologous cell lines, and use of cells in which preimplantation embryo development is known to take place. In addition, there is rather convincing evidence of a chemical dialogue between the developing embryo and the maternal endometrium.108,109 Coculture with human endometrial epithelial cells has been noted to be beneficial to blastocyst development, presumably owing to the induction of embryonic paracrine secretion.110 Furthermore, endometrial cells may be cryopreserved so that a proper cellular confluence can be timed in order to allow a beneficial effect for the early developing embryo. Coculture of embryos on autologous endometrial cells prior to transfer in patients with repeated failures of implantation was first reported by Jayot et al.111 With this approach, these investigators reported a pregnancy rate of 21% vs 8% in patients’ previous cycles. These investigators used a mixture of stromal and epithelial cells following 1 month of subculture and multiple tissue flask passages. Nieto et al112 used cryopreserved autologous endometrial (predominantly epithelial) cells and reported a positive effect on the proportion of embryos with minimal or no fragmentation. Simon et al113 further developed a coculture system using

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autologous endometrial epithelial cells that were previously cryopreserved. In 168 cycles in patients with a history of implantation failure (>3 previous failed cycles), a 49.2% blastocyst formation, 11.8% implantation rate, and a 20.2% pregnancy rate were achieved using a day 6 transfer approach. We have used an autologous endometrial coculture system incorporating both stromal and epithelial cells in equal proportions. It is highly likely that endometrial stromal cells also play a significant role in implantation. Our system isolates endometrial stromal and epithelial cells through differential sedimentation, obtaining cell lines of >90% purity. Cells are then cryopreserved and subsequently thawed in synchrony with the patient’s IVF cycle so that a developing monolayer of both epithelial and stromal cells is available by the time the fertilized oocytes have reached the pronuclear stage. Zygotes are then placed on coculture and incubated until day 3, when transfer is undertaken. The initial trial using our autologous endometrial coculture system was undertaken in women who had a history of at least one previously failed IVF-ET attempt with poor embryo quality (defined as <6 cells or
Table 45.1

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to the patient irrespective of the culture system. From this study, although it was found that an approximately equal number of embryos were transferred from either group, embryos derived from autologous endometrial coculture had a statistically lower percentage of fragmentation and higher mean number of blastomeres at the time of transfer (Table 45.1). A second trial utilized autologous endometrial coculture in patients with a history of at least one previously failed IVF-ET attempt with poor embryo quality.61 In this trial, all available embryos were allocated to coculture. Again, it was noted that coculture resulted in a significant improvement in the mean number of blastomeres compared to that in the patient’s previous noncoculture cycle. The implantation and clinical pregnancy rates in these coculture cycles were 15% and 29%, respectively. In subsequent trials, we have noted that enhancement of cleavage rates and the lowering of the degree of fragmentation associated with autologous endometrial coculture appears to be related to the timing of the initial endometrial biopsy (Tables 45.2, 45.3).114 In particular, better results were obtained when the endometrial biopsy was obtained in the mid to late luteal phase as opposed to the early luteal phase of the menstrual cycle. With this in mind, we have further optimized our coculture system.

Characteristics of human embryos developed on autologous endometrial coculture and conventional medium

Embryo characteristics

Coculture

Conventional medium

No. of embryos Mean (± SD) No. of blastomeres (day 3) Mean (± SD) % of fragmentation No. of embryos transferred Mean (± SD) No. of blastomeres (transfer)

203 5.9 ± 1.5 21 ± 13 90 7.4 ± 1.3

186 5.5 ± 1.4 24 ± 11 83 6.7 ± 1.9

Wilcoxon’s matched-pairs test

0.19 0.045 0.032

Table 45.2 Cleavage characteristics of human embryos developed on autologous endometrial coculture (AECC) and conventional medium (CM) according to timing of the endometrial biopsy

All patients (n = 79) Early luteal (n = 33) Mid/late luteal (n = 46)

No. of blastomeres AECC

No. of blastomeres CM

p Value

6.2 ± 1.4 6.0 ± 1.6 6.3 ± 1.2

5.5 ± 1.3 5.6 ± 1.4 5.5 ± 1.2

0.0015 0.19 0.0024

Table 45.3 Degree of fragmentation of human embryos developed on autologous endometrial coculture (AECC) and conventional medium (CM) according to timing of the endometrial biopsy

All patients (n = 79) Early luteal (n = 33) Mid/late luteal (n = 46)

% fragmentation AECC

% fragmentation CM

17.7 ± 12.3 19.4 ± 13 16.5 ± 12

21.6 ± 11 20 ± 11 22.8 ± 11

p Value 0.04 0.87 0.012

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Complications Prophylactic salpingectomy Some investigators have expressed concern that salpingectomy prior to IVF may impair ovarian response. The mechanism in which salpingectomy might cause reduced ovarian response is not clear, but unilateral or bilateral removal of the fallopian tubes may have a detrimental effect on the ovarian arterial blood supply. Lass et al115 have demonstrated that there were fewer follicles and, consequently, fewer oocytes retrieved from the ipsilateral ovary in women who had previously undergone a unilateral salpingectomy. Other investigators have not demonstrated diminished ovarian responses in women who had undergone bilateral salpingectomy as compared to a tubal factor control group.116 For women with already suspected diminished ovarian reserve, however, the potential detrimental effect of unilateral or bilateral salpingectomy on ovarian response must be considered. In these cases, either interruption of tubal uterine patency or ultrasound-guided drainage of hydrosalpinges might also be considered.117,118 Moreover, salpingectomy is not a procedure without the recognized complications of operative laparoscopy and/or laparotomy in addition to the rare complications of subsequent interstitial119 or abdominal pregnancies.120

Assisted hatching The risk of injury to the embryo during the performance of assisted hatching techniques should be minimal in experienced hands. Because the breach in the zona pellucida may reduce some of the embryo’s natural defenses to bacteriologic and other pathogenic organisms, many investigators have advocated the concurrent use of corticosteroids and antibiotics in this setting. In a short series, Cohen et al reported that the implantation rate of partial zona dissected embryos reached 28% (11 out of 39) in patients who received immunosuppressive treatment, whereas implantation rates were only 7% in patients who did not (2 out of 31).121 Nevertheless, there are roughly an equal number of reports describing a positive action of corticosteroids as there are those that do not in the literature.122,123 Another concern regarding zona manipulation procedures is a possible increased rate of monozygotic twins.124,125 This risk has been attributed to the use of small openings in the zona, which may be prone to bisecting the embryo during the hatching process. While some reports have suggested that the increased risk of monozygotic twinning seen after assisted hatching reflects just the overall increased monozygotic twinning seen with assisted reproduction techniques,126 other reports have suggested otherwise.127 Lastly, because assisted hatching increases the implantation rate of embryos that otherwise may be of

poor prognosis and unable to escape from the zona pellucida, it was feared that the technique could result in the implantation of poor-quality embryos destined to abort. Fortunately, an increase in spontaneous abortions has not been seen in contemporary large trials using the technique.68–70,73–75

Preimplantation genetic diagnosis (aneuploidy screening) Potential adverse effects of PGD for aneuploidy focus mostly on the likelihood of misdiagnosis. A drawback of blastomere analysis at the cleavage stage is that the result may not be representative of the whole embryo, due to the high frequency of chromosomal mosaicism.128,129 Thus, haploid or aneuploid mosaicism could lead to genetic misdiagnosis and transfer of chromosomally abnormal embryos. An analysis of two blastomeres could theoretically decrease the likelihood of misdiagnosis and improve the detection rate of mosaic embryos. There remains less clinical experience with two blastomere biopsies, however, at the present time. In addition, the actual biologic significance of early cleavage-stage embryonic mosaicism remains unclear. Some investigators have suggested that abnormal cells may be subsequently eliminated or diverted to the trophectoderm.130 Therefore, detecting and discarding mosaic embryos, which is the current preferred approach, might lead to the loss of potentially normal embryos. Although FISH is relatively efficient, FISH failure or misinterpretation can also occur. Harper et al131 reported that a clear FISH signal is obtained in 97% of fixed blastomere nuclei. Interpretation of FISH signals can also be complicated by overlapping probe signals. In an early trial, Munné et al30 reported that PGD using FISH for the common aneuploidies was associated with an error rate of 5.4%. Lastly, it is still under debate which chromosomes need to be tested for aneuploidy.34 The question of whether embryo biopsy might adversely affect implantation and live birth rates as well as its possible impact on birth defects or more subtle developmental problems in the children must be further investigated. Polar biopsy diagnosis is less invasive, although is hampered by the inability to detect paternally derived chromosomal abnormalities as well as abnormalities derived from post-fertilization events.

Blastocyst culture and transfer One of the intriguing questions regarding the use of blastocyst culture and transfer is whether some of the remaining arrested embryos would have otherwise implanted had they been transferred earlier. Blastocyst development still does not exceed 55%, even with the new sequential culture media. While it seems that a higher proportion of embryos which fail

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to develop to the blastocyst stage in culture are chromosomally abnormal, it is possible that some may still not progress because of suboptimal laboratory conditions. In addition, limited information is available on the use of blastocyst culture and transfer in either unselected patient groups or in patients who are not high responders to gonadotropin therapy where small numbers of blastocysts may preclude selection of the ‘best embryos’ and offer no significant advantage. In addition, there is also the risk that a particular patient may have no blastocysts available for transfer. These latter risks may also be potentially increased in patients with a history of repeated implantation failure, many of whom may also be marginal responders to gonadotropin therapy.

Coculture methods Although the use of well-characterized animal cells such as Vero cells in human IVF-ET has been documented to be safe, it presents certain medical and ethical challenges. One potential risk is the transmission of infectious agents, including possibly those that may not ordinarily infect humans. The risk of transmission of infectious agents with the use of accessory homologous cells in human IVF-ET also exists. Regulatory agencies have recommended screening and testing for human immunodeficiency virus (HIV), hepatitis B, and hepatitis C for all donors of reproductive cells and tissue. In addition, the risks of syphilis as well as transmissible spongiform encephalopathies, including Creutzfeldt–Jakob disease (CJD), must also be considered. Use of autologous cells averts these infectious risks. Since various cellular preparations and protocols exist, however, laboratories employing coculture technologies are obligated to assess whether their particular method is embryotrophic and enhances clinical outcomes. A coculture system may occasionally result in poor cellular proliferation and an increased fraction of non-viable cells. Clinical judgment is required in these instances to ensure that the best environment for human embryos is being provided.

Future directions and controversies Technologies for PGS for aneuploidies in women with diminished IVF-ET prognoses are evolving, although their clinical utility has not been fully defined. In addition, methods to culture embryos to the blastocyst stage using sequential culture media are relatively new. In particular, little is known regarding whether the latter technique may help women with repeated implantation failure. Results with assisted hatching and coculture methodologies are variable, although on the whole seem to result in improved clinical outcomes in women with repeated implantation failure.

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An additional significant amount of focus has been placed on potential immunological causes of repeated implantation failure. Much work has been performed attempting to associate antiphospholipid antibodies and in vitro fertilization failure. The proposed mechanism of such failure includes abnormal implantation, abnormal placentation, and early embryonic compromise. Intravenous immunoglobulin and antithrombogenic therapy, including aspirin and heparin, have been proposed as treatments.132,133 Although an association between antiphospholipid abnormalities and IVF failure has been shown in some retrospective studies,134,135 recent prospective studies have failed to reveal an association.136 Additional work has focused on the relationship between acquired and inherited thrombophilias and recurrent implantation failure, which similarly has been shown to have an association in some retrospective studies,137 but not others.138 Certain micromanipulation techniques have recently been described that attempt to ‘rescue’ poorquality embryos. These include microsurgical embryonic fragment removal and cytoplasmic transfer.139,140 Results of these techniques, however, are very preliminary and have not been studied systematically in a controlled fashion. In addition, techniques such as cytoplasmic transfer present certain theoretical risks (transfer of third-party mitochondrial DNA) that need to be carefully considered. The interpretation that cytoplasmic transfer is a form of gene therapy has currently resulted in the technique receiving a heightened level of regulatory scrutiny in the United States. Finally, there is a recent renewed interest in the techniques of embryo transfer and variables that may affect success.141 Significantly different outcomes with varying embryo transfer catheters, embryo transfer methods, and physician experience have all highlighted the importance of optimal embryo transfer techniques. The use of ultrasound-guided embryo transfer rather than blind catheter insertion is currently attracting increased interest. In fact, a recent report suggests that transvaginal ultrasound-guided embryo transfer improves outcomes in patients with repeated implantation failure.142 A recent controlled study at our institute, however, did not reveal an advantage of this technique by experienced operators.

Conclusion Although treatment of patients with a history of repeated implantation failure has been historically discouraging, new techniques and methodologies are being developed that provide this difficult group of patients with a better prognosis. If severe tubal factor and bilateral hydrosalpinges are visible by ultrasonography, patients seem to clearly benefit from prophylactic salpingectomy. Most studies have found that assisted hatching, whether globally or selectively used, provides clinical benefits. PGD techniques for the detection of aneuploidic embryos have considerable

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promise, although their precise clinical roles need to be further defined. Blastocyst culture and transfer may offer some theoretic advantages in patients with previous IVF failures, particularly in patients who are good responders to gonadotropin therapy. Lastly, coculture methods have also shown promise in improving both embryo quality and clinical outcomes in patients with previous IVF failures. In particular, we have found that the use of autologous cryopreserved endometrial cells offers significant advantages as a coculture method.

Appendix: autologous endometrial coculture technique Endometrial tissue is obtained during a luteal phase endometrial biopsy performed in a cycle before the patient’s IVF procedure with the use of a Pipelle Endometrial Suction Curette (Unimar, Wilton, CT, USA). The sample is transferred to the laboratory in a sterile container filled with normal saline solution. A small portion of each endometrial biopsy is also placed in 10% neutral buffered formalin solution for histological assessment. All tissue samples have revealed secretory morphologic changes ranging from cycle day 16 to cycle day 25. The remaining tissue is then minced into small pieces (1–2 mm2) and washed with Hank’s balanced salt solution (HBSS) (Gibco BRL, Grand Island, NY, USA) supplemented with 5000 µg per 100 ml of penicillin–streptomycin (Gibco BRL) to remove excess red blood cells and mucus. The tissue is then enzymatically digested using four steps into separate glandular epithelial and stromal cells. The method involves a slight modification to previously published differential sedimentation techniques developed in our laboratory.143 Initially, we incubate the tissue pieces for 5 minutes at 37°C in a shaking water bath in 10 ml of HBSS containing 0.2% collagenase type 2 (Sigma, St. Louis, MO, USA) and 5000 µg per 100 ml of penicillin–streptomycin. Cell clumps are then dispersed by brisk aspiration through a sterile transfer pipette. The digested tissue pieces are then allowed to settle by differential sedimentation at unit gravity for 5 minutes. After sedimentation, the supernatant (containing a mixture of single stromal cells and small intact glands) is transferred into a separate 15 ml polyethylene test tube and centrifuged at 400 × g for 5 minutes. The pellet is then resuspended in RPMI medium 1640 (Gibco BRL) supplemented with 10% patient’s serum (RPMI-10% serum) and 5000 µg per 100 ml of penicillin–streptomycin. The above steps are repeated four times, resulting in a combined 4 ml of single stromal cells mixed with small glands. This stroma and small gland sample undergoes another differential sedimentation at unit gravity for 45 minutes to separate most small glands from the stromal cells. The supernatant (containing the stroma-enriched fraction) is centrifuged at 400 × g for 5 minutes and the cell pellet resuspended in RPMI-10%

serum. A small aliquot of the final sample is diluted 1:1 with 0.4% trypan blue stain (Gibco BRL) with cell yield and viability determined quantitatively on a hemocytometer. Tissue culture flasks (25 cm2) are then seeded with approximately 5 × 105 cells. The pellet which remains after four digestions contains predominately intact glands mixed with undigested connective tissue and stromal clumps. The glandular epithelial cells are purified by further resuspending this pellet in 10 ml of HBSS. After approximately 30 seconds, the largest fragments (stromal clumps and undigested tissue) settle on the bottom of the 15 ml test tube while the top 8 ml contains glands and single stromal cells. The top 8 ml (which has a typical snowflake appearance) is then transferred to another 15 ml test tube and allowed to settle for 30 minutes at unit gravity. This sedimentation allows most of the glands to form a pellet at the bottom of the test tube while leaving the remaining single stromal cells in the supernatant that is removed and discarded. This glandular-enriched pellet is then resuspended in RPMI-10% serum and plated into one –three 25 cm2 tissue culture flasks, depending on a gross estimate of the yield. The seeded tissue flasks are maintained at 37°C in 5% CO2 air atmosphere, with the culture medium changed every 2–3 days. After about 1 week, the cells generally reach confluence. Although not representing entirely purified cellular populations, immunostaining studies using monoclonal antipancytokeratin and desmin antibodies have revealed >90% endometrial epithelial and stromal cells, respectively, in the respective cell cultures at time of confluence. After confluence is achieved, the cells are then released with trypsin-ethylenediamine tetraacetic acid (EDTA) (Gibco BRL). The cells are cryopreserved in a 15% glycerol solution at −70°C overnight and then transferred to liquid nitrogen storage. Approximately equal mixtures of glandular and stromal cells are thawed on the estimated day prior to administration of human chorionic gonadotropin (hCG) during the patient’s subsequent IVF-ET treatment cycle. Cell count and viability are determined, and approximately 3 × 105 cells (both glandular and stromal) are seeded into a four-well tissue culture plate containing 1 ml of Ham’s F-10 medium (Gibco BRL) supplemented with 15% patient’s serum. Approximately 75% confluence is achieved at the time embryos are placed into the coculture system. Following identification of fertilization, zygotes are removed from the insemination droplet and allocated to growth in conventional medium (human tubal fluid + 15% maternal serum) or autologous endometrial coculture incorporating Ham’s F-10 medium supplemented with 15% maternal serum. In studies where embryos were allocated to either conventional media or coculture, the morphologically best embryos were transferred back to the patient 72 hours after retrieval, irrespective of the culture system.

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After embryo transfer, the coculture cells are fixed in 4% paraformaldehyde. Immunostaining of these coculture cells using a monoclonal antipancytokeratin antibody (Sigma, St. Louis, MO) have typically shown 25–50% glandular epithelial cells per coculture well.

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32. Hardy K, Martin KL, Leese HJ, Winston RML, Handyside AH. Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage. Hum Reprod 1990; 5: 708–14. 33. Gianaroli L, Magli MC, Munné S, Fortini D, Ferraretti AP. Advantages of day 4 embryo transfer in patients undergoing preimplantation genetic diagnosis of aneuploidy. J Assist Reprod Genet 1999; 16: 170–5. 34. Bahce M, Cohen J, Munné S. Preimplantation genetic diagnosis of aneuploidy: were we looking at the wrong chromosomes? J Assist Reprod Genet 1999; 16: 176–81. 35. Wells D, Sherlock JK, Handyside AH, Delhanty JDA. Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genomic hybridization. Nucleic Acids Res 1999; 27: 1214–18. 36. Voullaire L, Slater H, Williamson R, Wilton L. Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization. Hum Genet 2000; 106: 210–17. 37. Wilton L, Williamson R, McBain J, et al. Birth of a healthy infant after preimplantation confirmation of euploidy by comparative genomic hybridization. N Engl J Med 2001; 345: 1537–41. 38. Voullaire L, Wilton L, McBain J, et al. Chromosome abnormalities identified by comparative genomic hybridization in embryos from women with repeated implantation failure. Mol Hum Reprod 2002; 8: 1035–41. 39. Munné S, Dailey T, Sultan KM, et al. The use of first polar bodies for preimplantation diagnosis of aneuploidy. Hum Reprod (Mol Hum Reprod vol. 1) 1995; 10: 1014–20. 40. Verlinsky Y, Cieslak J, Friedine M, et al. Pregnancies following pre-conception diagnosis of common aneuploidies by fluorescent in situ hybridization. Hum Reprod (Mol Hum Reprod vol. 1) 1995; 10: 1923–7. 41. Verlinsky Y, Cieslak J, Ivakhnenko V, et al. Preimplantation diagnosis of common aneuploidies by first- and second-polar body FISH analysis. J Assist Reprod Genet 1998; 15: 285–9. 42. Menezo Y, Hazout A, Dumont M, Herbaut N, Nicollet B. Co-culture of embryos on Vero cells and transfer of blastocysts in the human. Hum Reprod 1992; 7(Suppl 1): 101–6. 43. Gardner DK, Vella P, Lane M, et al. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 1998; 69: 84–8. 44. Gardner DK, Schoolcraft WB, Wagley L, et al. A prospective randomized trial of blastocyst culture and transfer in in vitro fertilization. Hum Reprod 1998; 13: 3434–40. 45. Jones GM, Trounson AO, Lolatgis N, Wood C. Factors affecting the success of human blastocyst development and pregnancy following in vitro fertilization and embryo transfer. Fertil Steril 1998; 70: 1022–9. 46. Meldrum DR. Blastocyst transfer – a natural evolution. Fertil Steril 1999; 72: 216–17.

47. Gras LR, Gianaroli L, Magli MC, et al. High rates of aneuploidy in human embryos that fail to grow to blastocysts in vitro (Abstr). Presented at the 11th World Congress on in vitro Fertilization and Human Reproductive Genetics, Sydney, Australia, 1999. 48. Harlow GM, Quinn P. Development of mouse preimplantation embryos in vivo and in vitro. Aust J Biol Sci 1982; 35: 187–93. 49. Jung T, Fischer B. Correlation between diameter and DNA or protein synthetic activity in rabbit blastocysts. Biol Reprod 1988; 39: 1111–16. 50. Carney EW, Foote RH. Effect of superovulation, embryo recovery, culture system and embryo transfer on development of rabbit embryos in vivo and in vitro. J Reprod Fertil 1990; 89: 543–51. 51. Quinn P, Kerin JF, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985; 44: 493–8. 52. Yeung WSB, Lau EYL, Chan STH, Ho P-C. Coculture with homologous oviductal cells improved the implantation of human embryos – a prospective randomized clinical trial. J Assist Reprod Genet 1996; 13: 762–7. 53. Nieto FS, Watkins WB, Lopata A, Baker HW, Edgar DH. The effects of coculture with autologous cryopreserved endometrial cells on human in vitro fertilization and early embryo morphology: a randomized study. J Assist Reprod Genet 1996; 13: 386–9. 54. Plachot M, Antoine JM, Alvarez S, et al. Granulosa cells improve human embryo development in vitro. Hum Reprod 1993; 8: 2133–40. 55. Quinn P, Margalit R. Beneficial effects of coculture with cumulus cells on blastocyst formation in a prospective trial with supernumerary human embryos. J Assist Reprod Genet 1996; 13: 9–14. 56. Sakkas D, Jaquenoud N, Leppens G, Campana A. Comparison of results after in vitro fertilized human embryos are cultured in routine medium and in coculture on Vero cells: a randomized study. Fertil Steril 1994; 61: 521–5. 57. Barmat LI, Worrilow KC, Payton BV. Growth factor expression by human oviduct and buffalo rat liver coculture cells. Fertil Steril 1997; 67: 775–9. 58. Fukui Y, McGowan LT, James RW, et al. Factors affecting the in vitro development of blastocysts of bovine oocytes matured and fertilized in vitro. J Reprod Fertil 1991; 92: 125–31. 59. Menezo Y, Guerin JF, Czyba JC. Improvement of human early embryo development in vitro by coculture on monolayers of Vero cells. Biol Reprod 1990; 42: 301–6. 60. Barmat LI, Liu H-C, Spandorfer SD, et al. Human preembryo development on autologous endometrial coculture versus conventional medium. Fertil Steril 1998; 70: 1109–13. 61. Barmat LI, Liu H-C, Spandorfer SD, et al. Autologous endometrial co-culture in patients with repeated failures of implantation after in vitro fertilization-embryo transfer. J Assist Reprod Genet 1999; 16: 121–7. 62. de Wit W, Gowrising CJ, Kuik DJ, et al. Only hydrosalpinges visible on ultrasound are associated with

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reduced implantation and pregnancy rates after in vitro fertilization. Hum Reprod 1998; 13: 1696–701. Dechaud H, Daures JP, Arnal F, et al. Does previous salpingectomy improve implantation and pregnancy rates in patients with severe tubal factor infertility who are undergoing in vitro fertilization? A pilot prospective randomized study. Fertil Steril 1998; 69: 1020–5. Strandell A, Lindhard A, Waldenstrom U, et al. Hydrosalpinx and IVF outcome: a prospective, randomized multicentre trial in Scandinavia on salpingectomy prior to IVF. Hum Reprod 1999; 14: 2762–9. Strandell A, Lindhard A, Waldenstrom U, Thornburn J. Hydrosalpinx and IVF outcome: cumulative results after salpingectomy in a randomized controlled trial. Hum Reprod 2001; 16: 2403–10. Johnson NP, Mak W, Sowter MC. Laparoscopic salpingectomy for women with hydrosalpinges enhances the success of IVF: a Cochrane review. Hum Reprod 2002; 17: 543–8. Dechaud H, Anahory T, Aligier N, et al. Salpingectomy for repeated embryo nonimplantation after in vitro fertilization in patients with severe tubal factor infertility. J Assist Reprod Genet 2000; 17: 200–6. Schoolcraft WB, Schlenker T, Gee M, et al. Assisted hatching in the treatment of poor prognosis in vitro fertilization candidates. Fertil Steril 1994; 62: 551– 4. Stein A, Rufas O, Amit S, et al. Assisted hatching by partial zona dissection of human pre-embryos in patients with recurrent implantation failure after in vitro fertilization. Fertil Steril 1995; 63: 838–41. Meldrum DR, Wisot A, Yee B, et al. Assisted hatching reduces the age-related decline in IVF outcome in women younger than age 43 without increasing miscarriage or monozygotic twinning. J Assist Reprod Genet 1998; 15: 418–21. Janssens R, Carle M, De Clerk E, et al. Can zona pellucida thickness predict the implantation rate (abstr)? Hum Reprod 1994; 9(Suppl 4): 78. Edirisinghe WR, Ahnonkitpanit V, Promviengchai S, et al. A study failing to determine significant benefits from assisted hatching: patients selected for advanced age, zonal thickness of embryos, and previous failed attempts. J Assist Reprod Genet 1999; 16: 294–301. Magli MC, Gianaroli L, Ferraretti AP, et al. Rescue of implantation potential in embryos with poor prognosis by assisted zona hatching. Hum Reprod 1998; 13: 1331–5. Chao K-H, Chen S-U, Chen H-F, et al. Assisted hatching increases the implantation and pregnancy rate of in vitro fertilization (IVF)-embryo transfer (ET), but not that of IVF-tubal ET in patients with repeated IVF failures. Fertil Steril 1997; 67: 904–8. Lanzendorf SE, Nehchiri F, Mayer JF, et al. A prospective, randomized, double-blind evaluation of assisted hatching in patients of advanced maternal age. Hum Reprod 1998; 13: 409–13. Seif MMW, Edi-Osagie ECO, Farquhar C, et al. Assisted hatching on assisted conception (IVF &

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ICSI). Cochrane Database Syst Rev 2006; (1): CD001894. Primi M-P, Senn A, Montag M, et al. A European multicentre prospective randomized study to assess the use of assisted hatching with a diode laser and the benefit of an immunosuppressive/ antibiotic treatment in different patient populations. Hum Reprod 2004; 19: 2325–33. Balaban B, Urman B, Alatas C, et al. A comparison of four different techniques of assisted hatching. Hum Reprod 2002; 17: 1239–43. Blake DA, Forsberg AS, Johansson BR, Wikland M. Laser zona pellucida thinning – an alternative approach to assisted hatching. Hum Reprod 2001; 16: 1959–64. Mantoudis E, Podsiadly BT, Gorgy A, et al. A comparison between quarter, partial and total laser assisted hatching in selected infertility patients. Hum Reprod 2001; 16: 2182–6. Petersen CG, Mauri AL, Baruffi RL, et al. Implantation failures: success of assisted hatching with quarter-laser zona thinning. Reprod Biomed Online 2004; 10: 224–9. Verlinsky Y, Cieslak J, Ivakhnenko V, et al. Prevention of age-related aneuploidies by polar body testing of oocytes. J Assist Reprod Genet 1999; 16: 165–9. Gianaroli L, Magli MC, Munné S, et al. Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy? Hum Reprod 1997; 12: 1762–7. Gianaroli L, Magli MC, Ferraretti AP, et al. Preimplantation genetic diagnosis increases the implantation rate in human in vitro fertilization by avoiding the transfer of chromosomally abnormal embryos. Fertil Steril 1997; 68: 1128–31. Munné S, Magli C, Cohen J, et al. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum Reprod 1999; 14: 2191–9. Gianaroli L, Magli MC, Ferraretti AP, Munné S. Preimplantation diagnosis for aneuploidies in patients undergoing in vitro fertilization with a poor prognosis: identification of the categories for which it should be proposed. Fertil Steril 1999; 72: 837–44. Staessen C, Platteau P, Van Assche E, et al. Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized controlled trial. Hum Reprod 2004; 19: 2849–58. Mastenbroek S, Twisk M, van Echten-Arends J, et al. In vitro fertilization with preimplantation genetic screening. N Engl J Med 2007; 357: 9–17. Pehlivan T, Rubio C, Rodrigo L, et al. Impact of preimplantation genetic diagnosis on IVF outcome in implantation failure patients. Reprod Biomed Online 2002; 6: 232–7. Munné S, Sandalinas M, Escudero T, et al. Improved implantation after preimplantation genetic diagnosis of aneuploidy. Reprod Biomed Online 2003; 7: 91–7. Taranissi M, El-Toukhy T, Verlinsky Y. Influence of maternal age on the outcome of PGD for aneuploidy screening in patients with recurrent

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Textbook of Assisted Reproductive Technologies implantation failure. Reprod Biomed Online 2005; 10: 628–32. Gardner DK, Schoolcraft WB. No longer neglected: the human blastocyst. Hum Reprod 1998; 13: 3289–92. Cruz JR, Dubey AK, Patel J, et al. Is blastocyst transfer useful as an alternative treatment for patients with multiple in vitro fertilization failures? Fertil Steril 1999; 72: 218–20. Guerif F, Bidault R, Gasnier O, et al. Efficacy of blastocyst transfer after implantation. Reprod Biomed Online 2004; 9: 630–6. Barrenetxea G, De Laurruzea AL, Ganzabal T, et al. Blastocyst culture after repeated failure of cleavage-stage embryo transfers: a comparison of day 5 and day 6 transfers. Fertil Steril 2005; 83: 49–53. Levitas E, Lunenfeld E, Har-Vardi I, et al. Blastocyst-stage embryo transfer in patients who failed to conceive in three or more day 2–3 embryo transfer cycles: a prospective, randomized study. Fertil Steril 2004; 81: 567–71. Schoolcraft WB, Gardner DK, Lane M, et al. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril 1999; 72: 604–9. Menezo YJ, Sakkas D, Janny L. Co-culture of the early human embryo: factors affecting human blastocyst formation in vitro. Microsc Res Tech 1995; 32: 50–6. Wiemer KE, Hoffman DI, Maxson WS, et al. Embryonic morphology and rate of implantation of human embryos following coculture on bovine oviductal epithelial cells. Hum Reprod 1994; 61: 105–10. Wiemer KE, Garrisi J, Steuerwald N, et al. Beneficial aspects of co-culture with assisted hatching when applied to multiple-failure in vitro fertilization patients. Hum Reprod 1996; 11: 2429–33. Hu Y, Maxson WS, Hoffman DI, et al. Coculture of human embryos with buffalo rat liver cells for women with decreased prognosis in in vitro fertilization. Am J Obstet Gynecol 1997; 177: 358–63. Bongso A, Ng SC, Sathananthan H, et al. Improved quality of human embryos when co-cultured with human ampullary cells. Hum Reprod 1989; 4: 706–13. Yeung WS, Lau EY, Chan ST, Ho PC. Coculture with homologous oviductal cells improved the implantation of human embryos – a prospective randomized control trial. J Assist Reprod Genet 1996; 13: 762–27. Plachot M, Antoine JM, Alvarez S, et al. Granulosa cells improve human embryo development in vitro. Hum Reprod 1993; 8: 2133–40. Quinn P, Margalit R. Beneficial effects of coculture with cumulus cells on blastocyst formation in a prospective trial with supernumerary human embryos. J Assist Reprod Genet 1996; 13: 9–14. Fabbri R, Porcu E, Marsella T, et al. Human embryo development and pregnancies in an homologous granulosa cell coculture system. J Assist Reprod Genet 2000; 17: 1–12. Carrell DT, Peterson CM, Jones KP, et al. A simplified coculture system using homologous, attached

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cumulus tissue results in improved human embryo morphology and pregnancy rates during in vitro fertilization. J Assist Reprod Genet 1999; 16: 344–9. Cross JC, Werb Z, Fisher SJ. Implantation and the placenta: key pieces of the development puzzle. Science 1994; 266: 1508–17. Liu H-C, He Z-H, Mele CA, et al. Human endometrial stromal cells improve embryo quality by enhancing the expression of insulin-like growth factors and their receptors in cocultured human preimplantation embryos. Fertil Steril 1999; 71: 361–7. Tazuke SI, Giudice L. Growth factors and cytokines in the endometrium, embryonic development, and maternal embryonic interactions. Semin Reprod Endocrinol 1996; 14: 231–45. Jayot S, Parneix I, Verdaguer S, et al. Coculture of embryos on homologous endometrial cells in patients with repeated failures of implantation. Fertil Steril 1995; 63: 109–14. Nieto NS, Watkins WB, Lopata A, et al. The effects of coculture with autologous cryopreserved endometrial cells on human in vitro fertilization and early embryo morphology: a randomized study. J Assist Reprod Genet 1996; 13: 386–9. Simon C, Mercader A, Garcia-Velaso J, et al. Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure. J Clin Endocrinol Metab 1999; 84: 2638–46. Spandorfer SD, Barmat LI, Navarro J, et al. Importance of the biopsy date in autologous endometrial cocultures for patients with multiple implantation failures. Fertil Steril 2002; 77: 1209–13. Lass A, Ellenbogen A, Croucher C, et al. The effect of salpingectomy on ovarian response to superovulation in an in vitro fertilization embryo transfer program. Fertil Steril 1998; 70: 1035–8. Verhulst G, Vandersteen N, Van Steirteghem AC, Devroey P. Bilateral salpingectomy does not compromise ovarian stimulation in an in vitro fertilization/embryo transfer program. Hum Reprod 1994; 9: 624–8. Van Voorhis BJ, Sparks AE, Syrop CH, Stovall DW. Ultrasound-guided aspiration of hydrosalpinges is associated with improved pregnancy and implantation rates after in vitro fertilization cycles. Hum Reprod 1998; 13: 736–9. Kontoravdis A, Makrakis E, Pantos K, et al. Proximal tubal occlusion and salpingectomy result in similar improvement in in vitro fertilization outcome in patients with hydrosalpinx. Fertil Steril 2006; 86: 1642–9. Raziel A, El RR, Wardimon J, Arad D, et al. Ultrasonographic diagnosis of post salpingectomy interstitial pregnancy. Case report and review of the literature. Acta Obstet Gynecol Scand 1989; 68: 85–6. Fisch B, Peled Y, Kaplan B, et al. Abdominal pregnancy following in vitro fertilization in a patient with previous bilateral salpingectomy. Obstet Gynecol 1996; 88: 642–3. Cohen J, Malter H, Elsner C, et al. Immunosuppression supports implantation of zona pellucida dissected human embryos. Fertil Steril 1990; 53: 662–5.

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Repeated implantation failure: the preferred therapeutic approach 122. Kemeter P, Feichtinger W. Prednisolone supplementation to Clomid and/or gonadotrophin stimulation for in vitro fertilization – a prospective randomized trial. Hum Reprod 1986; 1: 441–4. 123. Catt JW, Ryan JP, Saunders DM, O’Neill C. Shortterm corticosteroid treatment does not improve implantation for embryos derived from subzonal insertion of sperm. Fertil Steril 1991; 61: 565–6. 124. Slotnik RN, Ortega JE. Monoamniotic twinning and zona manipulation: a survey of U.S. IVF centers correlating zona manipulation procedures and high-risk twinning frequency. J Assist Reprod Genet 1996; 13: 381–5. 125. Herschlag A, Paine T, Cooper GW, et al. Monozygotic twinning associated with mechanical assisted hatching. Fertil Steril 1999; 71: 144–6. 126. Schacter M, Raziel A, Friedler S, et al. Monozygotic twinning after assisted reproductive techniques: a phenomenon independent of micromanipulation. Hum Reprod 2001; 16: 1264–9. 127. Schieve LA, Meikle SF, Peterson HB, et al. Does assisted hatching pose a risk for monozygotic twinning in pregnancies conceived through in vitro fertilization? Fertil Steril 2000; 74: 288–94. 128. Harper JC, Coonen E, Handyside AH, et al. Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos. Prenat Diagn 1995; 15: 41–9. 129. Munné S, Lee A. Rosenwaks Z, et al. Diagnosis of major chromosome aneuploidies in human preimplantation embryos. Hum Reprod 1993; 8: 2185–91. 130. Reubinoff BE, Shushan A. Preimplantation diagnosis in older patients. To biopsy or not to biopsy? Hum Reprod 1996; 11: 2071–8. 131. Harper JC, Coonen E, Ramaekers FC, et al. Identification of the sex of human preimplantation embryos in two hours using an improved spreading method and fluorescent in situ hybridization (FISH) using directly labeled probes. Hum Reprod 1994; 9: 721–4. 132. Coulam CB, Krysa LW, Bustillo M. Intravenous immunoglobulin for in vitro fertilization failure. Hum Reprod 1994; 9: 2265–9.

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133. Sher G, Feinman M, Zouves C, et al. High fecundity rates following in vitro fertilization and embryo transfer in antiphospholipid antibody seropositive women treated with heparin and aspirin. Hum Reprod 1994; 9: 2278–83. 134. Birkenfeld A, Mukaida T, et al. Incidence of autoimmune antibodies in failed embryo transfer cycles. Am J Reprod Immunol 1994; 31: 65–8. 135. Coulam CB, Kaider BD, Kaider AS, et al. Antiphospholipid antibodies associated with implantation failure after IVF/ET. J Assist Reprod Genet 1997; 14: 603–8. 136. Denis AL, Guido M, Adler RD, et al. Antiphospholipid antibodies and pregnancy rates and outcome in in vitro fertilization patients. Fertil Steril 1997; 67: 1084–90. 137. Qublan HS, Eid SS, Ababneh HA, et al. Acquired and inherited thrombophilia: implication in recurrent IVF and embryo transfer failure. Hum Reprod 2006; 21: 2694–8. 138. Martinelli I, Taioli E, Ragni G, et al. Embryo implantation after assisted reproductive procedures and maternal thrombophilia. Haematologica 2003: 88: 789–93. 139. Alikani M, Cohen J, Tomkin G, et al. Human embryo fragmentation in vitro and its implications for pregnancy and implantation. Fertil Steril 1999; 71: 836–42. 140. Cohen J, Scott R, Schimmel T, et al. Birth of infant after transfer of anucleate donor oocyte cytoplasm into recipient eggs. Lancet 1997; 350: 186–7. 141. Schoolcraft WB, Surrey ES, Gardner DK. Embryo transfer: techniques and variables affecting success. Fertil Steril 2001; 76: 863–70. 142. Anderson RE, Nugent NL, Gregg AT, et al. Transvaginal ultrasound-guided embryo transfer improves outcome in patients with previous failed in vitro fertilization cycles. Fertil Steril 2002; 77: 769–75. 143. Liu HC, Tseng L. Estradiol metabolism in isolated human endometrial epithelial gland and stromal cells. Endocrinology 1979; 104: 1674–81.

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46 Ultrasound in ART Marinko M Biljan In the course of preparing for this third edition of the Textbook of Assisted Reproductive Technologies Dr Marinko Biljan, the author of Chapter 46, sadly passed away. He will be greatly missed by colleagues and friends worldwide. We would like to dedicate the chapter that he wrote as a memorial to his work.

Introduction In the last 20 years the use of gray-scale and Doppler ultrasound scanning has emerged as an indispensable tool in the assessment of pelvic structures, monitoring of follicular growth, endometrial development, and pelvic circulation in both natural and stimulated cycles. Additionally, procedures performed under ultrasound guidance, such as oocyte collection1 and more recently embryo transfer,2 have to a large extent contributed to the simplification of in vitro fertilization (IVF) procedures and facilitated easier, more economical, and affordable treatment.3 This chapter reviews: various ultrasound techniques currently in use; the value of performing a baseline ultrasound scan prior to the commencement of infertility therapy; its use in the assessment of pelvic morphology; and prediction of a patient’s response to ovarian stimulation. The use of ultrasound in the assessment of follicular maturity by assessing follicular size and perfusion, and the benefits of ultrasound assessment of the endometrial structure and uterine and subendometrial perfusion in the evaluation of its receptivity are also examined. Finally, the value of ultrasoundcontrolled embryo transfer is discussed.

Ultrasound techniques Real-time gray-scale two-dimensional ultrasound Pelvic structures can be visualized by the use of either transabdominal or transvaginal ultrasound probes. The transabdominal approach, which was favored in the early 1980s in infertility treatment, has now been largely made obsolete. In order to obtain an adequate image of pelvic structures by a transabdominal scanner, the bowels have to be displaced from the pelvis by a full bladder. This approach suffers from a number of disadvantages. First, the patient may feel significant discomfort because of her full bladder, and this is often exacerbated during the ultrasound examination because of the pressure applied to the lower abdomen. Secondly, because sound waves are attenuated and artifacts are caused by the abdominal subcutaneous

tissues, a precise characterization of pelvic structures is sometimes not possible. Candidates for infertility treatment may be especially difficult to scan because they commonly have lower abdominal surgical scars (adipose tissue and collagen impair ultrasound penetration), as well as periadnexal adhesions which limit the bowel displacement. Currently, transabdominal ultrasound scanning is reserved almost exclusively for ultrasound-controlled embryo transfer procedures.2 Additionally, it is used in very rare cases where ovaries are located high in the pelvis and are, therefore, not accessible to transvaginal scanning. More recently, the transvaginal approach for ultrasound scanning has been used. Since the pelvic structures are in close proximity to the vaginal vault, higher-frequency ultrasound probes (e.g. 7 MHz) can be used, thus providing better resolution and greater precision in the measurement of the follicular diameter and endometrial thickness. The vaginal approach avoids the need for a full bladder and bypasses the problems of attenuation and the artifacts associated with obesity. In a direct comparison of the two techniques, it was found that the transvaginal approach allowed increased visualization and resolution when compared with the transabdominal approach.4,5 In the recent years, the cost and complexity of three-dimensional (3D) ultrasound machines has been steadily dropping, allowing more researchers to investigate its value in the assessment of pelvic organs.6–8 A 3D approach seems to allow a more precise assessment of the endometrial cavity.8 It is, however, still too early to judge whether the additional financial and time investments in 3D imaging are going to be justified by an improvement in the outcome of assisted conception procedures.

Doppler ultrasound In recent years the assessment of pelvic vascularization by means of Doppler ultrasound has become an integral part of an ultrasound examination. Doppler ultrasound uses the physical characteristic of sound which, when directed against a moving object, is

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Fig 46.1 Pulsed Doppler ultrasound. Flow velocity waveform (FVW) of uterine artery is demonstrated.

reflected backwards and in the process undergoes a change in frequency. This so-called Doppler frequency shift is proportional to the relative velocity of the target object. Blood flow towards the insonating ultrasound beam causes a positive frequency shift, whereas flow away from the probe causes a negative frequency shift. The two types of Doppler equipment used in infertility work are the pulsed Doppler and color Doppler. Both of them are used in combination with standard ultrasound imaging. Pulsed Doppler ultrasound enables the frequencies from individual blood vessels to be displayed in a graphic form (the flow velocity waveform [FVW], and thus allows blood flow velocities to be measured at specific locations along the path of the transmitted ultrasound beam (Fig 46.1). Color Doppler allows blood flow studies to be performed more rapidly and accurately by displaying flow in two dimensions, and thus permits small blood vessels that are virtually undetectable with conventional Doppler techniques to be easily seen, allowing the characteristic FVW from these vessels to be quickly studied. Color Doppler ultrasound machines embody a computer which interprets a positive frequency shift as a red color and a negative frequency shift as a blue color. These colors are superimposed onto the real-time gray-scale ultrasound image so that the blood vessels supplying the individual structures and organs can be identified (Fig 46.2). In general, blood flow studies have been confined to arteries, as Doppler studies of the venous circulation provide no information of flow impedance and it is assumed that changes in venous circulation are a poor predictor of functional changes in organ perfusion. Every major artery in the body has its own characteristic FVW. The maximum outline, i.e. the shape, of the FVW

indicates the degree of resistance to flow in the artery under investigation. The absence of Doppler frequency shifts during the diastolic phase of the cycle is typically found in large arteries, e.g. the external iliac artery, supplying high-resistance vascular beds. In contrast, high end-diastolic velocities are usually present in smaller arteries that supply organs such as the uterus and ovaries. The FVW is most easily quantified by calculating an index of resistance of impedance to blood flow. The indices most commonly used clinically are the A/B index, the resistance index (RI), and the pulsatility index (PI). As all three indices are based on the ratio between the peak systolic and enddiastolic velocities, they are all independent of the angle of insonation. This is important since FVW analysis can, therefore, be used for blood flow studies even in small arteries which are not clearly visualized and which have an undefined angle of insonation. Of the three indices, the use of PI is favored at our center because it has been demonstrated to correlate most closely with changes in blood flow volume9 and can be used even when there is an absence of diastolic velocities or reverse flow in the diastolic phase. The two vessels that have been studied in relation to infertility have, therefore, been the uterine and ovarian arteries. In two-dimensional (2D) color Doppler studies, information concerning the vascularization and blood flow in uterus, ovaries, and follicles is observed. It is obtained from a single artery lying in a 2D plane, which is subjectively chosen. To accurately measure the blood flow velocity, the angle of the Doppler beam should be known. In the pelvic organs the arteries are frequently thin and tortuous, which makes an accurate measurement very difficult. A recent technical development, 3D

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Fig 46.2 Color Doppler ultrasound enables clear visualization of vascular structures in the pelvis. Here, the right uterine artery is clearly visualized.

power Doppler ultrasonography, is less angle dependent. It enables the mapping and quantifying of the power Doppler signal within the entire volume of interest.10–13 A number of new parameters have been invented to describe 3D-measured vascularization. Vascularization index, expressed as a percentage, is defined as the ratio of color voxels to all voxels in a defined volume; it represents the amount of vessels in a tissue. Flow index represents the mean intensity of the color voxels and indicates an average intensity of flow. Vascular flow index is the mean color value in all the voxels as a measure of both flow and vascularization. Finally, the mean gray value in the gray voxel expresses the mean echogenicity or brightness of the examined segment.10 Doppler results can be significantly affected by a patient’s activity prior to examination, as well as the time of the day when the investigation is performed. In an interesting study, Dickey and colleagues14 examined the influence of patient position on Doppler readings. In their study, the patients were first examined in the recumbent and subsequently in the upright position. After standing for 9–14 minutes, the uterine artery blood flow decreased by an average of 34% and the RI increased by 70%. In addition, the number of cycles with absent end-diastolic flow increased. Zaidi and colleagues15 have shown that the time of the day when Doppler measurements are made could also have a major impact on results. These authors found that blood flow in the uterine arteries follows a circadian rhythm, with the PI values being lowest during the early morning hours and increasing towards the evening. To obtain consistent and comparable data it is, therefore, important to allow patients to remain in a recumbent position and to perform investigations on all patients at approxiately the same time of the day.

Ultrasound scan and Doppler assessment prior to treatment Ultrasonic assessment of pelvic structures and Doppler evaluation of pelvic vascular perfusion prior to assisted reproductive technologies (ART) is of paramount importance. It enables a clinician to assess uterine, tubal, and ovarian morphological appearance, and detect abnormalities which may contribute to a patient’s infertility. Additionally, the assessment of ovarian volume, appearance, and vascularization enable a better prediction of patient response to ovulation induction medication. To avoid a distortion of ovarian volume caused by a developing follicle, a baseline scan is usually performed between days 1 and 5 of the menstrual cycle. The investigation should be performed in a systematic manner. The condition of each pelvic structure should be clearly documented using a standardized form (Fig 46.3). At the end of the procedure the patency of the fallopian tubes can be verified with a positive echo contrast medium.16 We record baseline ultrasound pictures using an electronic image grabber. All images are stored in high resolution (1200 × 800 pixels) jpeg format on a server. On request, images are forwarded either to the patient or to the referring physician by e-mail. This system of storage allows for a rapid access to images, and decreases the bulk of the patient’s chart.

Uterus The evaluation of pelvic structures usually starts with the assessment of the uterus. It should be visualized in its longest longitudinal plane. At this level the

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BASE-LINE ULTRASOUND DATE: ____________________

ADDRESSOGRAPH LMP:

____________________

HYCOSI

LEFT OVARY

L. UT. ART

Left tube

:

Patent/Not patent

Right tube

:

Patent/Not patent

Uterus

:

______________

Medication :

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Pain

:

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Date

:

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RIGHT OVARY

R. UT. ART

L. OVAR

R.OVARY

PI Vmax Tmax

COMMENTS:

__________________________________________________________________

____________________________________________________________________________________________________________ ____________________________________________________________________________________________________________ DOCTOR:

Fig 46.3

__________________________________________________________________

Standard form used to document results of baseline scan at the Montreal Fertility Center.

maximal length from the cervix to the fundus, the length of the uterine cavity, and the maximal uterine thickness are measured. The angle between cervix and uterine body is assessed and documented. The length of the uterine cavity and the angle between the cervix and uterine body can be of considerable value when doing cervical catheterization prior to embryo transfer or intrauterine insemination. In our practice, we usually generate an electronic file of the ultrasonic image of the uterus in its longitudinal plane, with measurements, and store it in the patient’s electronic file for further reference (Fig 46.4). Special attention should be paid to any uterine abnormalities. Of these, a uterine septum is most frequently

seen, and most easily correctable by a simple surgical procedure.17 Additionally, a uterine septum has been related to high miscarriage18 and perhaps lower implantation rates,19 and therefore should be removed prior to the treatment. In order to detect a uterine septum, the uterus should be scanned in transverse sections, and signs of division of the uterine cavity should be observed. Unlike a bicornuate uterus, in a septate uterus at least 5 mm of uterine wall can be observed above the highest point of the uterine cavity. Moreover, in a septate uterus, none or only a minimal indentation of the fundal serous surface is present. By using this technique of ultrasound assessment, a uterine septum can be detected with 100% sensitivity and 80% specificity.20 The

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Fig 46.4 Transvaginal scan of the uterus (longitudinal view). Measurement of the angle between the cervix and uterine body and the assessment of the uterine cavity length are demonstrated.

Fig 46.5 A uterine fibroid distorts the endometrial lining.

accuracy of detection of a septate uterus can be improved with the use of saline instillation.21 This is a relatively simple technique whereby saline is used as a nonechogenic contrast. Saline is injected slowly transcervically and the uterine cavity is observed simultaneously. As the uterine cavity is stretched, smaller uterine malformations and endometrial polyps are also more clearly visible. The disadvantage of this procedure is the moderate discomfort to the patient caused by distention of the uterine cavity. To decrease patient discomfort we recommend premedication of the patients undergoing saline instillation with 500 mg naproxen (Naprosyn, Roche, Canada) suppository 2 hours prior to the procedure. Some researchers suggested a higher detection rate of uterine septa and better distinction between bicornuate

uterus and uterine septum could be obtained by 3D ultrasound.22,23 However, with the higher cost of 3D ultrasound machines, and only a marginal improvement in detection rate, at present 3D ultrasound does not appear to have a major diagnostic impact on clinical practice. Additional attention should be paid to the presence of uterine fibroids, especially submucosal and perhaps intramural, which probably decrease implantation rate.24 Submucous fibroids typically distort the uterine cavity by interrupting the continuity of the smooth endometrial surface (Fig 46.5). Prior to any infertility treatment, we advocate hysteroscopic removal of all fibroids which distort the uterine cavity, regardless of their size.

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Fig 46.6 The ovary is shown in two perpendicular planes. Assessment of the largest diameters in both planes allows a more accurate assessment of ovarian volume.

Fig 46.7 Ultrasound image of an enlarged polycystic ovary with multiple small cysts scattered around the periphery, and increased highly echogenic stroma. The volume of this ovary is 13.75 cm3.

Ovaries Examination of ovaries follows the assessment of the uterus. Ovaries are usually found by directing the ultrasound probe 2–3 cm lateral to the cervix. When examining ovaries, attention should be paid to ovarian size, structure, and relation to the uterus. Ovarian volume should always be measured in two perpendicular planes. This is obtained by finding the largest cross section of the ovary, freezing the image, and in a separate image rotating the probe by 90°. Most of the software packages supplied with ultrasound machines allow the calculation of ovarian volume from these two images (Fig 46.6). Thereafter, ovarian structure is carefully examined, searching for ultrasonic

features of polycystic ovaries, and the presence of ovarian cysts.

Polycystic ovaries The precise diagnosis of polycystic ovaries depends on the finding of multiple follicular cysts and increased stroma in ovaries that are usually, but not always, enlarged25 (Fig 46.7). The advent of ultrasound scanning has shown that polycystic ovaries are much more common than was previously believed. Adams and colleagues26 reported that polycystic ovaries were found in 26% of women with amenorrhea, 87% with oligomenorrhea, and 92% with idiopathic hirsutism.

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Fig 46.8 A functional ovarian cyst. Note anechogenic contents and clearly defined edges of the cyst.

Fig 46.9 Two endometriomas of the ovary showing multiple echoes within the cysts.

With regard to assisted conception, patients with polycystic ovaries are prone to develop ovarian hyperstimulation syndrome (OHSS). In a study of 15 patients who developed moderate to severe hyperstimulation, out of 1302 patients undergoing ovarian stimulation for IVF, polycystic ovaries were identified in eight of 15 patients.27 Moreover, a diagnosis of polycystic ovaries is perhaps of prognostic value with regard to the outcome of assisted conception. Engmann and colleagues28 compared the outcome of 46 patients (97 cycles) with polycystic ovaries but no signs of polycystic ovary syndrome treated by IVF, with that of 145 women (332 cycles) with normal ovarian morphology on ultrasound examination. Significantly more oocytes were recovered from the patients with polycystic ovaries than from the control group, but the fertilization, cleavage, and miscarriage rates were similar in both groups. Interestingly, these

authors found that after 3 cycles of treatment patients with polycystic ovaries had significantly higher chances of achieving a pregnancy (odds ratio [OR] = 1.69, 95% confidence interval [CI] 0.99–2.90) and achieving a live birth (OR=1.82, 95% CI 1.05–3.16). Our current practice in patients with polycystic ovaries undergoing ovarian stimulation for assisted conception includes a reduced dose of gonadotropins and an increase in the frequency of ultrasonic monitoring to minimize the risk of development of OHSS.29

Ovarian cysts Ovarian cysts should be assessed for their volume and texture. Most commonly found are functional ovarian cysts, characterized by sharp edges and anechogenic contents (Fig 46.8) and endometriotic cysts, which contain more echogenic material (Fig 46.9).

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Fig 46.10 Stromal blood flow in the polycystic ovary on day 2 of the menstrual cycle is 12 cm/s2 in this image. Increased stromal blood flow is a predictor of a good response to ovulation induction.

Functional ovarian cysts Functional ovarian cysts are defined as any intraovarian sonolucent structure measuring >15 mm in the mean diameter causing elevation of serum estradiol (E2) above 150 pm/l.30 In patients undergoing ovulation induction, who have a functional ovarian cyst, in order to avoid the negative effect the estrogen-producing ovarian cyst has on the pituitary ovarian axis, we normally advise that treatment be delayed until a subsequent cycle when the cyst has disappeared. In the context of assisted reproduction, however, the effect of ovarian cysts on cycle outcome is controversial. While some studies suggest very poor outcome of cycles where functional cysts were detected, including high cancellation and low pregnancy rates,31–34 others have failed to report a difference in any outcome measures when comparing patients who did, with those who did not develop functional ovarian cysts.35–39 Biljan et al have prospectively followed 51 patients during their IVF treatment.30 Thirty of these patients developed functional ovarian cyst(s). Patients who developed a cyst required a significantly longer period of time to achieve pituitary suppression (21 vs 7 days), had significantly lower follicle-stimulating hormone (FSH) levels at the time of initiation of gonadotropin therapy, required more ampules of gonadotropin to achieve ovarian stimulation (45 vs 41 ampules), developed fewer follicles (13 vs 17.5), and had lower cumulative embryo scores (28 vs 36). However, there were no significant differences in the implantation (23.5% vs 17.2%) and pregnancy rates (37.2% vs 29.2%) between patients who developed cysts when compared with those who did not. Based on this evidence we do not cancel treatment cycles where ovarian cysts are detected. However, in view of

a more profound ovarian suppression, we consider increasing the dose of gonadotropins in the stimulation protocol.

Endometriotic cysts If a unilateral moderate-sized endometriotic cyst is identified on the baseline ultrasound scan in a patient with both her ovaries intact, who is undergoing IVF therapy, the cyst should generally be ignored. At the time of oocyte recovery, the cyst should be left intact if possible, because the drainage of the cyst at that time considerably increases the risk of infection. If an endometriotic cyst is inadvertently drained, or drainage is necessary in order to allow access to the ovarian follicles, then antibiotic coverage should be used.

Assessment of intraovarian blood flow Following the assessment of ovarian volume and structure, we proceed with the determination of ovarian stromal circulation. To do so, arteries within ovarian stroma are visualized with the color Doppler technique, avoiding arteries close to the ovarian surface (Fig 46.10). The FVWs are obtained by placing the Doppler gate over the colored areas and activating the pulsed Doppler function. When the highest signal is found, it is recorded in three consecutive cycles and analyzed.

Fallopian tubes Morphology of fallopian tubes Normal fallopian tubes are very fine structures which do not contain a significant quantity of fluid and are,

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Fig 46.11 This sausage-shaped structure behind the right ovary is a hydrosalpinx.

therefore, not conducive to ultrasound examination. However, when pathologically filled with fluid (hydrosalpinx), fallopian tubes are easily detectable at ultrasound scan as sausage-like structures filled with fluid which is usually moderately echogenic (Fig 46.11). In the last several years a number of researchers have reported a reduction in pregnancy rates in patients with hydrosalpinx undergoing IVF treatment.40–44 Murray and colleagues45 reported significantly lower implantation rates in patients who had hydrosalpinges diagnosed either on hysterosalpingogram or laparosco- pically prior to IVF than other patients with tubal damage (2.8% vs 15.7%). Moreover, these authors reported a significant improvement in implantation rates (16.1%) if hydrosalpinges were removed prior to IVF. Several theories have been proposed to explain the pathophysiology involved in the negative effect of hydrosalpinges on IVF outcome. Free fluid in the endometrial cavity produced by drainage from the hydrosalpinx could interfere with embryo contact with the endometrium. Meyer and colleagues46 demonstrated that αvβ3 endometrial integrins were expressed at significantly lower levels in women with hydrosalpinges, and the level of αvβ3 endometrial integrins appeared to return to normal after surgical treatment of hydrosalpinges. It has also been indicated that hydrosalpingeal fluid has a direct toxic effect on murine embryos, even at a concentration of 10%.47 Whatever the mechanism, in spite of the lack of a sufficiently large prospective randomized trial in this area, there seems to be compelling evidence that the presence of hydrosalpinges leads to decreased pregnancy rates in IVF. It, therefore, seems reasonable to recommend to the patients to have either a distal salpingostomy to allow peritoneal drainage of intratubal fluid, or even better a bilateral salpingectomy prior to IVF treatment.

Patency of fallopian tubes Recently, the introduction of echo-positive media has enabled a more accurate assessment of tubal patency under ultrasound control. In a multicenter European study of 600 infertility patients, Campbell and colleagues reported a specificity of 87% and a sensitivity of 83.7% in the detection of tubal abnormality when using an echo-positive mixture of galactose microparticles in galactose solution.48 This compared favorably with results of hysterosalpingography. The assessment of tubal patency and uterine cavity abnormalities is, therefore, possible under ultrasound control and could soon replace hysterosalpingography as the method of choice for screening tubal disease.

The value of baseline ultrasound scan in predicting subsequent response to ovulation induction Ovarian volume and structure In the last several years a number of groups have attempted to correlate certain morphological and Doppler features on baseline ultrasound with subsequent response to ovulation stimulation. In a retrospective study, Syrop and colleagues49 examined ultrasonic images made on 188 patients undergoing assisted conception. Estimation of ovarian volume was based on two ultrasonic images taken in the sagittal and coronal planes. Patients who had part of an ovary removed were excluded from the study. Their results demonstrated a correlation between total ovarian volume and: peak E2 concentrations; number of eggs retrieved; number of embryos obtained; and clinical pregnancy rate. The major criticism with this study is the authors’ failure to exclude patients with polycystic ovaries (PCO). It is reasonable to believe

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that the majority of patients with large ovaries were in fact patients with PCO, who normally exhibit an exaggerated response to ovarian stimulation.29 A group from Finland50 studied the predictive value of ovarian volume and the number of small (antral) follicles (2–5 mm in diameter) seen at the baseline scan on ovarian response. They divided the 166 patients studied into three groups according to: the number of antral follicles; inactive (<5 follicles); normal (5–15); and a PCOlike category with more than 15 follicles. These authors confirmed previous observations showing that patients with smaller ovaries and fewer antral follicles on the initial ultrasound scan developed fewer follicles than patients with PCO. They also concluded that the number of antral follicles at the beginning of the cycle may be more representative of the actual functional ovarian reserve than the patient’s age. Danninger and colleagues51 found that, even after excluding patients with PCO, there was a relationship between the volume of ovaries and the likelihood of developing OHSS. They reported that women with larger ovaries developed more follicles, had more embryos for transfer, had a higher clinical pregnancy rate, and had a higher risk of developing OHSS. In a number of recent studies, using 3D ultrasound, it was confirmed that ovarian volume, and especially the number of antral follicles, had the best correlation to the success of IVF treatment.13,52–54 Jarvela and colleagues52 showed a 50% cancellation rate in the group of patients with fewer than 5 antral follicles on the initial ultrasound scan. Similarly, 3D ultrasound studies have confirmed previous 2D data on the value of the ovarian volume in the assessment of ovarian reserve.13,52 Smaller volume seems to be related to a decreased number of functional follicles in the ovaries and their capacity to respond to ovarian stimulation.

to establish whether there is any value of intraovarian circulation in predicting a patient’s response to gonadotropins.

Summary Baseline ultrasound and Doppler pelvic scanning are an important part of infertility investigations. The assessment of the uterus allows measurement of uterine size and length of uterine cavity, and detection of potentially correctable uterine anomalies and fibroids. The assessment of ovarian volume, the number of antral follicles, detection of PCO and ovarian cysts, and, perhaps, measurement of intraovarian flow allows, to a certain degree, prediction of a patient’s response to ovarian stimulation. The investigation of fallopian tubes allows detection of hydrosalpinges, which probably should be removed prior to treatment. Finally, the use of echogenic media allows the patency of fallopian tubes to be assessed. Currently, prior to deciding to proceed with an IVF cycle, we determine several crucial parameters. Patients older than 41 years old, or those with FSH levels above 13 IU/l, providing that the uterine cavity is normal, are offered egg donation. Patients over the age of 38 years old, or those with FSH levels above 10 IU/l, low number of antral follicles, or reduced ovarian volume are warned of a possible low response, and higher chances of cycle cancellation. If they decide to proceed, these patients are stimulated more aggressively.

Monitoring follicular development in ART Technique of follicular measurement, methods of recording, and frequency of monitoring

Ovarian stromal perfusion Zaidi and colleagues55 performed a study on 105 patients, 26 of whom had ultrasonic features of polycystic ovary syndrome (PCOS), undergoing IVF treatment. In that study, both ovarian morphology and blood flow were assessed during the early follicular phase of the IVF cycle. Poor ovarian response was defined as the development of 6 or fewer follicles, which was representative of 10% of patients exhibiting the worst response. This study showed a positive independent relationship between the ovarian stromal peak systolic blood flow velocity (PSV) at the time of baseline ultrasound scan, and the subsequent follicular response. Kupesic and Kurjak.13 using 3D ultrasound, reported that flow index measured at the time of pituitary suppression correlated with the number of oocytes collected. In a recent study, however, Jarvela and colleagues52 were unable to confirm the correlation between any intraovarian vascular parameters and a subsequent response to gonadotropins in IVF treatment. Further studies are required

The ultrasound technique provides more accurate information of follicle number and size than can be obtained by serum estrogen determinations alone.56 Under optimal conditions a follicle in the ovary can be visualized from a diameter of 2–3 mm. The follicles appear as echo-free structures amidst the more echogenic ovarian tissue. We measure the internal diameter of the follicle in two planes and the average diameter is then calculated (Fig 46.12). Follicles usually grow by 2–3 mm per day. We have completely eliminated the use of paper charts in recording follicular progress. Instead we use an electronic graph which allows entering follicular sizes, endometrial thickness, serum estradiol levels, details regarding egg collection and transfer, dose of medications used, and cycle outcome directly into the patient database. All computers are connected to the network, allowing access from any terminal in the center. Following the ultrasound scan, the patient has a consultation with her treating physician during which, with help of the computer, all relevant characteristics of the cycle,

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Fig 46.12 Multiple follicles in a stimulated ovary on the day of hCG administration. Each follicle is measured in two perpendicular planes. Then, the average of the four largest diameters is calculated.

including the number of follicles, dynamics of follicular growth, endometrial thickness, and changes in the type of ovulation regimen are visualized and discussed. Thereafter, the patient is seen by a nurse specialist, who can also access patient data from her terminal. At the end of the cycle a summary sheet containing all relevant information is printed and inserted into the patient chart (Fig 46.13). The advantages of this system include easy access to the patient chart and more accurate keeping of the center’s statistics. The frequency of ultrasound scans does depend on the type of ovarian stimulation regimen used and whether the patient has polycystic ovaries. In a typical long stimulation protocol, we perform the first ultrasound scan on day 6 of stimulation with gonadotropins. The frequency of scanning thereafter depends on the patient’s response. Typically, patients with a rapid response would require an ultrasound scan 1–2 days later, while patients with a somewhat slower response are seen on day 9 and as frequently as necessary thereafter. The scanning policy differs in patients who have signs of PCO on their baseline scan. These patients are scanned for the first time on day 4 of ovulation induction and thereafter depending on their initial response.

The value of follicular characteristics assessment in predicting oocyte quality Follicular size Since the beginning of IVF treatment the measurement of follicular size and the volume of follicular fluid have been recognized as possible predictors of oocyte quality.56 In a retrospective study on more than

6000 follicles from 1109 patients undergoing IVF, Wittmaack and colleagues57 investigated the effect of follicular size on collection, fertilization, and pregnancy rates. They found a relatively constant oocyte recovery in follicles measuring between 12.5 mm and 24 mm in diameter. Oocyte recovery rates were significantly decreased only in very small and large follicles. This study also showed a continuous increase in fertilization and cleavage rates with increasing follicular size. Only when follicular size exceeded 24 mm was there a small decrease in fertilization rate noted. On the basis of these data, it was concluded that a larger number of mature eggs would be retrieved if smaller follicles in a cohort were allowed to reach at least 13 mm in diameter. They, therefore, challenged the policy adopted by many centers where human chorionic gonadotropin (hCG) is given when three follicles reach 18 mm, suggesting that oocyte collection should be delayed until the majority of follicles reach maturity. To determine if there is an optimum time for the administration of hCG when the long protocol of gonadotropin-releasing hormone (GnRH) agonist is used in an IVF program, Tan and colleagues performed a randomized controlled trial involving 247 patients.58 In this study the first group of patients had hCG administered on the day when the largest follicle reached 18 mm in diameter, two other follicles were larger than 14 mm, and the levels of serum E2 were appropriate for the number of follicles. Patients in the other two groups had hCG administered 1 and 2 days later. The results of this study showed that patients who had hCG injection delayed for up to 2 days had higher serum E2 levels and a larger number of follicles >14 mm in diameter on the day of hCG administration.

Fig 46.13

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GON

Computerized printout of an IVF cycle summary as used at the Montreal Fertility Center.

IVF NO. 1 LMP Bus. start Bus. reduced hCG date hCG time hCG dose

: : : : : :

29/08/03 29/08/03 02/10/03 13/10/03 01:00 5000

OOCYTE COLLECTION Surgeon : BILJAN Assistant : Diane Embryologist : Hanane Date : 14/10/03 Time start : 13:59 Time finish : 14:28 Duration : 00:29 Hours since HCG: 36:59 Right Left No. follicles : 10 13 Punctured foll. : 8 12 Eggs retrieved : 8 12 No. of stabs : 2 2 Needle type : Dou Dou Discomfort : 2 2 Midazolam (mg) : 2.0 Fentonyl (µg) : 100

EST

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SEMEN ANALYSIS Liquification : Comple. Volume (ml) : 3.0 Color : Normal Agglutination : Neg. Debris : Neg. Density (mil/ml) : 73.0 Motility (%) : 60 Rapid (%) : 33 Cell count x10 ml : 0 Med.batch : 14/10/03 Embryologist : Hanane AFTER PREPARATION Density (mil/ml) : 32.2 Motility (%) : 80 Rapid (%) : 54.0 Ass. time : 14.30 Volume (ml) : 1.0

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26 25 24 23 22 21 20 19 18 17 16 ☺ 15 ☺ 14 ☺☺☺ ☺ 13 ☺ ☺☺☺ 12 11 ☺☺ 10 ☺ 9 8

1ST TRANSFER Surgeon : BILJAN Assistant : Diane Embryologist : Hanane Date : 17/10/03 Time start : 14:20 Time finish : 14:35 Duration : 00:15 Vag. disch. : Normal Cervix : Healthy Volcellum : No Mix.IVF/ICSI : 20/0 Fertilized : 13/0 Ass. hatching : No Cleaved : 11 Transferred : 2 Stored : 8 Donated : 0 Research : 0 Discarded : 0 Discomfort : Neg. Catheter : Wall. On catheter : Mucus Transfer : Easy Lut. support : IM prog.

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– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 13 Tu 14/10

2ND TRANSFER Surgeon : BILJAN Assistant : Diane Embryologist : Hanane Date : 19/10/03 Time start : 10:20 Time finish : 10:25 Duration : 00:05 Vag. disch. : Normal Cervix : Healthy Volcellum : No Transferred : 1 Discomfort : Neg. Catheter : Wall. On catheter : Mucus Transfer : Easy

13.6

11 Su 12/10

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– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 15 Th 16/10

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 16 Fr 17/10

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 17 Sa 18/10

09/10/66 07/03/58

OUTCOME Pregnancy test: Positive 1st ultrasound: Singleton FH+ 2nd ultrasound: Singleton FH+

14 We 15/10

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

31/10/03 13/11/03 27/11/03

18 Su 19/10

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Fig 46.14 Color Doppler ultrasound scan showing perifollicular blood flow.

However, the number of oocytes collected and embryos cleaved were comparable among the three groups studied, and there were no significant differences in the pregnancy rates observed. It would appear that optimal oocyte recovery and fertilization rates can be obtained from follicles between 14 and 24 mm in diameter. Oocyte recovery rates start to decrease after the follicles exceed 24 mm in diameter.

Perifollicular perfusion Although the diameter of the follicle is a relatively good predictor of oocyte maturity, it is not a perfect indicator of oocyte quality. Despite optimal follicular size and no impairment of semen quality, more than 20% of oocytes fail to fertilize.59 It would, therefore, be beneficial to have an additional test of oocyte quality available prior to hCG administration. Tan et al have reported a rapid rise in blood flow velocity in the perifollicular and ovarian stromal blood vessels at the time of the luteinizing hormone (LH) surge.60 These changes may be a result of neoangiogenesis occurring during late follicular development. A marked increase in the PSV around the follicle, in the presence of a relatively constant PI, could be a sign of follicle maturity and herald impending ovulation. Nargund and colleagues61,62 studied the relationship between follicular blood flow and the production of morphologically normal embryos. These authors investigated individual follicles, oocytes, and preimplantation embryos, rather than pooling data. Interestingly, by using this approach, a very strong relationship between peak systolic velocities, collection rates, embryo development, and implantation rates was found. From this study it appeared that harvesting oocytes from follicles with a peak systolic velocity of ≥10 cm/s (Fig 46.14) is significantly more likely to result in obtaining grade I embryos, which in turn are more

likely to implant. These findings were supported by another study where follicles were divided arbitrarily according to the percentage of vascularized surface.63,64 Oocytes obtained from highly vascularized follicles were of a higher quality, and were more likely to fertilize and result in pregnancy. In a similarly designed study, Huey and colleagues65 confirmed that perivascular flow reflects developing competence of the corresponding oocyte. Coulam and colleagues66 investigated the role of both quantitative and qualitative blood flow characteristics of perifollicular flow in a group of 107 patients considered to be at risk of cycle failure. In this group, only patients who had peak systolic perifollicular flow >10 cm/s and more than 75% of follicle vascularized achieved a pregnancy. The authors concluded that in patients where no adequate vascularization is observed, cycle cancellation should be considered. From the available data, it appears that assessment of perifollicular vascular perfusion could lead to a better selection of oocytes and ultimately a higher pregnancy rate. However, it is difficult to envision its practical value in patients who have abundant follicles. In this particular group of patients, it would be very time consuming to measure the vascularity of each individual follicle. Moreover, due to the large number of follicles, it may be difficult to determine the exact vascularization of each single follicle. The largest potential for use of perifollicular blood flow is in patients undergoing natural cycle IVF treatment. In a recent study, Vlaisavljevic and colleagues,67 using 3D ultrasound, observed perifollicular blood flow in 52 patients undergoing unstimulated cycles of IVF/ICSI (intracytoplasmic sperm injection). They found that the patients who subsequently had successful implantation had an increased percentage of perifollicular volume showing a blood flow, when compared with pati-ents who did not achieve implantation. The authors concluded that follicles

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Fig 46.15 The endometrium is clearly visualized in this picture with a thickness of 11.0 mm. It has a triple line appearance, which is believed to represent optimal uterine receptivity.

containing oocytes capable of producing a pregnancy have a distinctive and more uniform perifollicular vascular network. If these data are corroborated, in patients undergoing unstimulated IVF, where perifollicular flow is not satisfactory, perhaps, the egg collection should be canceled.

In the last several years there has been a considerable interest in the assessment of endometrial receptivity by using ultrasound scanning technology and Doppler assessment of uterine and endometrial vascularization.

Summary

Two anatomical parameters have been suggested for the evaluation of the endometrium by ultrasound: endometrial thickness and endometrial pattern.

Ultrasound monitoring of follicular growth is the most important tool in the assessment of progress in ovarian stimulation. With follicles which are less than 24 mm in size, with increasing size the likelihood of obtaining mature oocyte increases. However, there is no difference in the oocyte quality obtained from follicles between 18 and 22 mm in diameter. This allows more convenient and predictable planning of oocyte collection. Quantitative and qualitative assessment of perifollicular flow allow for a more accurate assessment of follicular competence. Follicles that have >75% of their surface perfused, or where PSV is >10 cm/s, appear to contain an oocyte of satisfactory quality.

Endometrium The introduction of new culture media and the capability of culturing embryos up to the blastocyst stage has resulted in a major improvement in the selection of embryos capable of implantation.68 Unfortunately, the understanding of endometrial characteristics compatible with successful pregnancy has not progressed with the same pace. The traditional method of assessing endometrial receptivity involves histological dating of the endometrium.69 The value of this invasive test is, however, rather restricted in assisted conception cycles due to its relatively low predictive value and the concern of performing a biopsy during the treatment cycle itself because of the associated bleeding.

Ultrasound

Endometrial thickness Endometrial thickness is defined as the maximal distance between the echogenic interfaces of the myometrium and the endometrium measured in the plane through the central longitudinal axis of the uterus (Fig 46.15). It is an easily measurable ultrasonic parameter, and it represents a bioassay of estrogenic activity. Using transvaginal scanning, Gonen and colleagues70 suggested that endometrial thickness, on the day before oocyte recovery, was significantly greater in pregnant than in nonpregnant women, and postulated that it may predict the likelihood of implantation. However Glissant and co-workers,71 Fleischer and colleagues,72 and Welker and co-workers73 found that the measurement of endometrial thickness had no predictive value for pregnancy. Moreover, Li and colleagues74 reported no correlation between endometrial thickness, measured by abdominal ultrasound, and histological dating of the endometrium. In their study of endometrial thickness, Dickey and colleagues75 found an increased rate of early miscarriage in a group of patients with very thin (<6 mm) or thick endometrium (>13 mm). In a retrospective analysis, Weissman and colleagues76 also reported decreased implantation and pregnancy rates and perhaps increased miscarriage rates in patients whose endometrium was >14 mm (Fig 46.16) at the time

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Fig 46.16 A very thick endometrium (15.5 mm) is, perhaps, an indicator of diminished receptivity.

of hCG administration. In a retrospective study by Dietterich and colleagues77 outcomes of 570 consecutive IVF cycles were analyzed according to endometrial thickness on the day of hCG injection. They observed a questionable decrease in pregnancy rate only when endometrial thickness was more than 18 mm. Yakin and colleagues78 found no correlation between increased endometrial thickness and pregnancy rates in IVF treatment, whereas Krampl and Feichtinger79 found no correlation between endometrial thickness and the likelihood of miscarriage. Imoedemhe and colleagues80 compared the endometrial thickness in three groups of patients who were prescribed three different ovulation induction regimens. They found that the endometrial thickness in all three groups of patients were similar and comparable to that observed in a group of spontaneously ovulating, fertile control patients, despite significantly higher serum estradiol concentrations in all the hyperstimulated cycles. Their findings suggest that there is a maximum endometrial response, inducible by estrogen, which is virtually achieved in the normal menstrual cycle. Friedler and colleagues81 reviewed 2665 assisted conception cycles from 25 reports. Eight reports found that the difference in the mean endometrial thickness of conception and nonconception cycles was statistically significant, while 17 reports found no significant difference. They concluded that results from various trials are conflicting and that insufficient data exist to describe a linear correlation between endometrial thickness and the probability of conception. The main advantage of measuring endometrial thickness lies in its high negative predictive value in cases where there is minimal endometrial thickness. Gonen and colleagues82 reported an absence

of pregnancies in donor insemination cycles where the endometrium did not reach at least 6 mm in diameter. Similarly, in a group of oocyte recipients, no pregnancies were reported in women who had an endometrial thickness of less than 5 mm in diameter, whereas several pregnancies occurred in patients with an endometrium thinner than 7.5 mm.83 While the chances of pregnancy are decreased if the endometrium measures <5 mm, it does not always preclude a pregnancy. Sundstrom described a successful pregnancy in a patient who’s endometrium measured only 4 mm.84 At our center, we observed two pregnancies in patients where the endometrium measured only 4 mm; one continued while the other resulted in a miscarriage at 11 weeks of gestation. In the last few years several studies have investigated the value of 3D ultrasound in the assessment of endometrial volume. By using this technique three orthogonal planes of uterus are displayed simultaneously, providing exact frontal, sagittal, and horizontal sections through the uterine cavity. The endometrial volume is measured by outlining the areas of at least 12 parallel sections. The definition of the lower end of the endometrial cavity is frequently difficult.85 Using this method several authors using the IVF or intrauterine insemination (IUI) model86–88 reported significantly lower pregnancy rates in patients who had endometrial volume <2.0 cm3 and no pregnancies in patients who had endometrial volume <1.2 cm3 on the day of the embryo transfer. With endometrial thickness >2.0 cm2 correlation to the pregnancy rate was lost, suggesting that, similar to data obtained in 2D studies, an increased endometrial thickness beyond a certain threshold does not further increase implantation rates.

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Fig 46.17 Flow velocity waveform (FVW) with a uterine artery pulsatility index (PI) of 2.52, which is consistent with good pregnancy rates. A number of studies have suggested that the likelihood of pregnancy is diminished if the uterine artery PI is greater than 3.0.

Endometrial pattern

Doppler studies

Endometrial pattern is defined as the relative echogenicity of the endometrium and the adjacent myometrium as demonstrated on a longitudinal ultrasonic scan. In principle, the central echogenic line represents the uterine cavity; the outer lines represent the basal layer of the endometrium, or the interface between the endometrium and myometrium. The relatively hypoechogenic regions between the two outer lines and the central line may represent the functional layer of the endometrium.89 If three regions are clearly visible, the endometrium is described as multilayered (Fig 46.15). If the endometrium is more echogenic and the central line is blurred or nonexistent, the endometrium is classified as nonmultilayered.90 Endometrial thickness is unrelated to endometrial pattern.70 Of 13 studies which examined the value of endometrial pattern in predicting pregnancy, only four failed to confirm its predictive value. It is, however, important to emphasize that a poor endometrial pattern does not exclude pregnancy. Many authors have demonstrated that pregnancies can occur in patients with a nonmultilayered pattern of endometrium, albeit at a lower frequency.75,91 The endometrial pattern does not appear to be influenced by the type of ovarian stimulation and it is of prognostic value in both fresh IVF and frozen embryo transfer cycles. In an effort to provide more objective assessment of endometrial structure, Leibovitz and colleagues92 developed a computer program rating the degree of hypoechogenicity from 30% to more than 70%. Using this model, the authors described decreased implantation related to the degree of hypoechogenicity, irrespective of endometrial thickness.

Uterine arteries The uterine artery was the first vessel investigated in relation to implantation. Sterzik and colleagues93 reported that the RI measured on the day of embryo transfer was significantly lower in patients who subsequently became pregnant as compared with those who failed to achieve pregnancy. Steer and colleagues used transvaginal color Doppler to study the uterine arterial blood flow in 82 women undergoing IVF on the day of embryo transfer.94 The PI was calculated and the patients grouped according to whether the PI was low (1–1.99), medium (2–2.99) (Fig 46.17), or high (3.0). There were no pregnancies in the high PI group and the PI was significantly lower in the women who became pregnant as compared with those who did not. Zaidi and colleagues showed that an elevated PI of the uterine arteries maintains a similar prognostic value, even if it is performed at the time of hCG administration.95 These findings were confirmed by other authors. Coulam and colleagues found significantly more nonconceptional than conceptional cycles (p <0.001) in women where a uterine artery PI >3.3 was detected.96 Similarly, Bloechle and colleagues97 reported a significantly lower PI and RI in patients who achieved pregnancy in their IVF-ET program following pituitary suppression with goserelin and subsequent stimulation with recombinant FSH. Battaglia and colleagues98 reported a good correlation between PI, serum thromboxane levels, and the chances of achieving a pregnancy. Interestingly, Tekay and colleagues99 were not able to confirm data reported by other groups. In their study, which included only 30 nonselected patients, the authors found no difference

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Fig 46.18 Subendometrial blood flow is an additional parameter in the assessment of uterine receptivity.

in uterine perfusion between pregnant and nonpregnant patients. This difference could be attributed to an inconsistency in patient preparation and timing of Doppler investigations.

Subendometrial and endometrial blood flow With the appearance of more sensitive color Doppler machines much attention has recently been drawn to the potential value of subendometrial (Fig 46.18) and endometrial blood flow in predicting implantation. It has been postulated that local vascularization at the site of implantation is probably more important than global vascularization of the uterus measured by resistance in the uterine arteries. In the first study investigating the intrauterine circulation, Zaidi and colleagues studied 96 women undergoing IVF treatment on the day of hCG administration by transvaginal ultrasonography with color and pulsed Doppler ultrasound.100 These authors observed no pregnancies in the group of patients where subendometrial color flow and intraendometrial vascularization was absent. The importance of subendometrial flow was further investigated by Achiron and colleagues.101 They investigated subendometrial flow in 18 patients with premature ovarian failure (POF) and 12 healthy controls, and observed a decreased vascular impedance in the late follicular phase, with a gradual increase during the early and late luteal phase in both groups of patients. In the patients with POF, they observed a significantly higher vascular resistance in the early follicular phase. This difference disappeared after administration of hormone replacement therapy. These authors concluded that hormone replacement therapy enables normalization of subendometrial

blood flow and creates a vascular status compatible with pregnancy. Yang and colleagues102 adopted a semiqualitative approach in the assessment of endometrial flow. They investigated 95 patients who had endometrial thickness >10 mm on the day of hCG injection. These authors described endometrial power Doppler area (EPDA), defined as a part of the endometrium where vascular signal with velocities >5 cm/s were detected. They found that patients in whom EPDA <5 mm2 had significantly lower pregnancy and implantation rates. Interestingly, decreased EPDA was not reflected in impaired uterine PI, which in both groups of patients was normal. Some attempts have been made to use 3D endometrial and subendometrial flow to assess uterine receptivity. Most researchers calculate the vascularization index (VI), flow index (FI), vascularization flow index (VFI) in the thin hypoechoic layer around the endometrium. Wu and colleagues10 showed an excellent positive predictive value of 93.8% of VFI in predicting a positive outcome following IVF treatment. Contart and colleagues103 failed to confirm the above findings. In their study they found no correlation between subendometrial flow and implantation. Schild and colleagues,12 investigating the role of 3D Doppler performed at the start of ovarian stimulation, also found no correlation between any vascular indices and implantation rates. Chien and colleagues11 prospectively investigating 623 patients demonstrated the highest pregnancy rates in the patients who had both endometrial and subendometrial flow present at the time of embryo transfer. Finally, Kupesic and colleagues,104 comparing 2D and 3D Doppler assessment of the uterine environment, found no significant

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advantages of 3D imaging. In conclusion, 3D assessment of intrauterine vascularization is still an experimental tool. Time will show what its place in the assessment of the implantation potential will be.

paid to transferring embryos under ultrasound control. This can be done under transabdominal108–111 or transvaginal ultrasound112,113 guidance.

Transabdominal approach Motion analysis A novel method of evaluation of endometrial properties is by analyzing endometrial contractions. The test is done by digital acquisition of an ultrasound image of a cross section of endometrium at the speed of frames every 2 seconds for a period of 5–10 minutes. It has been shown that subendometrial contractions are more prominent in IVF than in a natural cycle, a phenomenon which could perhaps explain low implantation rates following embryo transfer.105 Fanchin and colleagues have reported that women with a higher frequency of subendometrial contractions at the time of embryo transfer had lower implantation rates.106 Recently, the same group107 reported that the contractions return to the level observed in a natural cycle 6 days following hCG injection. They postulated that this uterine quiescence may be one of the reasons for an increased implantation rate following the blastocyst transfer. Motion analysis is still a very new mode of endometrial assessment. Research thus far has been limited to very few centers, perhaps because of difficulties in the setup of the system and the length of the procedure. Further research is required prior to giving a definite judgment regarding the value of this procedure in routine clinical practice.

Summary An endometrial thickness of <7 mm or endometrial volume <2 cm3 and perhaps endometrial, thickness >14 mm, absence of multilayered endometrium, and uterine artery PI >3.0 at the time of hCG administration are signs of impaired implantation. Additionally, the absence of subendometrial or reduction in the endometrial vascularized area may help distinguish between patients with normal and abnormal implantation potential. At our center, an attempt is made to prolong ovulation induction until endometrial thickness of >7 mm is achieved. If pregnancy is not achieved, in a subsequent cycle the ovulation induction regimen is changed to allow for a better endometrial development.

Ultrasound-guided embryo transfer In spite of recent improvements in the development of culture media allowing the successful growth of embryos up to the blastocyst stage,68 the rate of pregnancy per blastocyst is still far from 100%. While this can be partly attributed to chromosomal problems with an embryo, an additional problem could be in a relatively primitive method used for traditional embryo transfer. Recently, much attention has been

Embryo transfer is performed with the patient having a full bladder. Following a standard preparation of the cervix, the endometrial cavity is visualized on a sagittal plane under real-time transabdominal ultrasound guidance with typically a 3.5 MHz probe. The trial embryo catheter is advanced through the endometrial cavity and positioned 1–2 cm from the fundus, and the depth of insertion is noted. When an optimal position is ascertained, embryos are loaded into a separate inner catheter and delivered at the previously determined position.

Transvaginal approach This procedure is typically performed using a 7 MHz transvaginal ultrasound probe covered with a non-latex probe cover. Contact gel is placed only between a probe and a probe cover. Following the insertion of a sterile speculum the cervix is prepared in a standard manner and a trial catheter is inserted just beyond the internal os into the uterus. Thereafter, the probe is manipulated to allow a sagittal vision of the uterus. The catheter is advanced under ultrasound guidance 1–2 cm from the fundus and the depth of insertion is noted. Embryos are loaded into a separate inner catheter and inserted at the previously determined position. The value of ultrasound-controlled embryo transfer has been examined in two meta-analyses. Buckett2 analyzed 150 published studies and abstracts performed between 1986 and 2002. Among those he found four properly randomized and four quasi-randomized trials on transabdominally ultrasound-controlled vs traditional transfer and no randomized trials with the transvaginal approach. The conclusion of his meta-analysis was that transabdominally ultrasound-controlled embryo transfer leads to higher pregnancy rates (OR = 1.51; 95% CI 1.32–1.73). In their meta-analysis, Sallam and Sadek,114 analyzing 14 studies which fulfilled their criteria, also confirmed superior pregnancy rates in patients undergoing transabdominally ultrasound-controlled embryo transfer (OR = 1.42; 95% CI 1.17–1.73). In this meta-analysis no difference in ectopic pregnancy rates was observed (OR = 0.39; 95% CI 0.14–1.1). The exact reason as to why these increased pregnancy rates are obtained is not clear. However, avoidance of positioning embryos too low in the cavity or damaging the uterine cavity during the transfer have been put forward as possible explanations.

Future In the last 10 to 20 years we have witnessed an incredible improvement in the sensitivity of ultrasound technology. This has enabled us to measure follicles and endometrial

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parameters more precisely, and to assess blood flow not only in relatively large uterine arteries but also in smaller vessels closer to the site of oocyte maturation and embryo implantation. We have begun to understand the significance of ultrasound-derived parameters on the success of infertility treatment. It is anticipated that in coming years there will be additional improvements in the understanding and use of standard 2D ultrasound technology and more substantial and important role for 3D gray-scale and color Doppler ultrasound techniques. Ultrasound will remain the most important noninvasive tool in the initial assessment of patients, monitoring of their response to medication, and the determination of their chances of achieving a pregnancy. It will also facilitate the assessment of the effect of new treatments on the improvement in follicular maturation and embryo implantation.

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28. Engmann L, Maconochie N, Sladkevicius P, et al. The outcome of in-vitro fertilization treatment in women with sonographic evidence of polycystic ovarian morphology. Hum Reprod 1999; 14: 167–71. 29. MacDougall MJ, Tan SL, Balen A, Jacobs HS. A controlled study comparing patients with and without polycystic ovaries undergoing in vitro fertilization. Hum Reprod 1993; 8: 233–7. 30. Biljan MM, Mahutte NG, Dean N, et al. Effects of pretreatment with an oral contraceptive pill on the time required to achieve pituitary suppression with gonadotropin-releasing hormone analogues and on subsequent implantation and pregnancy rates. Fertil Steril 1998; 70: 1063–9. 31. Thatcher SS, Jones E, DeCherney AH. Ovarian cysts decrease the success of controlled ovarian stimulation and in vitro fertilization. Fertil Steril 1989; 52: 812–16. 32. Segal S, Shifren JL, Isaacson KB, et al. Effect of a baseline ovarian cyst on the outcome of in vitro fertilization-embryo transfer. Fertil Steril 1999; 71: 274–7. 33. Keltz MD, Jones EE, Duleba AJ, et al. Baseline cyst formation after luteal phase gonadotropin-releasing hormone agonist administration is linked to poor in vitro fertilization outcome. Fertil Steril 1995; 64: 568–72. 34. Ben-Rafael Z, Bider D, Menashe Y, et al. Follicular and luteal cysts after treatment with gonadotropinreleasing hormone analog for in vitro fertilization. Fertil Steril 1990; 53: 1091–4. 35. Feldberg D, Ashkenazi J, Dicker D, et al. Ovarian cyst formation: a complication of gonadotropinreleasing hormone agonist therapy. Fertil Steril 1989; 51: 42–5. 36. Karande VC, Scott RT, Jones GS, Muasher SJ. Nonfunctional ovarian cysts do not affect ipsilateral or contralateral ovarian performance during in vitro fertilization. Hum Reprod 1990; 5: 431–3. 37. Hornstein MD, Barbieri RL, Ravnikar VA, McShane PM. The effects of baseline ovarian cysts on the clinical response to controlled ovarian hyperstimulation in an in vitro fertilization program. Fertil Steril 1989; 52: 437–40. 38. Herman A, Ron-El R, Golan A, et al. Follicle cysts after menstrual versus midluteal administration of gonadotropin-releasing hormone analog in in vitro fertilization. Fertil Steril 1990; 53: 854–8. 39. Sampaio M, Serra V, Miro F, et al. Development of ovarian cysts during gonadotrophin-releasing hormone agonists (GnRHa) administration. Hum Reprod 1991; 6: 194–7. 40. Strandell A, Waldenstrom U, Nilsson L, Hamberger L. Hydrosalpinx reduces in vitro fertilization/ embryo transfer pregnancy rates. Hum Reprod 1994; 9: 861–3. 41. Andersen AN, Yue Z, Meng FJ, Petersen K. Low implantation rate after in vitro fertilization in patients with hydrosalpinges diagnosed by ultrasonography. Hum Reprod 1994; 9: 1935–8. 42. Kassabji M, Sims JA, Butler L, Muasher SJ. Reduced pregnancy outcome in patients with unilateral or bilateral hydrosalpinx after in vitro fertilization. Eur J Obstet Gynecol Reprod Biol 1994; 56: 129–32.

43. Vandromme J, Chasse E, Lejeune B, et al. Hydrosalpinges in in vitro fertilization: an unfavourable prognostic feature. Hum Reprod 1995; 10: 576–9. 44. Katz E, Akman MA, Damewood MD, Garcia JE. Deleterious effect of the presence of hydrosalpinx on implantation and pregnancy rates with in vitro fertilization. Fertil Steril 1996; 66: 122–5. 45. Murray DL, Sagoskin AW, Widra EA, Levy MJ. The adverse effect of hydrosalpinges on in vitro fertilization pregnancy rates and the benefit of surgical correction. Fertil Steril 1998; 69: 41–5. 46. Meyer WR, Castelbaum AJ, Somkuti S, et al. Hydrosalpinges adversely affect markers of endometrial receptivity. Hum Reprod 1997; 12: 1393–8. 47. Mukherjee T, Copperman AB, McCaffrey C, et al. Hydrosalpinx fluid has embryotoxic effects on murine embryogenesis: a case for prophylactic salpingectomy. Fertil Steril 1996; 66: 851–3. 48. Campbell S, Bourne TH, Tan SL, Collins WP. Hysterosalpingo contrast sonography (HyCoSy) and its future role within the investigations of infertility in Europe. Ultrasound Obstet Gynecol 1994; 4: 253. 49. Syrop CH, Willhoite A, Van Voorhis BJ. Ovarian volume: a novel outcome predictor for assisted reproduction. Fertil Steril 1995; 64: 1167–71. 50. Tomas C, Nuojua-Huttunen S, Martikainen H. Pretreatment transvaginal ultrasound examination predicts ovarian responsiveness to gonadotrophins in in vitro fertilization. Hum Reprod 1997; 12: 220–3. 51. Danninger B, Brunner M, Obruca A, Feichtinger W. Prediction of ovarian hyperstimulation syndrome of baseline ovarian volume prior to stimulation. Hum Reprod 1996; 11: 1597–9. 52. Jarvela IY, Sladkevicius P, Kelly S, et al. Quantification of ovarian power Doppler signal with three-dimensional ultrasonography to predict response during in vitro fertilization. Obstet Gynecol 2003; 104: 816–22. 53. Chang MY, Chiang CH, Chiu TH, et al. The antral follicle count predicts the outcome of pregnancy in a controlled ovarian hyperstimulation/intrauterine insemination program. J Assist Reprod Genet 1998; 15: 12–17. 54. Ng EH, Tang OS, Ho PC. The significance of the number of antral follicles prior to stimulation in predicting ovarian response in an IVF programme. Hum Reprod 2000; 15: 1937–42. 55. Zaidi J, Barber J, Kyei-Mensah A, et al. Relationship of ovarian stromal blood flow at the baseline ultrasound scan to subsequent follicular response in an in vitro fertilization program. Obstet Gynecol 1996; 88: 779–84. 56. Haning RV Jr, Austin CW, Kuzma DL, et al. Ultrasound evaluation of estrogen monitoring for induction of ovulation with menotropins. Fertil Steril 1982; 37: 627–32. 57. Wittmaack FM, Kreger DO, Blasco L, et al. Effect of follicular size on oocyte retrieval, fertilization, cleavage, and embryo quality in in vitro fertilization cycles: a 6-year data collection. Fertil Steril 1994; 62: 1205–10.

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Ultrasound in ART 58. Tan SL, Balen A, el Hussein E, et al. A prospective randomized study of the optimum timing of human chorionic gonadotropin administration after pituitary desensitization in in vitro fertilization. Fertil Steril 1992; 57: 1259–64. 59. Edwards RG. Conception in the Human Female. New York: Academic Press, 1980. 60. Tan SL, Zaidi J, Campbell S, et al. Blood flow changes in the ovarian and uterine arteries during the normal menstrual cycle. Am J Obstet Gynecol 1996; 175: 625–31. 61. Nargund G, Doyle PE, Bourne TH, et al. Ultrasound derived indices of follicular blood flow before hCG administration and the prediction of oocyte recovery and preimplantation embryo quality. Hum Reprod 1996; 11: 2512–17. 62. Nargund G, Bourne T, Doyle P, et al. Associations between ultrasound indices of follicular blood flow, oocyte recovery and preimplantation embryo quality. Hum Reprod 1996; 11: 109–13. 63. Chui DK, Pugh ND, Walker SM, et al. Follicular vascularity – the predictive value of transvaginal power Doppler ultrasonography in an in vitro fertilization programme: a preliminary study. Hum Reprod 1997; 12: 191–6. 64. Bhal PS, Pugh ND, Chui DK, et al. The use of transvaginal power Doppler ultrasonography to evaluate the relationship between perifollicular vascularity and outcome in in vitro fertilization treatment cycles. Hum Reprod 1999; 14: 939–45. 65. Huey S, Abuhamad A, Barroso G, et al. Perifollicular blood flow Doppler indices, but not follicular pO2, pCO2, or pH, predict oocyte developmental competence in in vitro fertilization. Fertil Steril 1999; 72: 707–12. 66. Coulam CB, Goodman C, Rinehart JS. Colour Doppler indices of follicular blood flow as predictors of pregnancy after in vitro fertilization and embryo transfer. Hum Reprod 1999; 14: 1979–82. 67. Vlaisavljevic VV, Relic M, Gavric Lovrec V, et al. Measurement of perifollicular blood flow of the dominant preovulatory follicle using threedimensional power Doppler. Ultrasound Obstet Gynecol 2003; 22: 520–6. 68. Gardner DK, Vella P, Lane M, et al. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 1998; 69: 84–8. 69. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril 1997; 1: 23. 70. Gonen Y, Casper RF, Jacobson W, Blankier J. Endometrial thickness and growth during ovarian stimulation: a possible predictor of implantation in in vitro fertilization. Fertil Steril 1989; 52: 446–50. 71. Glissant A, de Mouzon J, Frydman R. Ultrasound study of the endometrium during in vitro fertilization cycles. Fertil Steril 1985; 44: 786–90. 72. Fleischer AC, Herbert CM, Sacks GA, et al. Sonography of the endometrium during conception and nonconception cycles of in vitro fertilization and embryo transfer. Fertil Steril 1986; 46: 442–7. 73. Welker BG, Gembruch U, Diedrich K, et al. Transvaginal sonography of the endometrium during ovum pickup in stimulated cycles for in vitro fertilization. J Ultrasound Med 1989; 8: 549–53.

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74. Li TC, Nuttall L, Klentzeris L, Cooke ID. How well does ultrasonographic measurement of endometrial thickness predict the results of histological dating? Hum Reprod 1992; 7: 1–5. 75. Dickey RP, Olar TT, Curole DN, et al. Endometrial pattern and thickness associated with pregnancy outcome after assisted reproduction technologies. Hum Reprod 1992; 7: 418–21. 76. Weissman A, Gotlieb L, Casper RF. The detrimental effect of increased endometrial thickness on implantation and pregnancy rates and outcome in an in vitro fertilization program. Fertil Steril 1999; 71: 147–9. 77. Dietterich C, Check JH, Choe JK, et al. Increased endometrial thickness on the day of human chorionic gonadotropin injection does not adversely affect pregnancy or implantation rates following in vitro fertilization-embryo transfer. Fertil Steril 2002; 77: 781–6. 78. Yakin K, Akarsu C, Kahraman S. Cycle lumping or sampling a witches’ brew? Fertil Steril 2000; 73: 175. 79. Krampl E, Feichtinger W. Endometrial thickness and echo patterns. Hum Reprod 1993; 8: 1339. 80. Imoedemhe DA, Shaw RW, Kirkland A, Chan R. Ultrasound measurement of endometrial thickness on different ovarian stimulation regimens during in vitro fertilization. Hum Reprod 1987; 2: 545–7. 81. Friedler S, Schenker JG, Herman A, Lewin A. The role of ultrasonography in the evaluation of endometrial receptivity following assisted reproductive treatments: a critical review. Hum Reprod Update 1996; 2: 323–35. 82. Gonen Y, Calderon I, Direnfeld M, Abramovici H. The impact of sonographic assessment of the endometrium and meticulous hormonal monitoring during natural cycles in patients with failed donor artificial insemination. Ultrasound Obstet Gynecol 1991; 1: 122–6. 83. Abdalla HI, Brooks AA, Johnson MR, et al. Endometrial thickness: a predictor of implantation in ovum recipients? Hum Reprod 1994; 9: 363–5. 84. Sundstrom P. Establishment of a successful pregnancy following in-vitro fertilization with an endometrial thickness of no more than 4 mm. Hum Reprod 1998; 13: 1550–2. 85. Schield RL, Indefrei D, Eschweiler S, et al. Threedimensional endometrial volume calculation and pregnancy rate in an in vitro fertilization programme. Hum Reprod 1999; 14: 1255–8. 86. Raga F, Bonilla-Musoles F, Casan EM, et al. Assessment of endometrial volume by threedimensional ultrasound prior to embryo transfer: clues to endometrial receptivity. Hum Reprod 1999; 14: 2851–4. 87. Yaman C, Ebner T, Sommergruber M, et al. Role of three-dimensional ultrasonographic measurement of endometrial volume as a predictor of pregnancy outcome in an IVF-embryo transfer programme: a preliminary study. Fertil Steril 2000; 74: 797–801. 88. Zollner U, Zollner KP, Blissing S, et al. Impact of threedimensionally measured endometrial volume on the pregnancy rate after intrauterine insemination. Zentralbl Gynakol 2003; 125: 136–41. 89. Forrest TS, Elyaderani MK, Muilenburg MI, et al. Cyclic endometrial changes: US assessment with histologic correlation. Radiology 1988; 167: 233–7.

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90. Sher G, Herbert C, Maassarani G, Jacobs MH. Assessment of the late proliferative phase endometrium by ultrasonography in patients undergoing in vitro fertilization and embryo transfer (IVF/ET). Hum Reprod 1991; 6: 232–7. 91. Serafini P, Batzofin J, Nelson J, Olive D. Sonographic uterine predictors of pregnancy in women undergoing ovulation induction for assisted reproductive treatments. Fertil Steril 1994; 62: 815–22. 92. Leibovitz Z, Grinin V, Rabia R, et al. Assessment of endometrial receptivity for gestation in patients undergoing in vitro fertilization, using endometrial thickness and endometrium–myometrium relative echogenicity coefficient. Ultrasound Obstet Gynecol 1999; 14: 194–9. 93. Sterzik K, Grab D, Sasse V, et al. Doppler sonographic findings and their correlation with implantation in an in vitro fertilization program. Fertil Steril 1989; 52: 825–8. 94. Steer CV, Campbell S, Tan SL, et al. The use of transvaginal color flow imaging after in vitro fertilization to identify optimum uterine conditions before embryo transfer. Fertil Steril 1992; 57: 372–6. 95. Zaidi J, Pittrof R, Shaker A, et al. Assessment of uterine artery blood flow on the day of human chorionic gonadotropin administration by transvaginal color Doppler ultrasound in an in vitro fertilization program. Fertil Steril 1996; 65: 377–81. 96. Coulam CB, Bustillo M, Soenksen DM, Britten S. Ultrasonographic predictors of implantation after assisted reproduction. Fertil Steril 1994; 62: 1004–10. 97. Bloechle M, Schreiner T, Kuchler I, et al. Colour Doppler assessment of ascendant uterine artery perfusion in an in vitro fertilization-embryo transfer programme after pituitary desensitization and ovarian stimulation with human recombinant follicle stimulating hormone. Hum Reprod 1997; 12: 1772–7. 98. Battaglia C, Artini PG, Giulini S, et al. Colour Doppler changes and thromboxane production after ovarian stimulation with gonadotrophin-releasing hormone agonist. Hum Reprod 1997; 12: 2477–82. 99. Tekay A, Martikainen H, Jouppila P. Blood flow changes in uterine and ovarian vasculature, and predictive value of transvaginal pulsed colour Doppler ultrasonography in an in vitro fertilization programme. Hum Reprod 1995; 10: 688–93. 100. Zaidi J, Campbell S, Pittrof R, Tan SL. Endometrial thickness, morphology, vascular penetration and velocimetry in predicting implantation in an in vitro fertilization program. Ultrasound Obstet Gynecol 1995; 6: 191–8. 101. Achiron R, Levran D, Sivan E, et al. Endometrial blood flow response to hormone replacement therapy in women with premature ovarian failure: a

transvaginal Doppler study. Fertil Steril 1995; 63: 550–4. 102. Yang JH, Wu MY, Chen CD, et al. Association of endometrial blood flow as determined by a modified colour Doppler technique with subsequent outcome of in vitro fertilization. Hum Reprod 1999; 14: 1606–10. 103. Contart P, Baruffi RL, Coelho J, et al. Power Doppler endometrial evaluation as a method for the prognosis of embryo implantation in an ICSI program. J Assist Reprod Genet 2000; 17: 329–34. 104. Kupesic S, Bekavac I, Bjelos D, Kurjak A. Assessment of endometrial receptivity by transvaginal color Doppler and three-dimensional power Doppler ultrasonography in patients undergoing in vitro fertilization procedures. J Ultrasound Med 2001; 20: 125–34. 105. Fanchin R, Ayoubi JM, Olivennes F, et al. Hormonal influence on the uterine contractility during ovarian stimulation. Hum Reprod 2000; 15(Suppl 1): 90–100. 106. Fanchin R, Righini C, Olivennes F, et al. Uterine contractions at the time of embryo transfer alter pregnancy rates after in vitro fertilization. Hum Reprod 1998; 13: 1968–74. 107. Ayoubi JM, Epiney M, Bioschi PA, et al. Comparison of changes in uterine contraction frequency after ovulation in the menstrual cycle and in in vitro fertilization cycles. Fertil Steril 2003; 79: 1101–5. 108. Strickler RC, Cristianson C, Crane JP, et al. Ultrasound guidance for human embryo transfer. Fertil Steril 1985; 43: 54–61. 109. Leong M, Leong C, Tucker M, et al. Ultrasoundassisted embryo transfer. J In Vitro Ferti Embryo Transf 1986; 3: 383–5. 110. Coroleu B, Carreras O, Veiga A, et al. Embryo transfer under ultrasound guidance improves pregnancy rates after in vitro fertilization. Hum Reprod 2000; 15: 616–20. 111. Prapas Y, Prapas N, Hatziparasidou A, et al. Ultrasound-guided embryo transfer maximizes the IVF results on day 3 and day 4 embryo transfer but has no impact on day 5. Hum Reprod 2001; 16: 1904–8. 112. Kojima K, Nomiyama M, Kumamoto T, et al. Transvaginal ultrasound-guided embryo transfer improves pregnancy and implantation after IVF. Hum Reprod 2001; 16: 2578–82. 113. Anderson RE, Nugent NL, Gregg AT, et al. Transvaginal ultrasound-guided embryo transfer improves outcome in patients with previously failed in vitro fertilization cycles. Fertil Steril 2002; 77: 769–75. 114. Sallam HN, Sadek SS. Ultrasound-guided embryo transfer: a meta-analysis of randomized controlled trials. Fertil Steril 2003; 80: 1042–6.

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47 Sperm-recovery techniques: clinical aspects Herman Tournaye, Patricio Donoso

In the past 10–20 years, several changes have taken place in clinical andrology. Gradually, empirical treatments have been replaced by techniques of assisted reproduction – intrauterine insemination, in vitro fertilization, and intracytoplasmic sperm injection. In particular, the introduction of intracytoplasmic sperm injection (ICSI) in 19921,2 completely changed the clinical approach towards male infertility by offering a novel opportunity for parenthood to azoospermic men. A single spermatozoon can be injected into an oocyte and result in normal fertilization, embryonic development, and implantation. Not only ejaculated spermatozoa can be used but also epididymal or testicular spermatozoa can be used for ICSI. Testicular spermatozoa can be retrieved in some patients with nonobstructive azoospermia (NOA) because of the persistence of isolated foci of active spermatogenesis. The first pregnancies using epididymal and testicular spermatozoa in men with obstructive azoospermia (OA) and NOA were published in 1993 and 1995, respectively.3–6 Surgical retrieval of spermatozoa for ICSI has currently become a routine technique in clinical andrology. Several techniques are available to retrieve epididymal or testicular spermatozoa. Although there is no real method of choice, some guidelines may be given in order to make the best choice for a specific clinical setting. ICSI has also reinforced the role of nonsurgical techniques to retrieve sperm in men suffering from anejaculation.

Azoospermia: what’s in a name? Most azoospermic patients suffer from primary testicular failure (60%).7,8 Because these patients do not show any clinical sign of obstruction, they are often referred to as patients with NOA. However, in a few cases, azoospermia without any obstruction is the result of a hypogonadotropic hypogonadism, i.e. a lack of adequate hormonal stimulation to support spermatogenesis. These patients have an early maturation arrest in spermatogenesis. Treatment with follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG) will restore spermatogenesis and

they do not, in the first instance, need assisted reproduction.9 In general, these patients are not referred to as suffering from NOA. Azoospermic patients with primary testicular failure show either a germ cell aplasia (Sertoli cell only), a maturation arrest, or tubular sclerosis and atrophy at their testicular histopathology. Germ cell aplasia may be iatrogenic, as it may result from irradiation or chemotherapy, or it may be congenital, because of a genetic disorder such as Klinefelter’s syndrome or a deletion on the long arm of the Y chromosome. In many cases, however, the cause of germ cell aplasia remains unknown. Many patients with primary testicular dysfunction, however, are now assumed to have testicular dysgenesis syndrome, a congenital developmental disorder causing spermatogenic failure, maldescence of the testis (cryptorchidism), and eventually hypospadias in more severe forms of the spectrum of this disorder.10 They also have a higher risk of developing testis carcinoma.10 Men with NOA may also show maturation arrest at testicular histology. Maturation arrest may be caused by viral orchitis, irradiation, and/or chemotherapy and Yq deletions. Other causes include systemic illness or exposure to gonadotoxins, but here too, idiopathic maturation arrest is most common. Again, testicular dysgenesis syndrome may yet explain these cases. Fewer men with NOA will show tubular sclerosis and atrophy on testicular histology. This may be the result of testicular torsion, vascular injury, or infection, but it is also a common finding in Klinefelter’s syndrome patients. Many studies on assisted reproduction technologies (ART) with testicular spermatozoa or spermatids use inadequate definitions, often based on the absence or presence of clinical signs of obstruction. According to World Health Organization (WHO) guidelines for ART, the diagnosis of ‘nonobstructive azoospermia’ should be made according to the histopathological findings, rather than on the basis of clinical indicators such as FSH levels or testicular size.11,12 Testicular failure is found in a third of normogonadotropic azoospermic men with normally

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sized testes; however, small testicular size or elevated FSH does not preclude normal spermatogenesis. Whenever testicular biopsy shows a normal spermatogenesis or a mild hypospermatogenesis, an obstruction of the excretory ducts is present. In a substantial subgroup of these men, however, no clinical signs of obstruction will be present. An accurate distinction between these two types of azoospermia is particularly relevant since spermatozoa can be retrieved in almost all patients with OA and mild hypospermatogenesis but only in up to 50% of unselected patients with NOA when no preliminary selection of patients on the basis of histopathology has been performed.13

Does my patient need surgical sperm recovery? In patients with obstructive azoospermia, fertility can be restored by surgical correction, i.e. vasoepididymostomy, vasovasostomy, or periurethral resection. When surgery has failed or is not indicated, e.g. patients with congenital bilateral absence of the vas deferens (CBAVD), surgical sperm recovery procedures are indicated. Most methods described for surgical sperm recovery are simple techniques. However, in some patients with azoospermia, even these simple techniques are not indicated. When after appropriate analysis the diagnosis of azoospermia is made, an appropriate clinical work-up is necessary in order to define the exact cause of the azoospermia and to define the best treatment option. If azoospermia is the result of a primary testicular failure caused by hormonal deficiency, such as hypogonadotropic hypogonadism, then hormone replacement therapy must be proposed. Often the diagnosis of azoospermia is made without centrifuing the semen. Centrifugation at 1800 × g for at least 5 minutes may reveal spermatozoa in the pellet, which may be used for ICSI.14 In a series of 49 patients with NOA, it was shown that, in 35% of patients, spermatozoa could be recovered from the ejaculate for ICSI.14 In cases of NOA, it may therefore be worthwhile to perform centrifugation of an ejaculate before embarking on a surgical recovery procedure to retrieve spermatozoa. Only when no spermatozoa are found in the pellet after centrifugation, or when only immotile nonviable spermatozoa are found, is surgical sperm recovery indicated in order to avoid performing ICSI with spermatozoa with DNA damage.

Anejaculation does not equal azoospermia Surgical sperm retrieval methods have been proposed as a means of obtaining spermatozoa for assisted reproduction in men with anejaculation, i.e. the absence of antegrade or retrograde ejaculation. However, given the efficiency of assisted ejaculation in these men, surgical

methods are only to be considered when penile vibrostimulation (PVS) or electroejaculation (EEJ) has failed. Epididymal or testicular sperm-recovery procedures are often proposed to anejaculatory patients because no PVS or EEJ is available.15 When these first-line recovery methods are unavailable, it is even preferable to refer anejaculatory patients, especially patients with spinal cord injuries, to specialized services where assisted ejaculation can be performed and semen can be cryopreserved. Vibro- or electrostimulation are noninvasive techniques that may be performed without any anesthesia in paraplegic men. Since scrotal hematoma may take a long time to heal in such men, surgical sperm retrieval techniques are indicated only where these noninvasive techniques fail to produce an ejaculate that may be used for ICSI. Even here, vas deferens aspiration may be preferable because of its low risk of iatrogenic obstruction.16,17 The ejaculates, even when oligoasthenoteratozoospermic, can be cryopreserved for later use. Testicular sperm retrieval must be considered only where primary testicular failure is present in an anejaculatory patient or when techniques of assisted ejaculation have failed to produce an ejaculate that can be used for ICSI. It is preferable in such patients to refrain from epididymal sperm aspiration techniques because of their higher risk of iatrogenic epididymal obstruction. Psychogenic anejaculation may be encountered unexpectedly during treatments with ART, e.g. ICSI. Here too, assisted ejaculation may be useful, rather than surgical methods, in order to obtain spermatozoa if treatment by sildenafil citrate has failed to overcome the problem of an acute erectile dysfunction.18 In some anejaculatory patients, prostatic massage – a simple alternative noninvasive method – can be used in order to obtain spermatozoa for ART.19

Ejaculation induced by penile vibratory stimulation and electroejaculation Anejaculation may be psychogenic or may result from spinal cord injury or retroperitoneal lymph node dissection. These causes represent almost 95% of etiologies. Diabetic neuropathy, multiple sclerosis, Parkinson’s disease, and aortoiliac, colorectal, or bladder neck surgery are less frequently encountered causes. Occasionally, anejaculation is drug associated: antidepressive, antipsychotic, and antihypertensive medication may induce anejaculation. Given the low efficiency of medical treatments for inducing ejaculation in anejaculatory men, PVS (Fig 47.1 and Protocol 2 in Appendix) and EEJ (Fig 47.2 and Protocol 1 in Appendix) may be considered as the first-line treatments for anejaculation.20 PVS is recommended because it is still less invasive and less expensive than EEJ and because semen quality has been reported to be much better after PVS than after EEJ, especially in men with spinal cord injury.21

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Fig 47.1 Penile vibrostimulation (PVS). The vibrator should deliver a high peak-to-peak amplitude of at least 2.5 mm and a frequency of about 100 Hz. The vibrating part is applied to the posterior glans penis and frenulum.

Fig 47.2 Electroejaculation (EEJ). The patient is in lateral decubitus and a stimulatory probe is gently introduced in the rectum with the electrodes facing the prostate.

PVS will restore ejaculation in half of the anejaculatory patients when a high amplitude (at least 2.5 mm amplitude) is used.21 The amplitude of a vibrator is the distance over which the vibrating part is moving up and down. The frequency of vibration should be around 100 Hz. Each patient scheduled for PVS should undergo a complete neurological and uroandrological examination. PVS needs an intact spinal cord up to the lumbosacral level. In spinal cord-injured men, PVS is less successful in case of lower cord lesions. When the patient has a transsection at T6 or higher, an increase in blood pressure because of autonomic dysreflexia may occur during a PVS procedure. Close monitoring of the blood pressure is thus indicated. Whenever acute hypertension develops, 10–20 mg of nifedipine should be administered sublingually. In spinal cordinjured patients with a history of autonomic dysreflexia, 10 mg of nifedipine should be given preventively about 15 minutes before starting PVS. The patient is instructed to drink 500 ml of water containing 600 mg of sodium bicarbonate the morning of the procedure in order to alkalinize the urine. After emptying, the bladder is washed with a buffered sperm preparation medium. About 50 ml of this medium is left in the bladder. The vibrating part of the vibrator is placed on the posterior glans penis and frenulum. The position can be slowly changed in order to find a reactive trigger-point. When no ejaculation is obtained within 10 minutes, the procedure should be discontinued. Although less frequent than with EEJ, retrograde ejaculation may occur during PVS. Flushing, goose skin, and spasms of the abdominal muscles and legs may indicate ejaculation. In general, spermatozoa can be obtained in approximately 55% of men; however, in spinal cord-injured men with lesions above T11, the retrieval rate will be reaching 88%.15

High-amplitude penile vibrostimulators have become affordable, and therefore couples, infertile because of anejaculation, can use PVS at home for attempting pregnancy or to improve semen quality by regular ejaculation. Home-use PVS may not be indicated in spinal cord-injured men with lesions above T6 because of the risk of autonomic dysreflexia. Electroejaculation is the treatment of choice if PVS fails. EEJ is a technique initially introduced to obtain spermatozoa from endangered species. In the late 1980s the technique was introduced successfully in the clinic too.22 Patients should receive the same work-up and preparation as for PVS. For EEJ, patients with no spinal cord injury or patients with incomplete spinal cord lesions need general anesthesia. Sympathicolytic agents should not be used during anesthesia. As for PVS, spinal cord-injured men with lesions at T6 or above must be closely monitored for autonomic dysreflexia and pretreated whenever indicated (see above). The patient is placed in lateral decubitus. Because of the risk of rectal burning by the heating of the EEJ probe, it is recommended to use equipment with a built-in temperature sensor. The EEJ probe is introduced in the rectum with the electrodes facing the prostate. In spinal cord-injured men it may be recommended to perform a preliminary digital rectal examination and an anoscopy. A repetitive electrical stimulus of maximum 5 V is applied for about 2–4 seconds each stimulus. When no ejaculation, either antegrade or retrograde, is obtained, the voltage may be gradually increased. With a few exceptions, ejaculation occurs at voltages lower than 20 V. During the stimulation an assistant collects the antegrade fraction. After the procedure, anoscopy is repeated to ensure no rectal lesions have occurred. The patient is placed in the lithotomy position and the bladder is washed in order to recover any retrograde fraction. In 80–95% of patients,

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Azoospermia

Centrifuge at 1800 × g for 15 min

No motile spermatozoa

Motile spermatozoa

Obstructive azoospermia? No need for SRT

Fig 47.3

Proven by previous work-up

Suspected

Microsurgical reconstruction possible?

Scrotal exploration

Microsurgical reconstruction possible?

No

Yes

No

PESA with cryopreservation

Vasovasostomy or Vasoepididymostomy

MESA with cryopreservation

Treatment algorithm for patients with obstructive azoospermia.

No see Fig 47.7

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spermatozoa can be recovered.15,23 According to the quality of the specimen obtained, either intrauterine insemination or assisted reproduction by ICSI can be performed. In anejaculatory men, and especially in spinal cord-injured men, both semen quality and sperm function may be deteriorated because of accumulation of reactive oxygen species, denervation, male accessory gland infection, postinfectious partial obstruction, or postinfectious primary testicular failure. Therefore, the introduction of ICSI has dramatically changed the perspective of patients suffering from anejaculation.24 In a small retrospective study, prostatic massage, EEJ, and testicular sperm extraction were compared in terms of establishing a pregnancy by ICSI. It was shown that the three techniques resulted in similar pregnancy and live birth rates.19

Methods for retrieving epididymal or testicular spermatozoa If no motile spermatozoa can be obtained from the ejaculate after centrifugation, a sperm retrieval procedure has to be performed. At present, different methods are available to obtain spermatozoa from the vas deferens, epididymis, or testicular mass.25 The method of choice will depend merely on the surgical skills and the techniques available in a given setting. If sperm has to be retrieved on an outpatient basis, techniques should be adopted that are compatible with local or locoregional anesthesia. In case of OA, several methods are available. Fig 47.3 shows the algorithm currently used in our setting. If OA is expected, but either the cause or the site of the obstruction is unknown, a scrotal exploration must be performed. A scrotal exploration may not only reveal the cause and site of the obstruction and confirm the diagnosis of OA but also provide the possibility of

Fig 47.4 Microsurgical epididymal sperm aspiration (MESA). The epididymis is exposed and epididymal fluid is collected after a microincision in a dilated tubule.

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performing reconstructive surgery. If no surgical correction is feasible, we prefer to perform a microsurgical epididymal sperm aspiration (MESA) during the exploration (Fig 47.4, Protocol 7 in Appendix). The epididymal spermatozoa that are obtained can be easily cryopreserved for later use without jeopardizing the outcome after ICSI.26 If, however, previous work-up has shown that microsurgical reconstruction is not possible, then a percutaneous epididymal sperm aspiration (PESA) may be performed (Fig 47.5, Protocol 3 in Appendix). Although there have been some concerns that this blind method may cause epididymal damage and fibrosis27 this issue is not important where reconstruction is not possible. After PESA, epididymal sperm may not always be obtained.28 In this case, testicular spermatozoa may be obtained either by fine-needle aspiration (FNA) or by open testicular biopsy of the testis (Fig 47.6, Protocols 4 and 5, respectively, in

Fig 47.5 Percutaneous epididymal sperm aspiration (PESA). The epididymis is palpated and epididymal fluid is collected after a blind percutaneous puncture with a 19- or 21-gauge needle.

Fig 47.6 Fine-needle aspiration (FNA) of the testis. Using a fine 21-gauge butterfly needle filled with a minute volume of sperm preparation medium, the testicular mass is punctured and an aspirate is collected.

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Appendix). Both methods are similar in terms of outcome,29 but the numbers of sperm obtained after open biopsies are much higher. For this reason, open testicular biopsy may be preferred whenever cryopreservation is desired. Alternative methods of testicular aspiration have been described that yield higher numbers of spermatozoa.30 In these aspiration techniques, needles with a larger diameter are used in order to obtain tissue cylinders. Compared with FNA, these alternative methods are less patient friendly and need

local or locoregional anesthesia. Sometimes they even need to be combined with a small incision by a sharp blade in the scrotal skin. Their main advantage is that cryopreservation is easy and efficient because of the higher numbers of sperm obtained. Fig 47.7 shows our current algorithm for patients with non-obstructive azoospermia willing to undergo ICSI treatment. Testicular sperm extraction (TESE) is the most frequently used technique for NOA, with an average

Nonobstructive azoospermia

Proven by preliminary single biopsy

Suspected

With sperm found?

Perform testicular biopsy

No

Yes

Sperm found?

Schedule for ICSI with TESE

Sample dilated tubules under microscope

Sperm found?

Yes

Yes

No

Cryopreserve and schedule for ICSI with TESE or frozen TESE

Sample dilated tubules under microscope

No Sperm found?

Schedule for ICSI with TESE or frozen TESE

Perform multiple biopsies at random

No

Yes

Sperm found?

Consider artificial insemination by donor or adoption

Fig 47.7

Treatment algorithm for patients with nonobstructive azoospermia.

No

Yes

Schedule for ICSI with TESE or frozen TESE

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Fig 47.8 Multiple testicular sampling by open excisional testicular biopsy (TESE). Small tissue specimens are taken from the testicular mass while avoiding vascular injuries when incising the tunica albuginea.

sperm retrieval rate of 50%. The appropriate number of biopsies to be taken remains controversial. Although single testicular biopsy was initially proposed as the best approach,31–33 it is currently recommended to take multiple samples from different sites of the testis since a patchy distribution of spermatogenesis throughout the testis has been identified.5,34–36 In addition, it has been shown that TESE with multiple biopsies results in a higher chance of finding motile spermatozoa.36 Concerning the best location to perform the biopsy, two small descriptive studies have reported opposite results:36,37 Hauser et al36 found no differences in the sperm retrieval rate between three testicular sites, whereas Witt et al37 concluded that the midline portion of the testis enabled the highest retrieval rate. If a preliminary single biopsy has shown focal spermatogenesis with testicular spermatozoa present, the patient and his partner may be scheduled for ICSI with a TESE performed on the day of the oocyte retrieval or the day before. The excisional biopsy may be scheduled under local anesthesia. When a preliminary single biopsy has not shown the presence of testicular spermatozoa, a testicular sperm retrieval procedure with multiple biopsies has to be proposed (Fig 47.8, Protocol 6 in Appendix).38,39 Because multiple biopsies may lead to extensive fibrosis and devascularization,40,41 multiple excisional biopsies may be taken under an operating microscope at ×40 and ×80 magnification.42 This microsurgical approach aims at sampling the more distended tubules in order to limit testicular damage. This technique may be very useful in cases of Sertoli cell-only syndrome with focal spermatogenesis, but is useless in cases with maturation arrest, where there is generally no difference in diameter of tubules with or without focal spermatogenesis. Doppler ultrasound before TESE has also been used to help

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identify regions with better vascularization where spermatozoa would most likely be found.43 In theory this method could help reduce the number of biopsies required to retrieve motile spermatozoa, and hence minimize testicular injury. Nonetheless, more research is required in this area to establish the added value of this technique. When sperm are found, the tiny samples may be frozen for later use with ICSI. If only a few spermatozoa are available or only a tiny amount of tissue is cryopreserved with only a few spermatozoa observed, we always ask the patient to be on standby on the day of ovum pickup in case no spermatozoa can be observed after thawing. If NOA is suspected from the clinical findings, a testicular biopsy is performed preferentially under general anesthesia. If sperm are found after taking a single biopsy, the tissue may be cryopreserved for ICSI in the future. When no sperm are found, the microsurgical approach may be performed. If after microsurgical tubule sampling no sperm is found, then small multiple biopsies are taken at random. The number of biopsies taken depends on the volume of the testis. It has been shown that in about half of patients, spermatozoa can be observed when more than a single tissue specimen has been taken.36 Only small tissue pieces should be taken and avoid cutting the arterioles as much as possible in order not to cause too much devascularization. The retrieval of testicular spermatozoa in these difficult cases may be facilitated by using erythrocyte lysing buffer 44 and enzymatic digestion. 45 Some authors have reported that scheduling the testicular recovery procedure 1 day before the ovum pickup, 46 or the use of motility stimulants, e.g. pentoxifylline, may facilitate the retrieval of motile spermatozoa from the tissue.47 In about half of unselected patients with NOA, no testicular spermatozoa will be found25,35,39 and no accurate clinical parameters are available by which to predict finding sperm before the biopsy.35,39,48 To date, only histopathology has been shown to predict the probability of finding sperm (sensitivity 86% and specificity 93%) in subgroups of NOA men.49 There still exists some controversy about the role of inhibin B for predicting successful sperm recovery.50,51 Although based on a limited series, Yq deletion testing may have a predictive role: testicular sperm recovery failed in all azoospermic men with AZFb deletions.52 A special subgroup of patients with NOA are patients with Klinefelter’s syndrome. Again, in half of these patients spermatozoa may be recovered for ICSI.53,54 Pregnancies have been obtained after ICSI with testicular spermatozoa from 47,XXY nonmosaic Klinefelter’s syndrome.53–56 Age is an important parameter for predicting sperm recovery in this group of patients, as shown by Okada et al, with a cut-off value of 35 years old.57 However, it is important to combine

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ICSI with preimplantation genetic diagnosis,58 because of the risk of aneuploidy in the embryos obtained in these patients.59,60 Oncological patients are another subgroup. Patients undergoing a potentially sterilizing chemotherapy must bank their semen before starting any treatment. However, they may be azoospermic at the time of cancer diagnosis because of spermatogenic depression due to factors related to the malignancy. Yet these patients may be offered sperm recovery and banking before starting chemotherapy by vasal or epididymal sperm aspiration during orchiectomy61 or testicular sperm extraction (onco-TESE).62,63 In cases where no semen was banked before starting chemotherapy, patients with postchemotherapy azoospermia may also benefit from testicular sperm extraction.63–66 Less invasive methods have been proposed in order to obtain testicular spermatozoa from patients with NOA, i.e. testicular aspiration (TESA). The main advantages of this technique are its simplicity and low cost, and that it is minimally invasive and produces less postoperative pain than TESE under local anesthesia.25 However, several prospective controlled studies have shown that the sperm recovery rate (SRR) is significantly lower than with excisional biopsies.25,67–70 An additional disadvantage is that frequently there are no surnumerary spermatozoa to cryopreserve because of the limited number retrieved.25,67 Furthermore, in patients with a history of cryptorchidism, testicular aspiration is contraindicated. These patients have a higher risk of developing a testicular cancer from carcinoma in-situ cells and an excisional biopsy must therefore be performed in order to verify for carcinoma in-situ.71 TESA has also been used as a mapping technique prior to TESE, aiming to identify the areas in which sperm are present,72,73 and resulting in a high retrieval rate (95% of the cycles). In addition, in 20% of the cases TESE was not required. Color Doppler ultrasound before TESA has been examined as a tool to recognize areas with better vascularization where foci of spermatogenesis could be found, and so far shows promising results in small studies.74,75 Nevertheless, further research is needed to determine if this technique can enhance sperm retrieval rates in patients undergoing TESA. The main complications of testicular retrieval techniques are hematoma, fibrosis, and testicular atrophy. Microdissection TESE seems to be the safest technique as regards postoperative complications, followed by TESA. Nevertheless, recent animal studies have raised concerns of the possible long-term consequences of TESA because of an increased disturbance of the tubular architecture compared with TESE. Furthermore, the fact that conventional TESE performed by a skilled surgeon achieves high rates of sperm retrieval even after two or three repeated biopsies reinforces this strategy as a safe procedure.76–79

A successful testicular sperm recovery: what’s next? In patients with normal spermatogenesis, pregnancy rates after ICSI using testicular spermatozoa are comparable to those obtained after ICSI using epididymal spermatozoa.79 However, a recent metaanalysis showed a significantly higher fertilization rate (relative risk [RR] = 1.18; 95% confidence interval [CI] 1.13–1.23) and clinical pregnancy rate (RR = 1.36; 95% CI 1.10–1.69) for OA as compared to NOA.80 This metaanalysis does not include an earlier published study comprising an even larger series than that reported, which showed significant lower fertilization and pregnancy rates in NOA when compared with OA.81 The reasons for these findings are currently unclear, but may be associated with a deficient meiosis in NOA.82 There was no difference in either implantation or miscarriage rates between these two groups. When fresh and frozen–thawed epididymal sperm cycles were compared, no significant difference was observed in fertilization or implantation rates; however, a significantly higher clinical pregnancy rate was reported for fresh cycles (RR = 1.20; 95% CI 1.0–1.42).80 In azoospermic men with primary testicular failure, significant differences do exist between various reports, mainly because of differences in patient selection, sample size of the study, and definition of NOA.11,83,84 Limited evidence is available of the influence of the sperm retrieval technique on the pregnancy rate. Although it has been suggested that TESA results in higher implantation and pregnancy rates than TESE because of a lesser degree of spermatogenetic impairment in these patients,85 others have found no significant difference in the fertilization, implantation, and clinical pregnancy rates between TESA and TESE.86 Preimplantation genetic aneuploidy screening (PGS) has been proposed as a way to improve embryo selection in azoospermic men because of a higher frequency of aneuploid and mosaic embryos.87 PGS could be particularly important under the framework of a single embryo transfer policy, where a higher chance of selecting a chromosomally abnormal embryo was reported when morphological criteria had solely been used.88 Nevertheless, more evidence is required to establish the value of this technique in these couples. Few data are available about the pregnancy outcome and the neonatal data of children born after ICSI with testicular sperm in patients with an NOA.89,90 Although based on small sample sizes, these data have not shown any difference between pregnancies after the use of testicular sperm from NOA men compared with OA men. Thus, patients should be counseled that treating sterility because of NOA has many limitations: firstly, there are limitations in the chances to recover testicular spermatozoa; and secondly, there are limitations in the outcome after ICSI itself.

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Appendix Protocol 1: electroejaculation Indication Anejaculation refractory to penile vibratory stimulation (see Protocol 2).

Patient preparation In spinal-cord injured men a preliminary microbiological examination of the urine has to be performed. No rectal preparation (such as clysma)! Fluid intake is restricted to 500 ml in the 12 hours preceding the procedure. The patient has to empty the bladder before EEJ. In spinal cord-injured men with lesions at T6 or higher, monitoring of blood pressure is mandatory. Nifedipine 10–20 mg may be given to prevent autonomic dysreflexia-related hypertension. The patient wears a top only. He is placed in the lithotomy position. The penal region is cleansed with antiseptic solution (e.g. HAC, Zeneca: hospital antiseptic concentrate, which contains chlorhexidine).

The EEJ procedure The tip of a Nelaton bladder catheter is dipped into sterile liquid mineral oil as used in IVF. After instillation of 10 ml of sperm preparation medium into the urethra, the catheter is gently introduced into the bladder. The bladder is emptied and the urinary pH is measured. The bladder is then washed with 200 ml of medium. After emptying, 50 ml of the medium is left in the bladder for collecting retrograde-ejaculated sperm. The patient is put into lateral decubitus. In spinal cord-injured men, an assistant should control leg spasm during the procedure. Electrostimulation is performed using equipment with a built-in temperature sensor. After digital rectal examination and anoscopy, a standard probe is gently inserted into the rectum. Care is taken to orientate the electrodes anteriorly. Electrostimulations are repeated, each stimulation lasting for 2–4 seconds. Baseline voltage should be 5 V and voltage can be increased or maintained according to the patient’s reaction. In the case of acute hypertension in patients with spinal cord lesions at T6 or higher, the procedure must be discontinued until blood pressure is again under control. An assistant collects the antegrade fraction in a sterile container containing buffered sperm-washing medium. The pendulous and bulbar urethra are continuously massaged by the assistant during the procedure. With the aid of a 1 ml syringe, ejaculated drops are flushed into the container. When no antegrade ejaculation is observed, indirect signs such as spasms

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of the lower abdominal muscles and legs and the appearance of goose bumps may indicate (retrograde) ejaculation. When ejaculation discontinues, the probe is removed and anoscopy is performed again to check for rectal lesions. Then the patient is put again in the lithotomy position. The bladder is recatheterized and emptied into a sterile container in order to collect any retrograde fraction. The bladder is flushed with 100 ml of medium until the flushing medium remains clear. The collected fractions are transported to the andrology laboratory for identification of spermatozoa and further preparation. Centrifugation of the retrograde suspension may be necessary or open biopsy under local anesthetic should be performed.

Dressing after Disposable underpants.

Patient care postoperation None.

Requirements A runner Two assistants Seager Model 14 Electroejaculator (Dalzell Medical System, The Plains, VA, USA) Anoscope Manual manometer Nelaton catheter ch 14 (Cat. No. 110) pH indicator strip (Merck, Germany) Mineral oil (Sigma) Cleansing solution (3.5% HAC) Syringe, 50 cc (BS-50 ES Terumo) Syringe, Norm-Ject Cook 1 ml (K-ATS-1000) 100 ml modified Earle’s balanced salt solution with HEPES, 0.4 Heparin Novo, and 2.25% human serum albumin Gauze squares 10 × 10.

Protocol 2: penile vibratory stimulation Indication Anejaculation.

Patient preparation As for EEJ.

The PVS procedure The patient empties his bladder before PVS and the urinary pH is measured. PVS is performed using highamplitude equipment.

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The antegrade fraction is collected into a sterile container containing buffered sperm-washing medium. When no ejaculation occurs after 5 minutes, PVS is discontinued. Then the patient is put again in the lithotomy position. When no antegrade ejaculation is observed, but indirect signs are present (goose bumps, muscular spasms), the bladder is catheterized and emptied into a sterile container in order to collect any retrograde fraction (see above). The collected specimens are transported to the andrology laboratory for identification of spermatozoa and further preparation. When PVS fails to induce ejaculation, EEJ has to be performed.

Patient care postoperation None.

Requirements Ferticare Personal vibrostimulator (Multicept ApS, Denmark) Manual sphygmomanometer pH indicator strip (Merck, Germany) Cleansing solution (3.5% HAC) Syringe, Norm-Ject Cook 1 ml (K-ATS-1000) 50 ml modified Earle’s balanced salt solution with HEPES, 0.4 Heparin Novo, and 2.25% human serum albumin.

Protocol 3: percutaneous epididymal sperm aspiration (PESA)

anesthetic – 1–2 ml of 2% lidocaine (without epinephine) – is injected in the spermatic cord in order to obtain locoregional anesthesia and into the scrotal skin.

The PESA procedure A 19 or 21 G needle is used. Attached is a 10 ml syringe. The epididymis is held firmly between two fingers of one hand and the needle is inserted with the other hand perpendicular to the epididymis. The needle is inserted into the epididymal mass and then gently withdrawn under slight suction. Care is taken not to move the needle in order to minimize contamination with blood and prevent epididymal damage. The embryologist/nurse brings an 1.5 ml Eppendorf micro test tube filled with culture medium. The needle is placed in the micro test tube and rinsed several times with the medium. The micro test tube is then passed to the embryologist for identification of spermatozoa. Centrifugation of the suspension may be necessary. The procedure can be repeated if not enough sperm are retrieved. However, if after two aspirations there is no success, then an aspiration of the testis or open biopsy under local anesthetic should be performed.

Dressing after Gauze squares and disposable underpants.

Patient care postoperation The man is told that there may be some pain, but it should be minimal. Autaminophen can be taken. If more is required, then he should contact the clinic.

Indication All cases of obstructive azoospermia with normal spermatogenesis, e.g. congenital absence of the vas deferens, failed vasectomy reversal (CBAVD patients: read caveat in MESA section – Protocol 7).

Patient preparation The man is given hibitane soap, to wash the area the night before and the morning of the operation. He is also asked to shave the area. Mepevidine 1 mg/kg i.m. and midazolam 2.5 mg i.m. may be given. The man has to empty the bladder before surgery. The man is fully draped, with the operation site obscured to him. He wears a top only. The operation site cleansed with antiseptic solution (e.g. HAC, Zeneca: hospital antiseptic concentrate, contains chlorhexidine). The penis is held up out of the way with a swab fixed underneath the drape. A drape with a small hole of 5 cm in diameter in the middle covers the operation site. The testes are gently pulled through, to be in the field of the procedure. Local

Requirements A runner Drape with central hole Cleansing solution – not Betadine (povidoneiodine) Syringe 10 cc (BS-10 ES Terumo) Micro test tube 1.5 ml (Eppendorf 3810) (to be washed and sterilized first) with medium modified Earle’s balanced salt solution + HEPES + 0.4 Heparin Novo + 2.25% human serum albumin Gauze squares 10 × 10 (35813 Hartmann).

Protocol 4: fine-needle aspiration (FNA) of testis for sperm retrieval Indication All cases of obstructive azoospermia (OA) with normal spermatogenesis, e.g. congenital absence of the vas deferens, failed vasectomy reversal (CBAVD patients: read caveat in MESA section – Protocol 7).

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Patient preparation

Patient care post operation

As for PESA (see Protocol 3).

As for PESA (see Protocol 3).

The FNA procedure

Requirements

A 21G 3/4 inch butterfly needle is used. Attached is a 20 ml syringe. A small amount of culture medium is drawn up into the tubing and the majority expelled until only about 1–2 mm is left in the butterfly tubing. There may be no air in the fluid. The butterfly needle is inserted perpendicular to the testis, and a little away from the site of insertion of the needle used to inject the local anesthetic, as there is usually some blood at that site. The testis is held firmly in one hand, and the butterfly needle is inserted with the other. Care is taken not to move the butterfly needle in order to minimize contamination with blood and prevent testicular damage. The patient may feel some pain only when the needle enters the tunica. The operator or assistant now ‘pumps’ 5–10 times on the 20 ml syringe in order to generate suction to aspirate sperm. It is important to keep a slight negative pressure in order to make sure the aspirate is not pushed back into the testis. This is done by ensuring the plunger does not return all the way to the end. The butterfly needle tubing is then occluded near the needle and the butterfly subsequently removed with a smooth sharp movement in order to minimize tissue trauma and contamination with blood. Occluding the tubing prevents aspirating blood from the skin surface. With the tubing still occluded, the 20 ml syringe (must have rubber stop which may never be in contact with the medium) is removed, and a 1 ml syringe with the plunger partially withdrawn is attached. Otherwise, the 20 ml syringe may be used. The embryologist/nurse brings a dish containing 9 droplets of culture medium (1 central droplet surrounded by 8 droplets). The butterfly needle is placed in a droplet of culture medium, and the butterfly needle tubing released, thereby removing the negative pressure. A small amount of the aspirate and the culture medium in the butterfly needle is then injected into each droplet in turn: usually about 3–5 droplets will be used in this way. Fractionating the aspirate containing red blood cells will improve subsequent visualization under the microscope. The dish is then passed to the embryologist for identification of spermatozoa. The procedure can be repeated if not enough sperm are retrieved initially. However, if after three aspirations there is no success, an open biopsy under local anesthetic should be performed.

Dressing after As for PESA (see Protocol 3).

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A runner Drape with central hole Cleansing solution – not Betadine (povidoneiodine) Syringe 20 cc (BS-20 ES Terumo) Surflo Winged Infusionset CE 0197 21 G × 3/4 inch (SV-21BL Terumo) flushed with medium (modified Earle’s balanced salt solution + HEPES + 0.4 Heparin Novo + 2.25% human serum albumin) Syringe 1 cc (Air-Tite K-ATS-1000 Cook) Gauze squares 10 × 10 (35813 Hartmann) To transport sperm: tissue culture dishes (3200 Falcon Becton Dickinson) with droplets of medium (modified Earle’s balanced salt solution + HEPES + 0.4 Heparin Novo + 2.25% human serum albumin).

Protocol 5: open testicular biopsy under local anesthesia Indication Patients with obstructive azoospermia with normal spermatogenesis who wish to have testicular sperm cryopreserved. (CBAVD patients: read caveat in MESA section – see Protocol 7.)

Patient preparation As for PESA (see Protocol 3).

Procedure Approximately 5 ml of lidocaine (2%) is injected into the skin and the underlying layers up to the tunica albuginea. The testis is fixed in the left hand and a 1–2 cm incision is then made into the scrotum and down through the tissue made edematous by the lidocaine to the tunica. The testis must remain fixed in order not to lose the alignment of the scrotal incision with the incision into the tunica. With the sharp point of the blade, the tunica is opened and the incision slightly extended. Under gentle pressure with the left hand, testicular tissue will protrude through the incision. Using a curved pair of Mayo scissors, a small sample is excised and placed into a Petri dish filled with sperm preparation medium, e.g. Earle’s. Selective hemostasis with diathermy is performed since intratesticular bleeding may cause discomfort and fibrosis. The testicular tissue is rinsed in the medium and then placed into another Petri dish filled with medium. After hemostasis, the tunica is closed with

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3.0 Vicryl. The skin is closed with interrupted 3.0 Vicryl sutures. A clean gauze swab covers the suture site and disposable underpants are given for support.

Patient care postoperation As for PESA (see Protocol 3). The patient is told that the sutures will dissolve. There is increased risk of hematoma. The patient should report undue bruising or pain which is not alleviated with acetaminophen.

identify the best testis to explore. This is done by reading any previous histology reports and feeling the testis for size and consistency. If the testis is high or retracted, the chance of retrieving spermatozoa is lower.

Patient preparation As for PESA (see Protocol 3).

Procedure Requirements An assistant and a runner Monopolar pencil with needle and cord (E 2502 Valleylab) Tubeholder (1X) (708130 Mölnlycke) to fix cords on drape (pencilcord off foot end) Needleholder Mayo-Hegar (20-642-16 Martin) Straight Mayo scissors (11 180 15 Martin) Adlerkreutz pincet (12-366-15 Martin) Allis forceps (30-134-15 Martin) Kryle forceps (13-341-14 Martin) Micro Adson pincet (2×) (12-404-12 Martin) Micro Adson pincet (2×) (12-406-12 Martin) Adson pincet (31-09770 Leibinger) Adson pincet (31-09772 Leibinger) Metzenbaum scissors (11-264-15 Martin) Metzenbaum scissors (11-939-14 Martin) Knifehandle with blades No. 15 (0505 SwannMorton) Swabs 10 × 10 (35813 Hartmann) Vicryl 3/0 (JV 497 Ethicon Johnson/Johnson) Tissue culture dishes 2× (3102 Falcon Becton Dickinson) with medium (modified Earle’s balanced salt solution + HEPES + 0.4 Heparin Novo + 2.25% human serum albumin) Local anesthesia: Syringe 20 cc CE 0197 (BS-20 ES Terumo) Needle 18 G (NN 1838 S Terumo) Needle 26 G (NN 2613 R Terumo) Xylocaine (lidocaine) 2% (Astra Pharmaceuticals).

Protocol 6: testicular biopsy under general anesthesia

Biopsies taken at random As for under local anesthetic (see Protocol 4). The main difference is that a larger scrotal incision is made, and the testis is delivered. If no sperm are observed in the wet preparation, multiple small incisions can be made and biopsies taken accordingly. The incisions must avoid the arterial blood supply. The contralateral testis may be explored as well.

Biopsies taken with operating microscope (microTESE) After scrototomy, the tunica albuginea is opened longitudinally with the sharp point of the blade avoiding the arterial blood supply. Then the testicular pulpa containing the tubuli seminiferi is exposed to a 40–80× magnification using an operating microscope. Care is taken to keep the tubuli wet by a constant drip of saline. Distended tubules are spotted and sampled by micro-scissors, avoiding the arterial blood supply. The tiny samples are placed into a Petri dish filled with sperm preparation medium, e.g. Earle’s. The testicular samples are rinsed in the medium and then placed into another Petri dish filled with medium. After controlling hemostasis, the tunica is closed with a continuous 7.0 ethilon suture. The skin is closed with interrupted 3.0 Vicryl sutures. A clean gauze swab covers the suture site and disposable underpants are given for support.

Patient care postoperation See open biopsy under local anesthesia (Protocol 5) and MESA (Protocol 7).

Indication All cases of nonobstructive azoospermia (NOA; primary testicular failure). When testicular biopsy is performed in such patients, a preliminary screening for deletions of the Yq region of the Y chromosome is preferable in the male partner, since deletions may be found in about 5–10% of patients with unexplained primary testicular failure. Before undertaking the procedure it is important to

Protocol 7: microsurgical epididymal sperm aspiration (MESA) Indication Patients with obstructive azoospermia (OA) with normal spermatogenesis who wish to have epididymal sperm cryopreserved. The main drawback of

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MESA is that it is an invasive and expensive procedure that requires a basic knowledge of epididymal anatomy and of microsurgical techniques. However, the major benefit of this procedure is its diagnostic power: a full scrotal exploration can be performed and whenever indicated a vasoepididymostomy may be performed concomitantly. Furthermore, the number of spermatozoa retrieved is high, which facilitates cryopreservation.

Caveat When MESA is performed in CBAVD patients, a preliminary screening for mutations of the cystic fibrosis (CF) gene is mandatory in both the male CBAVD patient and his partner, since mutations are found in 60–70% of CBAVD patients without congenital renal malformations. If the female partner is found to be a carrier of a CF gene mutation, preimplantation genetic diagnosis (PGD) should be proposed. Even where only the man is a carrier of a CF mutation, the couple has to be informed of the risk of having a boy with a genital CF phenotype – with CBAVD.

Patient preparation As for PESA (see Protocol 3).

MESA procedure MESA can be performed during any scrotal exploration, taking place even long before the ICSI treatment is scheduled or in a satellite center, e.g. by a surgeon not involved in assisted reproduction. Using an operating microscope, the epididymis is carefully dissected and, after hemostasis, using bipolar coagulation, a distended epididymal tubule is longitudinally opened by micro-scissors through a small opening in the serosa. The proximal corporal or distal head region of the epididymis is opened first. The epididymal fluid is aspirated by means of a disposable tip from an intravenous cannula mounted on a 1 ml syringe filled with 0.1 ml HEPES-buffered Earle’s medium supplemented with 0.4% human serum albumin. The aspirated epididymal fluid is then transferred into a Falcon test tube, filled with 0.9 ml of this Earle’s medium. When motile spermatozoa, as assessed by peroperative microscopic examination of the aspirates, are recovered, no further epididymal incision is made and a maximum of fluid is aspirated. If microscopic assessment does not show any motile sperm cells, a more proximal incision is made until motile sperm cells are found. In some instances centrifugation (1800× g, 5 minutes) of the epididymal aspirates is needed in order to observe spermatozoa under the microscope. In cases where no motile spermatozoa are recovered, a testicular biopsy is taken for sperm recovery (see below). The sperm suspension is further prepared and kept in the incubator until the moment of intracytoplasmic injection or cryopreservation.

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Patient care postoperation Same as for TESE under general anesthesia (see Protocol 6).

Requirements An assistant and a runner Needleholder Mayo-Hegar (20-642-16 Martin) Straight Mayo scissors (11 180 15 Martin) Monopolar pencil and cord (E 2502 Valleylab) Bipolar pincet and cord (4055 Valleylab) Tubeholders (2×) (708130 Mölnlycke) to fix cords on drape (bipolar cord off head end, pencilcord off foot end) Micro-scissors (OP 5503 V-Mueller) Micro-needleholder (GU 8170 V-Mueller) Jeweller’s forceps (3×) (E 1947 Storz) (72 BD 330 Aesculaep) Curved blunt scissors (11 939 14 Martin) 1 cc syringe (4×) (Air-tite K-ATS-1000 Cook) with or 22 ga medicut (8888 100 107 Argyle) or Cook aspiration CT (K Sal 400 300 Cook) Micro Adson pincet with teeth (2×) (12-406-12 Martin) Knife handle with blades, No. 15 (0505 SwannMorton) Knife handle with blades, No. 11 (0503 SwannMorton) NaCl 0.9% 500 ml (B1323 Baxter) with 2500 U.I. Heparin Novo (Heparine Novo Nordisk Pharma) Syringes 20 cc (2×) (SS 20 ES Terumo) with 22 ga Medicut tip (8888 100107 Argyle) Swabs 10 × 10 (35813 Hartmann) Tip cleaner (Surgikos 4315 Johnson-Johnson) Micro sponges (NDC 8065-1000-02 Alcon) Sutures: Ethilon 9/0 (W 1769 Ethicon) Vicryl 3/0 (JV 497 Ethicon) Microscope: surgical operating and diagnostic microscope, Wild M 691 with 180° positioning for doctor and assistant and optical eyepiece opposite each other (M 691 Leica ). Achromatic lens f = 200 mm (M 382162 Leica).

References 1. Palermo G, Joris K, Devroey P et al. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992; 340: 17–18. 2. Van Steirteghem AC, Nagy Z, Joris H, et al. Higher fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1993; 8: 1061–6. 3. Craft I, Benett V, Nicholson N. Fertilising ability of testicular spermatozoa. Lancet 1993; 342: 864. 4. Schoysman R, Vanderzwalmen P, Nijs M, et al. Pregnancy after fertilisation with human testicular spermatozoa. Lancet 1993; 342: 1237.

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5. Devroey P, Liu J, Nagy Z, et al. Pregnancies alter testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum Reprod 1995; 10: 1457–60. 6. Tournaye H, Camus M, Goosens A, et al. Recent concepts in the management of infertility because of non-obstructive azoospermia. Hum Reprod 1995; 10(Suppl 1): 115–19. 7. Jarow JP, Espeland MA, Lipshultz LI. Evaluation of the azoospermic patients. J Urol 1989; 142: 62. 8. Matsumiya K, Namiki M, Takahara S, et al. Clinical study of azoospermia. Int J Androl 1994; 17: 140–2. 9. Burgues S, Calderon MD. Subcutaneous self-administration of highly purified follicle stimulating hormone and human chorionic gonadotrophin for the treatment of male hypogonadotrophic hypogonadism. Spanish Collaborative Group on male hypogonatrophic hypogonadism. Hum Reprod 1997; 12: 980–6. 10. Skakkebaek NE. Testicular dysgenesis syndrome. Horm Res 2003; 60(Suppl 3): 49. 11. Tournaye H, Camus M, Vandervorst M, et al. Surgical sperm retrieval for intracytoplasmic sperm injection. Int J Androl 1997; 20(Suppl 3): 69–73. 12. Tournaye H. Gamete source and manipulation. In: Vayena E, Rowe PJ, David P, Griffin PD, eds. Current Practices and Controversies in Assisted Reproduction. Oxford: Oxford University Press, 2002. 13. Tournaye H, Joris H, Verheyen G, et al. Sperm parameters, globozoospermia, necrozoospermia and ICSI outcome. In: Filicori M, ed. Treatment of Infertility: the New Frontiers. New York: Communications Media for Education, 1998: 259–68. 14. Ron-El R, Strassburger D, Friedler S et al. Extended sperm preparation: an alternative to testicular sperm extraction in non-obstructive azoospermia. Hum Reprod (1997); 12: 1222–6. 15. Kafetsoulis A, Brackett N, Ibrahim E, Attia G, Lynne C. Current trends in the treatment of infertility in men with spinal cord injury. Fertil Steril 2006; 86: 781–9. 16. Hirsh A, Mills C, Tan SL, et al. Pregnancy using spermatozoa aspirated from the vas deferens in a patient with ejaculatory failure due to spinal injury. Hum Reprod 1993; 8: 89–90. 17. Hovatta O, Reima I, Foudila T, et al. Vas deferens aspiration and intracytoplasmic sperm injection of frozen–thawed spermatozoa in a case of anejaculation in a diabetic man. Hum Reprod 1996; 11: 334–5. 18. Tur-Kaspa I, Segal S, Moffa F, Massobrio M, Meltzer S. Viagra for temporary erectile dysfunction during treatments with assisted reproductive technologies. Hum Reprod 1999; 14: 1783–4. 19. Engin-Ustun Y, Korkmaz C, Duru NK, Baser I. Comparison of three sperm retrieval techniques in spinal cord-injured men: pregnancy outcome. Gynecol Endocrin 2006; 22: 252–5. 20. Kamischke A, Nieschlag E. Update on medical treatment of ejaculatory disorders. Int J Androl 2002; 25: 333–44. 21. Brackett N. Semen retrieval by penile vibratory stimulation in men with spinal cord injury. Hum Reprod Update 1999; 5: 216–22.

22. Halstead LS, Vervoort S, Seager S. Rectal probe electrostimulation in the treatment of anejaculatory spinal cord injured men. Paraplegia 1987; 25: 120–9. 23. Seager SW, Halstead LS. Fertility options and success after spinal cord injury. Urol Clin North Am 1993; 20: 543–8. 24. Hultling C, Rosenlund B, Levi R, et al. Assisted ejaculation and in-vitro fertilization in the treatment of infertile spinal cord-injured men: the role of intracytoplasmic sperm injection. Hum Reprod 1997; 12: 499–502. 25. Tournaye H. Surgical sperm recovery for intracytoplasmic sperm injection: which method is to be preferred? Hum Reprod 1999; 14(Suppl 2): 71–81. 26. Tournaye H, Merdad T, Silber S. No differences in outcome after intracytoplasmic sperm injection with fresh or with frozen–thawed epididymal sperm. Hum Reprod 1999; 14: 101–6. 27. Girardi SK, Schlegel P. MESA: review of techniques, preoperative considerations and results. J Androl 1996; 17: 5–9. 28. Gorgy A, Meniru GI, Bates S, Craft IL. Percutaneous epididymal sperm aspiration and testicular sperm aspiration for intracytoplasmic sperm injection under local anesthesia. Assisted Reprod Rev 1998; 8: 79–93. 29. Tournaye H, Clasen K, Aytoz A, et al. Fine needle aspiration versus open biopsy for testicular sperm recovery: a controlled study in azoospermic patients with normal spermatogenesis. Hum Reprod 1998; 13: 901–4. 30. Morey AF, Deshon GE Jr, Rozanski TA, Dresner ML. Technique of biopsy gun testis needle biopsy. Urology 1993; 42: 325–6. 31. Silber S, Nagy Z, Devroey P, Tournaye H, Van Steirteghem AC. Distribution of spermatogenesis in the testicles of azoospermic men: the presence or absence of spermatids in the testes of men with germinal failure. Hum Reprod 1997; 12: 2422–8. 32. Roosen-Runge EC. Quantitative investigations on human testicular biopsies. I. Normal testis. Fertil Steril 1956; 7: 251–61. 33. Steinberg E, Tjioe DY. A method for quantitative analysis of human seminiferous epithelium. Fertil Steril 1968; 19: 960–7. 34. Gil-Salom M, Minguez Y, Rubio C, et al. Efficacy of intracytoplasmic sperm injection using testicular spermatozoa. Hum Reprod 1995; 10: 3166–70. 35. Tournaye H, Liu J, Nagy Z, et al. Correlation between testicular histology and outcome after intracytoplasmic sperm injection using testicular sperm. Hum Reprod 1996; 11: 127–32. 36. Hauser R, Botchan A, Amit A, et al. Multiple testicular sampling in non-obstructive azoospermia – is it necessary? Hum Reprod 1998; 13: 3081–5. 37. Witt MA, Richard JR, Smith SE, Rhee EH, Tucker MJ. The benefit of additional biopsy sites when performing testicular sperm extraction in non-obstructive azoospermia. Fertil Steril 1997; 67: S79–80. 38. Tournaye H, Camus M, Goossens A, et al. Recent concepts in the management of infertility because of non-obstructive azoospermia. Hum Reprod 1995; (Suppl 1): 115–19. 39. Ezeh UIO, Moore HDM, Cooke ID. Correlation of testicular sperm extraction with morphological, biophysical and endocrine profiles in men with

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azoospermia due to primary gonadal failure. Hum Reprod 1998; 13: 3066–74. Schlegel P, Su LM. Physiological consequences of testicular sperm extraction. Hum Reprod 1997; 12: 1688–92. Ron-El R, Strauss S, Friedler S, et al. Serial sonography and colour flow Doppler imaging following testicular and epididymal sperm extraction. Hum Reprod 1998; 13: 3390–3. Schlegel PN, Li PS. Microdissection TESE: sperm retrieval in non-obstructive azoospermia. Hum Reprod Update 1998; 4: 439. Herwig R, Tosun K, Pinggera GM, et al. Tissue perfusion essential for spermatogenesis and outcome of testicular sperm extraction (TESE) for assisted reproduction. J Assist Reprod Genet 2004; 21: 175–80. Nagy P, Verheyen G, Tournaye H, Devroey P, Van Steirteghem A. An improved treatment procedure for testicular biopsy offers more efficient sperm recovery: case series. Fertil Steril 1997; 68: 376–9. Crabbé E, Verheyen G, Tournaye H, Van Steirteghem A. The use of enzymatic procedures to recover testicular sperm. Hum Reprod 1997; 12: 1682–7. Angelopoulos T, Adler A, Krey L, et al. Enhancement or initiation of testicular sperm motility by in vitro culture of testicular tissue. Fertil Steril 1999; 71: 240–3. Tasdemir I, Tasdemir M, Tavukçuogˇ lu S. Effect of pentoxifylline on immotile testicular spermatozoa. J Assisted Reprod Genet 1998; 15: 90–2. Tunc L, Kirac M, Gurocak S, et al. Can serum inhibin B and FSH levels, testicular histology and volume predict the outcome of testicular sperm extraction in patients with non-obstructive azoospermia? Int Urol Nephrol 2006; 38: 629–35. Tournaye H, Verheyen G, Nagy P, et al. Are there any predictive factors for successful testicular sperm recovery? Hum Reprod 1997; 12: 80–6. Ballesca JL, Balasch J, Calafell JM, et al. Serum inhibin B determination is predictive of successful testicular sperm extraction in men with non-obstructive azoospermia. Hum Reprod 2000; 15: 1734–8. Vernaeve V, Tournaye H, Schiettecatte J, et al. Serum inhibin B cannot predict testicular sperm retrieval in patients with non-obstructive azoospermia. Hum Reprod 2002; 17: 971–6. Brandell RA, Mielnik A, Liotta D, et al. AZFb deletions predict the absence of spermatozoa with testicular sperm extraction: preliminary report of a prognostic genetic test. Hum Reprod 1998; 13: 2812– 15. Tournaye H, Staessen C, Liebaers I, et al. Testicular sperm recovery in nine 47,XXY Klinefelter patients. Hum Reprod 1996; 11: 1644–9. Tournaye H, Camus M, Vandervorst M, et al. Sperm retrieval for ICSI. Int J Andrology 1997; 20(Suppl 3): 69–73. Palermo GD, Schlegel PN, Sils ES. Births after intracytoplasmic sperm injection of sperm obtained by testicular sperm extraction from men with nonmosaic Klinefelter’s syndrome. N Engl J Med 1998; 338: 588–90. Ron-El R, Friedler S, Strassburger D, et al. Birth of a healthy neonate following the intracytoplasmic injection of testicular spermatozoa from a patient

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with Klinefelter’s syndrome. Hum Reprod 1999; 14: 368–70. Okada H, Goda K, Yamamoto Y, et al. Age as a limiting factor for successful sperm retrieval in patients with nonmosaic Klinefelter’s syndrome. Fertil Steril 2005; 84: 1662–4. Staessen C, Coonen E, Van Assche E, et al. Preimplantation diagnosis for X and Y normality in embryos from three Klinefelter patients. Hum Reprod 1996; 11: 1650–3. Guttenbach M, Michelmann HW, Hinney B, Engel W, Schmid M. Segregation of sex chromosomes into sperm nuclei in a man with 47,XXY Klinefelter’s karyotype: a FISH analysis. Hum Genet 1997; 99: 474–7. Staessen C, Tournaye H, Van Assche E, et al. Preimplantation diagnosis in 47,XXY Klinefelter patients. Hum Reprod Update in press. Baniel J, Sella A. Sperm extraction at orchiectomy for testis cancer. Fertil Steril 2001; 75: 260–2. Rosenlund B, Sjoblom P, Tornblom M, et al. In-vitro fertilization and intracytoplasmic sperm injection in the treatment of infertility after testicular cancer. Hum Reprod 1998; 13: 414–18. Schrader M, Muller M, Straub B, et al. Testicular sperm extraction in azoospermic patients with gonadal germ cell tumors prior to chemotherapy – a new therapy option. Urology 2003; 61: 421–5. Tournaye H. Storing reproduction for oncological patients. Mol Cell Endocrinol 2000; 27: 133–6. Chan PT, Palermo GD, Veeck LL, Rosenwaks Z, Schlegel PN. Testicular sperm extraction combined with intracytoplasmic sperm injection in the treatment of men with persistent azoospermia postchemotherapy. Cancer 2001; 15: 1632–7. Damani MN, Masters V, Meng MV, et al. Postchemotherapy ejaculatory azoospermia: fatherhood with sperm from testis tissue with intracytoplasmic sperm injection. J Clin Oncol 2002; 15: 930–6. Hauser R, Yogev L, Paz G, et al. Comparison of efficacy of two techniques for testicular sperm retrieval in nonobstructive azoospermia: multifocal testicular sperm extraction versus multifocal testicular sperm aspiration. J Androl 2006; 27: 28–33. Rosenlund B, Kvist U, Ploen L, et al. A comparison between open and percutaneous needle biopsies in men with azoospermia. Hum Reprod 1998; 13: 1266–71. Friedler S, Raziel A, Strassburger D, et al. Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia. Hum Reprod 1997; 12: 1488–93. Ezeh UIO, Moore HDM, Cooke ID. A prospective study of multiple needle biopsies versus a single open biopsy for testicular sperm extraction in men with non-obstructive azoospermia. Hum Reprod 1998; 13: 3075–80. Novero V, Goossens A, Tournaye H, et al. Seminoma discovered in two males undergoing successful testicular sperm extraction for intracytoplasmic sperm injection. Fertil Steril 1996; 65: 1015–54. Turek P, Givens CR, Schriock ED, et al. Testis sperm extraction and intracytoplasmic sperm injection guided by prior fine-needle aspiration mapping in

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Textbook of Assisted Reproductive Technologies patients with nonobstructive azoospermia. Fertil Steril 1999; 71: 552–7. Meng MV, Cha I, Ljung B, Turek P. Relationship between classic histological pattern and sperm findings on fine needle aspiration map in infertile men. Hum Reprod 2000; 15: 1973–7. Foresta C, Garolla A, Betella A, et al. Doppler ultrasound of the testis in azoospermic subjects as a parameter of testicular function. Hum Reprod 1998; 13: 3090–3. Belenky A, Avrech OM, Bachar GN, et al. Ultrasoundguided testicular sperm aspiration in azoospermic patients: a new sperm retrieval method for intracytoplasmic sperm injection. J Clin Ultrasound 2001; 29: 39–43. Friedler S, Raziel A, Schachter M, et al. Outcome of first and repeated testicular sperm extraction in patients with non-obstructive azoospermia. Hum Reprod 2002; 17: 2356–61. Kamal A, Fahmy I, Mansour R, et al. Outcome of repeated testicular sperm extraction and ICSI in patients with non-obstructive azoospermia. MEFSJ 2004; 9: 42–6. Vernaeve V, Verheyen G, Goosens A, et al. How successful is repeat testicular sperm extraction in patients with azoospermia? Hum Reprod 2006; 21: 1551–4. Nagy Z, Liu J, Janssenwillen C, et al. Comparison of fertilization, embryo development and pregnancy rates after intracytoplasmic sperm injection using ejaculated, fresh and frozen–thawed epididymal and testicular spermatozoa. Fertil Steril 1995; 63: 808–15. Nicopoullos J, Gilling-Smith C, Almeida P, et al. Use of surgical sperm retrieval in azoospermic men: a meta-analysis. Fertil Steril 2004; 82: 691–700. Vernaeve V, Tournaye H, Osmanagaoglu K, et al. Intracytoplasmic sperm injection with testicular spermatozoa is less successful in men with nonobstructive azoospermia than in men with obstructive azoospermia. Fertil Steril 2003; 79: 529–33.

82. Levron J, Aviram-Goldring A, Madgar I, et al. Sperm chromosome abnormalities in men with severe male factor infertility who are undergoing in-vitro fertilization with intracytoplasmic sperm injection. Fertil Steril 2001; 76: 479–84. 83. Schlegel PN, Palermo GD, Goldstein M, et al. Testicular sperm extraction with intracytoplasmic sperm injection for non-obstructive azoospermia. Urology 1997; 49: 435–40. 84. Palermo GD, Schlegel PN, Hariprashad JJ, et al. Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men. Hum Reprod 1999; 14: 741–8. 85. Mercan R, Urman B, Alatas C, et al. Outcome of testicular sperm retrieval procedures in non-obstructive azoospermia: percutaneous aspiration versus open biopsy. Hum Reprod 2000; 15: 1548–51. 86. Nassar ZA, Lakkis D, Sasy M, et al. Fine needle testicular sperm aspiration: an alternative to open testicular biopsy in patients with non-obstructive azoospermia. Fertil Steril 2001; 76: S137. 87. Platteau P, Staessen C, Michiels A, et al. Comparison of the aneuploidy frequency in embryos derived from testicular sperm extraction in obstructive and non-obstructive azoospermic men. Hum Reprod 2004; 19: 1570–4. 88. Donoso P, Platteau P, Papanikolaou EG, et al. Does PGD for aneuploidy screening change the selection of embryos derived from testicular sperm extraction in obstructive and non-obstructive azoospermic men? Hum Reprod 2006; 21: 2390–5. 89. Levron J, Aviram-Goldring A, Madgar I, et al. Sperm chromosome abnormalities in men with severe male factor infertility who are undergoing in-vitro fertilization with intracytoplasmic sperm injection. Fertil Steril 2001; 76: 479–84. 90. Vernaeve V, Bonduelle M, Tournaye H, et al. Pregnancy outcome and neonatal data on children born after ICSI with testicular sperm in obstructive and non-obstructive azoospermia. Hum Reprod 2003; 18: 2093–7.

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48 Gamete intrafallopian transfer (GIFT) and zygote intrafallopian transfer (ZIFT) Machelle M Seibel, Ariel Weissman

Gamete intrafallopian transfer Gamete intrafallopian transfer (GIFT) has emerged as one of the major assisted reproductive technologies (ART). It began in 1979 with a case report in which clomiphene citrate was given to a woman on cycle days 5–9 and artificial insemination performed on cycle day 12.1 A laparotomy was performed the following morning to reanastomose the ligated fallopian tubes. Six follicles were aspirated and the follicular fluid divided equally and transferred into each reopened tube. A normal, single, term delivery followed. Similar successes were reported in primates the following year.2 The first successful transfer of both sperm and oocytes was reported in 1983 in six patients with a history of pelvic inflammatory disease (PID). After ovulation induction, laparotomy and microsurgical repair was scheduled just prior to ovulation. Capacitated sperm and oocytes were transferred into the repaired fallopian tube and two patients conceived. One miscarried in the fifth postoperative week and the second continued to term without complication.3 The first United States center specializing in low ovum transfer also opened in 1983 in a Catholic hospital that wished to overcome the ethical objections to in vitro fertilization (IVF). Couples were instructed to have intercourse 24–30 hours after preovulatory injection of human chorionic gonadotropin (hCG) and a laparoscopic oocyte retrieval and transfer into the fallopian tubes. Because of poor results, moral theologians at the Pope John XXIII Medical-Moral Research and Education Center allowed semen also to be transferred into the fallopian tubes, provided the semen was collected in a special perforated condom.4 The first reported transfer of gametes in patients with unexplained infertility was by Asch et al in 1984.5 To ensure that fertilization occurred within the body of the patient, the gametes were separated by an air space, and transferred laparoscopically into the fimbriated ends of normal fallopian tubes. These same investigators subsequently used minilaparotomy to assure accurate gamete transfer and placement. In the years that followed, GIFT became a major assisted reproductive technique.

In the new millennium, IVF is performed almost exclusively by ultrasound and GIFT is typically performed by laparoscopy. It is sometimes difficult to remember that in the early 1980s both procedures required laparoscopy and in the 1970s both typically required laparotomy. In addition, IVF success rates were quite low and few centers had the technology and personnel to perform it. GIFT was an ideological breakthrough that was both simpler and more successful than IVF. Today, IVF is routine, less invasive, and as successful as GIFT. The primary contemporary significance of GIFT is religious and personal. As in the past, it still requires less laboratory equipment and less complexity than IVF and therefore remains an important procedure for a specific category of patients.

Patient selection Patients who undergo GIFT should have completed a full infertility evaluation. The presence of at least one patent tube has to be confirmed by either hysterosalpingogram (HSG) or laparoscopy. If HSG is done at most 6 months prior to GIFT it might improve fertility prospects. This effect could be attributed to proper selection of the fallopian tube for cannulation during transfer or some therapeutic effect of HSG.6 It is always necessary to address the couple’s insurance coverage, including restrictions that could be affected by combining these techniques.7 Having the ability to provide IVF as a back up becomes extremely important if unexpected scarring or adhesions will compromise the GIFT procedure. Success rates for GIFT are typically not broken down by diagnosis. In general, success rates for GIFT are comparable to those achieved through IVF. The most common indications are unexplained infertility, stages I and II endometriosis, cervical factor infertility, and oligospermia.8 GIFT is also often recommended after three failed cycles of ovulation induction, and for individuals who object to IVF for religious or personal reasons. Patients must have at least one patent fallopian tube, although some authors emphasize a need for both tubes to be normal. Because the rate of ectopic pregnancy

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among patients undergoing GIFT is less than 1%,9 one must assume that tubal disease is more likely to result in failure than it is in tubal pregnancy. Nevertheless, one must emphasize to patients the potential for ectopic pregnancy prior to treatment. Patients must also understand that unlike IVF, unless pregnancy occurs, GIFT does not address the question of whether the woman’s oocytes are fertilizable by the man’s spermatozoa. This may be of particular importance in cases such as unexplained, immunologic, or male factor infertility. To minimize this concern, some suggest that GIFT be performed only in couples who have had prior proof of fertilization. However, there can be no absolute conclusions drawn as supernumerary oocytes in a GIFT cycle inseminated in vitro demonstrate no correlation between IVF and the likelihood of GIFT pregnancies.

Controlled ovarian hyperstimulation As with IVF, controlled ovarian hyperstimulation (COH) is routinely used to achieve follicle growth and produce multiple preovulatory oocytes for oocyte transfer. As with IVF, no one protocol has proven to be more effective than another, although long-term gonadotropin-releasing hormone (GnRH) agonist protocols appear to yield higher pregnancy rates than short regimens.10,11 There is virtually no information on using natural cycles for GIFT.

The procedure Sperm collection and preparation Because the sperm and eggs are transferred into the fallopian tubes at the time of the oocyte retrieval, the sperm must be collected 2–2.5 hours earlier to allow time for sperm preparation. Patients who wish to comply with the doctrines of the Roman Catholic Church or Jewish Halacha can collect sperm during intercourse using a special condom that is perforated so that neither contraception nor masturbation is used. This allows any ensuing pregnancy to result as an extension of the conjugal act. Following liquefaction for 15–30 minutes at 37°C, the sperm are washed, centrifuged, and separated. Many different sperm-washing media are used. Some of the more common ones that have been used are: 1. Ham’s F-10 with 7.5% patient serum containing penicillin G, 75 mg/l; streptomycin sulfate, 75 mg/l; calcium lactate, 252 mg/l; and sodium bicarbonate, 2.1 g/l at a pH of 7.35 and osmolarity of 280–285 mOsm – Sigma Chemical, St Louis, MO, USA 2. Earle’s medium – Sigma Chemical, St Louis, MO, USA 3. Modified human tubal fluid (MHTF) – Irvine Scientific, Santa Ana, CA, USA 4. HEPES-buffered media if not using a CO2 incubator or isolette – Irvine Scientific, Santa Ana, CA, USA.

Each of these methods appears comparable, although transmission electron microscopic examination suggests that Percoll gradient centrifugation yield sperm of better morphologic quality. After preparation, the sperm are incubated in 95% air and 5% CO2 until it is time to load the transfer catheter. It is preferable to perform a semen analysis at the time of the procedure.

Oocyte retrieval Oocyte retrieval is performed 36 hours after hCG administration. The most common method of oocyte retrieval is via laparoscopy using general anesthesia (Fig 48.1). However, transvaginal ultrasound-guided oocyte retrieval is used to a limited extent (Fig 48.2). The oocytes may either be transferred by laparoscopy or with ultrasound (Fig 48.3). Some centers perform office laparoscopy under local anesthesia with good patient acceptance. Milki and Tazuke12 were first to perform transvaginal ultrasound-guided retrieval followed by GIFT, using a 5 mm laparoscope and two 3 mm trocars with local anesthesia and intravenous sedation. There is a theoretical benefit to using ultrasound for the oocyte retrieval, as it limits the exposure of the oocytes to the CO2 necessary to achieve pneumoperitoneum. However, pregnancy data do not support combining ultrasound retrieval with laparoscopic transfer. In addition, it is much more difficult to set up for a vaginal case and then to reprep for an abdominal case. Therefore, ultrasound should be avoided unless visualization of follicles at the time of laparoscopy is expected to be poor. The laparoscopic oocyte retrieval requires standard operative laparoscopy equipment and technique. In addition, a special cannula can be ordered to allow passage of the retrieval needles. If these are not available, a standard IVF needle can be used with a 10 cc syringe, although it is difficult to irrigate the follicles with this approach. We have found it easiest to use a single midline puncture site, two fingers above the symphysis, for the retrieval and a slightly higher lateral incision for the transfer. Some programs utilize two separate lateral sights for cannulating the fallopian tubes. However, comparable results can be obtained with cannulating only one tube. Care should be taken to observe any potential pathology and to determine the optimal site for placement of the trochar cannula. In some situations where laparoscopy is not available, minilaparotomy can be used both for oocyte retrieval and gamete transfer. A 2–3 cm transverse incision is made at the level where the uterine fundus reaches the anterior abdominal wall as determined by pelvic exam. It is often possible to see both ovaries and insert the needle directly into each follicle. Sometimes it is necessary to grasp the utero-ovarian ligament to bring the ovaries into view, or to bring them back into view if they recede following aspiration of the larger follicles. Following retrieval, the oocytes are placed into standard culture media to assess maturity. Clear follicular fluid from the current case can be used instead

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Flushing medium

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To suction pump

Fig 48.1 Laparoscopic oocyte retrieval (inset shows needle entering follicle).

Flushing medium

To suction pump

Fig 48.2 Ultrasound-guided transvaginal oocyte retrieval (inset shows needle entering follicle).

of culture medium. No particular method has proven more effective than the other, although it has been suggested that outcomes might be higher when follicular fluid is used to capacitate sperm and as a transfer medium rather than Ham’s F-10.13 This finding was most pronounced for male-factor infertility patients in whom the pregnancy rates were 44% for follicular fluid compared with zero for the Ham’s F-10 group. One pregnancy occurred with a motile sperm concentration of 1.5 × 106/ml. These findings suggest that GIFT can be used in cases of severe oligospermia. Not all would agree with this philosophy. However, IVF with intracytoplasmic sperm injection (ICSI) is

recommended in preference to GIFT for sperm counts below 1.5 × 106/ml.

Transfer A number of transfer catheters are designed specifically for GIFT, including GIFT/ZIFT catheter (Fertility Technologies, CCD 1309000); Deseret Intracath (No. 3132, Deseret Co, Sandy, UT); 16-gauge 24-inch Deseret Intracath; Semar Catheter (Wisap, Munich, Germany); Teflon embryo transfer catheter (5F) with side open tip cut off; Stirrable GIFT catheter (7F), and oocyte catheter (3F); 16-gauge end-hole Teflon catheter (HT

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Fig 48.3

eggs

medium air

air

5 mm

5 mm 5 mm

Fig 48.4

sperm air

medium... air

5 mm 10 mm

GIFT via laparoscopy.

5 mm 10 mm

remaining length

GIFT. Gametes are separated by air or medium.

Barnaby, Baltimore, MD); and Cook Catheter (No. NRT 5.0-VT-50-P-NS-GIFT, Cook, Melbourne, Australia). Loading the transfer catheter can be one of the most important aspects of the procedure, especially if it is done for religious reasons. A representative illustration is shown in Fig 48.4. A 5 mm air bubble should separate the eggs and sperm so that fertilization cannot begin before transfer. However, at least one report suggests that results may be higher if the gametes are allowed to mix prior to transfer.14 If both tubes are cannulated, it is advisable to use different catheters for each. Typically, 10 000 to 100 000 motile sperm per egg are transferred. However, this number may be increased to 200 000 or more per egg in cases of oligospermia. Many programs will not transfer more than three oocytes per fallopian tube, although some will transfer six and occasionally eight. Several countries have specific legislative limits on the number that can be used with GIFT.15 In one report, a 52.7% pregnancy rate per cycle was obtained when three oocytes were transferred vs 30.7% when only two were transferred.16 Another study17 performed retrospectively on 399 cycles found a three times higher

clinical pregnancy rate when four or more oocytes were transferred. This may well have been due to the fact that women who yield fewer eggs are, by definition, poor responders. Interestingly, adding additional oocytes beyond the four did not further increase pregnancy rates. As with IVF, the greater the number of oocytes transferred, the greater the multiple pregnancy rates.18 For this reason, it makes sense to fertilize and freeze all oocytes beyond the fourth to reduce the likelihood of higher-order gestations. Cannulating the fallopian tubes can typically be performed without difficulty with adequate preparation. The tube should be grasped on the antimesenteric border of the fallopian tube to identify the tubal lumen. Choose an angle that aligns the tube with the cannula. The magnification afforded by laparoscopy greatly helps achieve this task. Often, the cumulus complex is large enough to be seen within the transfer catheter. Inserting the catheter a distance of 2–3 cm is usually sufficient. If a metal cannula is used, it can be inserted 1–2 cm and the transfer catheter advanced another 1–2 cm. Often one can see the ampulla swell slightly following the

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Fig 48.5

injection of the gametes. The same can be repeated on the contralateral fallopian tube if both are to be used. If only one tube is going to be used, it has been recommended to perform the transfer on the side that provided the most oocytes. Success rates of 41.6% have been reported when the ipsilateral side was cannulated compared with 22.8% when the contralateral side was cannulated (p = 0.042, odds ratio [OR] = 2.39).19 While it is intriguing to speculate why this might occur, it must be realized that other investigators have found that pregnancy occurred in 40.3% of cycles when both tubes were used compared with 21.6% when only one tube was used.18 In order to avoid laparoscopy and general anesthesia, some authors have suggested using hysteroscopy to transfer the gametes. Under conscious sedation, Possati et al20 treated 27 patients with transvaginal ultrasound-guided oocyte retrieval followed by flexible hysteroscopic gamete transfer using a 30° hysteroscope that had a 4 mm outer diameter sheath and CO2 as a distention medium. Atropine 0.5 cc intramuscularly was administered 30 minutes prior to the procedure. The catheter was advanced 2–4 cm into the tubal lumen and the CO2 distention stopped 1 minute prior to injecting the gametes. A pregnancy rate of 25.5% per cycle was reported. Another report from Italy used a falloposcopic approach to deliver the gametes in 25 patients. Two lengths of catheter were used and pregnancy rates were the same whether the catheter was advanced 3 cm or 6 cm. The pregnancy rate was 28%, the miscarriage rate was 28.6%, and the live birth rate was 20%. This approach may have appeal because of its less invasive nature.21 Others have reported transvaginal ultrasound-guided GIFT with variable results.22 Success rates of 20% have been reported, but the procedure is technically quite difficult and results typically are lower than they are for laparoscopic GIFT. For these reasons, it has not gained widespread use despite its inherent appeal.

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Gamete uterine transfer.

There are also several reports of placing gametes directly into the uterine cavity (gamete uterine transfer or GUT) following transvaginal oocyte retrieval (Fig 48.5). The procedure is similar to intrauterine insemination (IUI) and therefore requires limited technical experience or equipment. Success rates of 15% have been reported, but series sizes are small.7 However, because of the potentially substantial cost reduction and risk, larger studies are both needed and necessary.

Results When GIFT was first introduced, it was available in only a few centers. Today, most reproductive centers offer GIFT as a procedure. Analyses of the trends in GIFT reveal that the number of cycles performed annually are declining. The 1995 National Survey reported that 6% of all ART cycles performed in the United States were GIFT. This declined to 5% in the 1996 National Survey and 3.2% in the 1997 survey23 (Fig. 48.6). The decline is in large part due to the need for general anesthesia and laparoscopy for GIFT, coupled with the fact that success rates for IVF are roughly comparable to those for GIFT. Data for 2004, the most recent available, show that GIFT represented only 0.1% of the 99 639 assisted reproduction cycles performed in that year. Zygote intrafallopian transfer (ZIFT) constitutes an additional 0.3% of cases, and combination of IVF with or without ICSI and either GIFT or ZIFT accounts for another 0.1%. Overall live births per retrieval for different types of 94 242 ART procedures, using fresh nondonor eggs or embryos in the year 2004, are:24 IVF with ICSI IVF without ICSI GIFT ZIFT Combined IVF/GIFT/ZIFT

30.9% 32.9% 23.3% 30% 34.8%

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18 16 14

Percentage

12 10 8 6 4 2

∗From 1996 United States only. % ZIFT

%GIFT

Despite the fact that GIFT requires gametes to be placed directly into the fallopian tubes, ectopic pregnancy rates are only 0.3%.9 Multiple births are not broken down for GIFT, but twins occur in roughly one in three deliveries and triplets or more occur in roughly 5% of deliveries. As with other forms of assisted reproduction, women > 40 years old experience worse outcomes and higher cancellation rates. However, one group recommended GIFT rather than donor oocyte IVF for women aged 40–42 years old with good ovarian reserve because it was less expensive (mean cost per infant US$22 924 vs US$30 457 for donor oocyte IVF).25 This, of course, must be established on a case-by-case basis. The cause of infertility leading to GIFT also has an impact on outcome. Those with mild endometriosis and unexplained infertility experience the greatest success rates, reaching 66% following three cycles. Those with male factor, cervical factor, and more severe endometriosis can anticipate success rates closer to 15%. Additionally, the presence of serum Chlamydia trachomatis immunoglobulin G (IgG) antibodies is also associated with a significantly lower implantation rate and a tendency toward a higher early pregnancy loss.26 Other investigators have found an association between the type of light source used at laparoscopy and pregnancy rates. Women treated with a halogen light source had a pregnancy rate of 50% in 22 cycles compared with 9% in 12 cycles using a xenon light source.27 Although the numbers are small, the finding that xenon emitted more ultraviolet light than halogen suggests an intriguing reason to consider this point further. Choice of general anesthesia is probably not a major factor, with the exception that halothane is associated with a poorer prognosis.28 Patients may, however, fare better with epidural anesthesia vs general anesthesia (40.9% vs 31%),29

2003

2001

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1997

1995

1993

1991

1989

0

Fig 48.6 Percentage of ZIFT and GIFT of all ART cycles in the United States and Canada* 1989– 2004.39,41–47,49–54

although at least one report could not demonstrate a difference in success rates whether GIFT was performed under local anesthesia with air vs CO2 pneumoperitoneum.30 As suggested earlier, semen parameters, maternal age, and number of oocytes transferred are major determinants of outcome. Several reports suggest that endometriosis at any stage adversely affects outcome.18,31 In one large study of 1826 GIFT cycles,32 women >40 years old were found to have a higher rate of cancellation and a significantly lower delivery rate (12.5%). Women aged 44–45 years old had a pregnancy rate of 4.2%. As with IVF, many centers obtain no pregnancies among women >43 years old. For this reason, it is common practice to transfer a greater number of oocytes into women >40 years old if larger numbers of oocytes are retrieved. Because GIFT implies that at least one fallopian tube is patent, some have questioned whether or not it has achieved better results than IUI. In one randomized study33 of 200 couples receiving either GIFT or human menopausal gonadotropin (hMG), followed by IUI or intercourse, the GIFT pregnancy rate was 26.7% vs 9.7%. The study excluded male factor and tubal infertility. However, it was flawed in that the ovulation induction protocol was more aggressive for GIFT patients and the patients receiving IUI or intercourse were not differentiated. Another similar study34 evaluated cumulative pregnancy rates for GIFT and pronuclear stage tubal transfer (PROST) used for male factor infertility vs IUI. The cumulative probability of achieving pregnancy after four or more cycles of superovulation-IUI was 0.41 compared with 0.74 for the GIFT/PROST group. Patients who initially underwent superovulation IUI without success and then completed three or more GIFT/PROST treatments saw their cumulative probability of achieving pregnancy rise to 0.80.

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One study35 of 2941 patients evaluated the pregnancy outcome for successful GIFT patients undergoing a subsequent procedure. The initial pregnancy rate was 31%, with 34.7% for those seeking a second GIFT pregnancy, and 42.7% for those seeking a third GIFT pregnancy. The time it took to become pregnant also shortened. The first GIFT cycle achieved pregnancy in 34.3% of patients, which increased to an initial pregnancy rate of 39.7% in the second pregnancy and 53.6% in the third pregnancy. These findings suggest that GIFT may have a positive effect on subsequent pregnancy rates and time to conception.

Summary GIFT was developed out of a desire to place gametes directly into their natural physiologic environment in order to enhance the potential for fertilization. It is not a procedure that can be used for all patients because at least one patent fallopian tube is required and severe oligospermia is a relative contraindication. In general, success rates for IVF and GIFT are comparable. Because GIFT requires general anesthesia and a laparoscopy in most instances, most centers prefer to focus the majority of their cases on IVF to reduce operative risk, time, and recovery, and to verify fertilization. As a result of these considerations, GIFT is now used only in niche situations,15 and it is likely that, over time, GIFT will become an even smaller percentage of ART.36 Nevertheless, GIFT will continue to be an important option for those individuals who either for personal or religious reasons are opposed to IVF, and for those centers that cannot afford or do not have the laboratory equipment, space, and technical expertise needed to perform IVF.

Zygote intrafallopian transfer The ability of tubal transfer of embryos to produce pregnancy and live birth was first demonstrated in a non-human primate model by Balmaceda et al.37 Soon thereafter, Devroey et al described the first successful ZIFT in humans.38 Early reports on ZIFT were encouraging, showing superior results over uterine embryo transfer (UET), mainly for the treatment of male factor and unexplained infertility. Despite its technical complexity and high cost compared to UET, ZIFT was gradually introduced into clinical practice. In 1991, ZIFT constituted 6.4% of all ART cycles in the United States and Canada.39 Subsequently, with the publication of randomized clinical trials that failed to show a clear advantage for ZIFT as compared to UET, along with the introduction of ICSI as a powerful clinical tool for the treatment of male factor infertility,40 the use of ZIFT has dramatically declined (see Fig. 48.6). In the year 2004, ZIFT represented only 0.3% of the 99 639 assisted reproduction cycles performed in that year.24

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Throughout the years 1989–1996, delivery rates per retrieval in North America were consistently superior with ZIFT as compared to UET39,41–47 (Fig. 48.7). In 1996, for example, 30.9% of all ZIFT cycles resulted in delivery, as compared to only 26% of retrievals followed by UET.47 The pregnancy rate per transfer for ZIFT in the combined 1991–1996 SART (Society for Assisted Reproductive Technology) database was 44.5% of 5379 transfers, which was significantly higher than the pregnancy rate per transfer of IVF-ET of 28.3% of 134 912 transfers (p <0.001).48 From 1997 and onwards, delivery rates for ZIFT and UET have become very similar49–54 (see Fig. 48.7) mainly due to improvements in the efficiency of IVF with UET. While the exact indications for the use of ZIFT were never clearly specified, the consistently high delivery rates observed with ZIFT throughout the years suggest that ‘there is something about ZIFT’ that should be further explored. Our objectives in this text are to summarize the world experience with ZIFT and to try to clarify the current role and indications for ZIFT among the assisted reproductive technologies.

Nomenclature Tubal transfer procedures are known by different names, according to the developmental stage of the embryos being transferred. When transfer is done at the pronuclear stage, the procedure is known as ZIFT (zygote intrafallopian transfer) or PROST (pronuclear stage tubal transfer). If transfer is done at the two-toeight-cell stage, the procedure is known as TET (tubal embryo transfer) or EIFT (embryo intrafallopian transfer). In the literature all the latter three are commonly referred to as ZIFT.

Advantages and disadvantages of ZIFT Compared with GIFT, ZIFT allows confirmation of fertilization and selection of only normally fertilized zygotes for transfer, whereas polyploid embryos can be discarded. ZIFT also allows for extended incubation of immature oocytes. On the other hand, ZIFT involves the extra work, facilities, and expenses associated with embryo culture. Compared with UET, ZIFT is less selective at the level of embryo quality, as the transfer occurs as soon as normal fertilization has been confirmed. Some pronuclear stage embryos, when left in culture, would either arrest from cleaving, or yield poor-quality embryos. With the ZIFT procedure, early embryo cleavage and development occur in the natural and physiological environment of the fallopian tube. The oviduct is certainly not a simple transport pipe. It is a metabolically active organ, which provides nutrients and growth factors for zygotes and cleavage-stage embryos. Although very little is known on embryo–tubal interactions at the early stages of embryonic development, the tubal environment may be potentially superior to the suboptimal

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35

Percentage of delivery

30 25 20 15 10

*From 1996 United States only. IVF-ET

ZIFT

conditions developed in artificial culture media and incubators, and thus facilitates the first steps of embryonic development. Indeed, efforts have been made to study the composition of human tubal fluid,55 and commercial culture media that attempt to mimic the tubal milieu are widely used. One of the indications for tubal transfers may be a suboptimal in vitro culture system.56 It has been suggested that this might have been one of the reasons why early retrospective studies comparing UET with GIFT and ZIFT found tubal transfer procedures so successful.56 An in vivo role has been suggested for the fallopian tube in preventing zona hardening, especially in couples with advanced female partner age.57 It has been demonstrated that co-culture on oviduct epithelial cells is efficient in promoting preimplantation embryo development.58–61 The specificity of human tubal cell secretions for embryo development is challenged by the development of co-culture techniques involving human endometrial cell62 or non-human genital tract cell layers yielding favorable results.63 Advances in culture media composition and laboratory conditions now allow for in vitro embryo culture to the blastocyst stage. Retrospective analysis of results obtained with ZIFT vs UET at the blastocyst stage after co-culture in an unselected population yielded comparable ongoing pregnancy rates per transfer, at about 28% for both groups.64 Furthermore, culture protocols using artificially prepared sequential media support human blastocyst development and implantation, irrespective of co-culture use.65 Thus, the beneficial role of the tubal lumen environment in supporting zygote and embryo development remains speculative and lacks a strong scientific basis. Other putative explanations must than be sought. One of the potential advantages of ZIFT over UET is a more appropriate mode and timing of embryo entry

GIFT

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0

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Fig 48.7 Delivery rates per retrieval following IVF-ET and IVF-ZIFT in the United States and Canada* 1989– 1996.39,41–47,49–54

into the uterus. ZIFT may overcome certain deleterious effects related either to uterine receptivity or the microtrauma and hypermotility at the time of transfer, which may lead to embryo expulsion. With ZIFT, embryos may reach the uterine cavity at the appropriate moment with better synchronization between embryonic and endometrial development. Clinical experience has shown that transcervical UET is a relatively simple procedure but is far from being perfect. Experimental studies with mock embryo transfer showed expulsion of methylene blue in 57% of transfers66 and movement of X-ray contrast medium towards the fallopian tubes and cervix/vagina in 38.2 and 20.6%, respectively.67 Furthermore, embryos have been found in the vagina following UET,68,69 and some UET techniques are more frequently associated with ectopic pregnancy.70,71 There is evidence that a greater frequency of uterine junctional zone contractions (JZC) on the day of embryo transfer is associated with a reduced pregnancy rate.72 Furthermore, interference with the endometrium by the transfer catheter at the time of UET may increase the JZC frequency73 and adversely affect implantation. These ill effects are probably prevented with ZIFT, as there is no direct contact between the transfer catheter and the uterine cavity. In addition, endometrial activity is minimal and progressively decreases during the luteal phase.74 A significant decrease in JZC frequency was observed from the day of hCG onward, with a marked decline between days hCG+4 and hCG+7.75 The virtual uterine quiescence observed at hCG+7, the time period around which blastocysts presumably arrive at the uterine cavity, may explain the relative efficiency of both tubal transfer procedures and UET at the blastocyst stage. We have recently studied the patterns of JZC during ZIFT in patients with repeated (>3) failed IVF

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cycles.76 Our hypothesis was that increased JZC frequency may be a causative factor in repeated failures of UET, and that ZIFT may be protective from increased uterine contractility. A high JZC frequency was observed just before anesthesia in all patients (4.5 ± 2.4/min), which decreased significantly after induction of general anesthesia (2.1 ± 1.6; p = 0.001). Contraction frequency was dramatically increased again with tubal manipulation during the actual ZIFT procedure (5.5 ± 2.6/min) as compared to the frequency after anesthesia (p = 0.001), and decreased again after completing the procedure (4.0 ± 1.4; p = 0.002). Interestingly, JZC frequency before anesthesia was significantly lower in patients who conceived as compared to those who did not (2.04 ± 0.6 vs 5.4 ± 2.6; p = 0.004). Thus, as with conventional UET, a correlation between increased baseline (before ZIFT) JZC frequency and cycle failure has been established. In contrast to our hypothesis, ZIFT failed to protect patients with increased baseline uterine contractility from cycle failure. Another potential problem limiting the success of UET procedures may be related to the presence of cervical microorganisms on embryo transfer catheters. It has been shown that implantation and clinical pregnancy rates were significantly lower in women with positive microbial catheter-tip cultures.77,78 Furthermore, prophylactic antibiotics administered at the time of oocyte retrieval were associated with both a reduction in positive microbiology cultures of transfer catheter tips 48 hours later and improved UET outcome.78 Since tubal transfer procedures bypass the cervical canal, the detrimental effects of inoculating the uterine cavity with cervical microorganisms may be avoided. Last but not least, another practical advantage associated with surgical ZIFT procedures is the diagnostic information provided by laparoscopy. Advantages and disadvantages of the ZIFT procedure are outlined in Table 48.1.

World experience with ZIFT As mentioned above, early reports on ZIFT were encouraging, showing superior results in terms of clinical pregnancy, implantation, and live birth rates, as compared to UET. Retrospective reports on ZIFT showed pregnancy rates per transfer ranging from 37% to 53%, as compared to only 12% to 28% for UET.79–84 Subsequently, several randomized controlled trials were conducted in order to evaluate the efficacy of ZIFT vs standard UET for the treatment of nontubal factor infertility. These prospective studies either lacked power calculation85–88 or initial sample size requirements have not been met.89,90 As discussed by Toth et al,86 in order to achieve a 20% increase in the clinical pregnancy rate (e.g. from 30% to 36%) a sample size of >900 patients would be required. Overall, all prospective trials failed to demonstrate any difference

Table 48.1

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Advantages and disadvantages of ZIFT procedures

Advantages • Confirmation of fertilization and selection of only normally fertilized zygotes for transfer • Embryo cleavage and development occur in the natural and physiological environment of the fallopian tube • Better synchronization between embryonic and endometrial development • Avoidance of suboptimal in vitro culture systems • Prevention of zona hardening, especially in couples with advanced female partner age • Prevention of microtrauma to the endometrium by uterine transfer catheters • Prevention of embryo expulsion following UET induced by subendometrial myometrial contractions • Prevention of the detrimental effects of cervical microorganisms associated with UET • Important diagnostic information provided by laparoscopy Disadvantages • Risks and complications associated with general anesthesia and endotracheal intubation • Risks and complications inherent with laparoscopy • Increased cost compared with uterine embryo transfer • Longer hospital stay compared with uterine embryo transfer • Lack of the ability to select the morphologically best-cleaving embryos compared with uterine embryo or blastocyst transfer

in the rates of implantation, clinical pregnancy, ongoing pregnancy, and miscarriage rate between ZIFT and standard UET. A recent meta-analysis48 using data from six randomized prospective trials85,87–91 has shown that cycle outcome with either ZIFT or UET is comparable. Sixty-six pregnancies resulted from 181 (36.5%) transfers in ZIFT, and 65 of 207 (31.4%) pregnancies in IVF-ET (OR = 1.23, 95% confidence interval [CI] 0.8–1.89) (Fig 48.8). One study87 used cryopreserved embryos, and a second study91 used donor eggs. Thus, the six studies included in the meta-analysis vary in terms of patient inclusion criteria, randomization methods, stimulation protocols, stage and number of embryos transferred, and main outcome measures reported. Considering the heterogeneity and sample size of the randomized trials, it can be concluded that the clinical efficacy of ZIFT has never been critically evaluated. The risk for ectopic pregnancy following ZIFT appears to be increased. Clayton et al92 assessed the ectopic pregnancy risk among women who conceived with ART procedures in US clinics in 1999–2001. Of 94 118 ART pregnancies, 2009 (2.1%) were ectopic. In comparison with the ectopic rate (2.2%) among pregnancies conceived with IVF and transcervical transfer of freshly fertilized embryos from the patient’s oocytes, the ectopic rate was significantly increased when ZIFT was used (3.6%) (OR = 1.65; 95% CI 1.13– 2.4). In our experience, of 96 pregnancies achieved by

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Peto OR (95% Cl)

WMD (95% Cl)

Age Etiology of infertility Stimulation protocol used Peak estradiol level Number of oocytes Number of zygotes/embryos transferred Implantation rate Ectopic pregnancy Number of pregnancies per retrieval Spontaneous abortion Ongoing pregnancy per transfer Multiple pregnancy 0.1 0.2

1

5 10 −10 −5

0

5

10

Fig 48.8 Summary of outcome measures, ZIFT vs IVF-ET. Lines indicate odds ratio (OR) and 95% confidence interval (CI). WMD, weighted means difference. From Habana and Palter,48 with permission.

ZIFT, there were 72 live birth (75%), 19 miscarriages (19.8%), and 5 ectopic pregnancies (5.2%).93

The ZIFT procedure ZIFT normally requires general anesthesia and endotracheal intubation. Intrafallopian transfer with local anesthesia and continuous sedation has also been described.94 ZIFT is performed 18–48 hours after oocyte aspiration using a three-puncture videolaparoscopic technique. After introducing the umbilical trocar and optical equipment, the abdominal cavity is surveyed. The sanguineous fluid, containing blood, follicular fluid, and pelvic fluid, is aspirated through a 5 mm midline suprapubic incision. The fallopian tube chosen for the transfer should be the one that is both most easily accessible and with the most healthy-looking appearance. Manipulating the large and hyperstimulated ovaries should be avoided, as they tend to rupture and bleed. A 3.5F 35-cm Teflon catheter (Patton Laparoscopic Catheter Set, Cook Ob/Gyn, Spencer, IN, USA) is loaded with 10–20 µl of medium containing the zygotes or embryos. Using a third paraumbilical puncture with a dedicated trocar stylet (Cook Ob/Gyn, Spencer, IN, USA), the catheter tip is gently introduced about 3 cm into the ampullary region through the fimbriated end of the fallopian tube, where the contents are slowly discharged.

Selection of zygotes for transfer During the ZIFT procedure, pronuclear embryos are normally selected for transfer based only on the visualization of two pronuclei 18–24 hours after egg retrieval and insemination. A universally accepted scoring system for zygote quality, like the ones that

exist for scoring embryos, is not available. Consequently, none of the groups that reported on their experience with ZIFT have used a systematic approach for selection of zygotes for tubal transfer. Scott and Smith have suggested that the morphology of the human zygote at 16–18 hours’ post insemination can be used as a positive predictor for the outcome of day 1, zygote UET.95 Pronuclear embryos could be successfully selected for transfer based on the positioning of their pronuclei, the alignment of their nucleoli, and the appearance of their cytoplasm. A high score was found to be associated with high rates of implantation and successful pregnancies. For a detailed description of zygote assessment the reader is referred to Chapter 15. It is reasonable to assume that the above-mentioned or similar scoring systems could be adapted and used for better selection of preembryos for tubal transfers. Selection of high-quality zygotes may allow limiting the number of zygotes being transferred. This may lead to a reduction in multiple pregnancy rates following ZIFT without compromising overall success. Clinics that regularly perform ZIFT should be encouraged to study carefully and document zygote morphology. Whether the incorporation of zygote-quality scoring systems into clinical practice will improve the overall outcome remains to be shown.

Transcervical tubal transfer procedures Transcervical tubal transfer procedures of zygotes or embryos spare patients from the risks and complications as well as the high cost inherent with laparoscopy under general anesthesia, while potentially retaining the therapeutic advantage of tubal transfer. Transcervical ZIFT may be especially attractive for high surgical risk and

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obese patients. Initial attempts at transcervical retrograde catheterization were made under ultrasound guidance, using modified embryo transfer catheters.96,97 Later, blind tactile tubal catheterization procedures with gametes98 and embryos99 have been successfully performed. However, the success of blind procedures as well as ultrasound-guided tubal catheterizations is limited to a great extent by the operators’ skills and ability to avoid uterine and tubal trauma. With transcervical ZIFT, it is rather difficult to negotiate the catheter through almost the entire tubal lumen, and to replace zygotes or embryos in the favorable environment of the ampullary portion of the tube. In a prospective study by Scholtes et al,100 the implantation rate after ultrasound-guided ZIFT was inferior as compared to laparoscopic ZIFT, with an implantation rate of 4 and 12%, respectively. Similar findings were obtained when nonsurgical and laparoscopic GIFT were compared.101 Hysteroscopic tubal transfers allow clear visualization of the tubal ostium as well as accurate estimation of the depth of catheter insertion into the fallopian tube. They are advantageous when a difficult laparoscopic tubal transfer due to pelvic adhesions is expected. The published experience with hysteroscopic transfer procedures is rather limited.102 The potential adverse effects of CO2 exposure on gametes and embryos, and blunt endometrial trauma have limited their use. Overall, efforts to develop transcervical methods of tubal transfer have not translated into higher pregnancy rates than those achieved with UET or laparoscopic ZIFT. Consequently, this approach has been almost completely abandoned.

Modifications of ZIFT: microinjected oocytes fallopian transfer (MIFT) A special subtype of transfer of gametes into fallopian tubes is the immediate transfer of injected oocytes into the fallopian tubes. The advantage of MIFT compared with ZIFT is the shorter time of in vitro culture (4 hours) without the loss of fertilization assessment.103 Sahebkashaf et al,104 in a multicenter study, presented a large series (4000 cycles) of this method (also known as ‘Rapid ICSI-ZIFT’), with a pregnancy rate of 46%. With MIFT, Vorsselmans et al103 reported an ongoing pregnancy rate and implantation rate of 29% and 11%, respectively. They demonstrated that the pregnancy rate in the MIFT procedure did not differ from UET in patients younger than 37 years old (29% vs 35%), although the implantation rate decreased significantly from 24% UET to 11% in MIFT. In a prospective, randomized clinical trial, Alleyassin et al105 compared unilateral vs bilateral transfer into the fallopian tubes after MIFT. The study population included 160 patients presenting with male factor infertility, and four injected oocytes were transferred into two tubes (study group) or one tube (control group). A total of 72 (45%) pregnancies were

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achieved, without significant difference between the two groups in the implantation rate, clinical pregnancy rate, and multiple pregnancy rate. It was therefore suggested that unilateral transfer is the preferred method of MIFT. A variant using hysteroscopic tubal transfer of ova after ICSI has been also described.106

Current potential indications for ZIFT Originally, ZIFT was advocated for all classes of nontubal factor infertility. The majority of patients who underwent ZIFT suffered from either male factor or unexplained infertility. Advances in laboratory and clinical practice have changed dramatically the prognosis for both male factor and unexplained infertility. A close look at the US Registry National Summary and Fertility Clinics reveals a more than double increase in the delivery rate per retrieval from only 14% in 198941 to 31.6% in 2004.24 Currently, delivery rates per retrieval are comparable for most categories of infertility.50 IVF-ICSI with UET is the treatment of choice for moderate to severe male factor, and IVF (with or without ICSI) followed by UET is highly efficient for unexplained infertility. Thus, as the original indications for ZIFT seem to be outdated, the role of tubal transfer procedures in ART is once again being debated.56,64,107,108 Who, in turn, are the patients that may still benefit from ZIFT?

ZIFT for patients with repeated implantation failure It has been demonstrated that pregnancy rates do not change over the first three IVF-ET cycles but decrease by 40% for four or more prior failed attempts.109 Thus, couples with repeated implantation failure (RIF) following UET represent one of the greatest challenges for the caring physician. Our own experience over two decades consistently shows that patients with multiple failed attempts of UET are the most likely to benefit from ZIFT. RIF patients may initially present with any of the known infertility categories, but the exact etiology for RIF is obscure in the vast majority of cases. A variety of interventions have been advocated for couples with RIF. None, however, seems to be consistently efficacious in achieving implantation and conception. For a detailed review of this topic the reader is referred to Chapter 45. In a case-control study, summarizing the experience of two Israeli ART centers, the outcome of ZIFT and UET in RIF patients was compared.110 Seventy patients suffering from either male factor or unexplained infertility who underwent ZIFT were compared with 70 control patients undergoing UET, matched for age, diagnosis, and duration of infertility. All patients had a normal uterine cavity with transfer of three to five normally cleaving embryos in at least three IVF-ET cycles and failure of implantation in all previous UET attempts. Patients in both groups had a similar number of oocytes retrieved and fertilized.

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Patients who underwent ZIFT had a mean of 4.8 ± 1.6 zygotes transferred, and patients who underwent UET had a mean of 4.2 ± 2.0 embryos replaced (not significantly different). A significantly higher clinical pregnancy rate and implantation rate was achieved with ZIFT (34.2% and 8.7%) as compared with UET (17.1% and 4.4%). The miscarriage rate was comparable for both groups. We have recently extended the ZIFT series by analyzing current data from our own center over 10 years.93 A total of 176 patients, <43 years old, with mean number of 8.5 ± 3.9 previous failed attempts underwent a total of 280 ZIFT cycles. Clinical pregnancy rate per transfer was 34.3%, with a live birth rate per transfer of 25.7%. This is remarkable because cycle outcome in the poor prognostic group of RIF patients became favorable with ZIFT, comparable to the results achieved for good prognosis patients, who have just embarked on IVF-ET therapy. Our results are encouraging, as they confirm the efficacy and reproducibility of ZIFT as a powerful clinical tool for the treatment of RIF. In contrast to the above observations, Aslan et al111 compared a total of 141 ZIFT cycles of 132 women with 145 UET cycles of 97 women in whom >5 embryos were transferred. All patients had five or more previous failed IVF cycles. The implantation rate was 6.5% vs 7.2%, clinical pregnancy rate was 22.7% vs 24.8%, and live birth rate was 21.2% vs 16.5% in ZIFT and ET groups, respectively. It was concluded that ZIFT is not superior to transcervical UET in RIF patients. The potential advantages of ZIFT have been outlined above (see Table 48.1). Our understanding of the mechanisms leading to superiority of ZIFT over UET in patients with RIF is fragmentary and incomplete. Avoidance of poor in vitro culture conditions is unlikely to be a major mechanism in view of the comparable outcome of ZIFT using pronuclear-stage and cleavage-stage embryos for tubal transfer (see below), as well as the favorable outcome with UET for goodprognosis patients in our center. In our opinion, the more relevant advantages of ZIFT are related to mechanical aspects of the procedure. With ZIFT, embryo expulsion from the uterine cavity, commonly observed following UET,68,69 is very unlikely. The embryo presumably enters the uterine cavity in the midluteal phase, when the frequency of junctional zone contractions has decreased, relative to the time of UET in the early luteal phase.75 Furthermore, transcervical transfer catheters may induce junctional zone contractions73 and inoculation of the uterine cavity with cervical microorganisms,77,78 which could both lead to embryo expulsion and interfere with implantation. Any of the above mechanisms as well as yet other undefined factors could be functioning, leading to the high success rates observed with ZIFT in RIF patients. At present, all should be best regarded speculative and should be further investigated in depth.

ZIFT in patients with difficult uterine transfers One condition where ZIFT is likely to be beneficial is in patients with cervical anatomy that renders negotiating the cervical canal during UET extremely difficult or even impossible. While the question whether the success rate of UET is negatively correlated with the difficulty of the transfer procedure remains under debate,112 ZIFT may spare the patient and the caring physician difficult, stressful, painful, and time-consuming transfers. Unfortunately, there are no data available to prove this concept. ZIFT is certainly indicated with congenital abnormalities of cervical anatomy such as congenital hypoplasia or atresia of the cervix, or following cervical irradiation or surgery.113,114

ZIFT for advanced maternal age Pregnancy and implantation rates decline progressively with advanced maternal age.39,42,44,46,47,50,115 Whether ZIFT can enhance the implantation potential of embryos originating from patients with advanced maternal age has been the subject of an ongoing controversy. It has been speculated that the tubal milieu may be conductive in rescuing marginal embryos in older patients. Batzofin et al conducted a retrospective analysis of 77 UET and 50 ZIFT cycles in women >40 years old.116 A similar number of zygotes/embryos were transferred in the two groups. Clinical pregnancy rates per transfer were 7% and 40%, and delivery rates per transfer were 3.5% and 28%, for UET and ZIFT, respectively. The superiority of ZIFT over UET was statistically significant. Pool et al84 retrospectively analyzed their experience with 114 ZIFT cycles over a 2-year period. No significant decline in the clinical pregnancy or delivery rate was observed with ZIFT in women aged 25–39 years old, approaching a 40% delivery rate overall for women aged 35–39 years old. Too few cases were completed for the age group >40 years old for valid statistical analysis. In contrast, Balmaceda et al117 retrospectively analyzed data from their clinic comparing GIFT and ZIFT by age group. Both pregnancy and implantation rates obtained with GIFT remained stable, whereas those achieved with ZIFT decreased dramatically with age. In our own experience, looking at cycle outcome by age group (unpublished work), it appears that pregnancy rates are fairly constant up to the age of 39 years old. Pregnancy rates per procedure were 43.4% and 39.4% for the age groups of <35 years old and 35– 39 years old, respectively. Clinical pregnancy rate in patients >39 years old was 12%, demonstrating only a marginal benefit for ZIFT in this age group. Clearly, more studies are warranted in order to determine the value of ZIFT in patients with advanced maternal age.

ZIFT following ICSI While the ZIFT procedure was originally advocated for couples with male factor infertility undergoing

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IVF, the introduction of ICSI dramatically changed the clinical management and prospect for such couples. Favorable pregnancy and delivery rates were reported for patients undergoing IVF-ICSI with UET for severe male factor infertility. Very little, however, is known of the added value of ZIFT in cycles where fertilization was achieved by means of ICSI. Boldt et al118 analyzed whether the mode of embryo transfer (ZIFT vs UET) affected the outcome in ICSI cycles. In a retrospective study, 82 ICSI cycles (42 ZIFT and 40 UET) were analyzed. The implantation and clinical pregnancy rates in ZIFT cycles (23.2% and 52.3% respectively) were significantly higher than in UET cycles (9.7% and 17.5%, respectively). Another retrospective analysis by La Sala et al, comparing UET with ZIFT of cleavage-stage ICSI-derived embryos, has yielded similar results.119 These data suggest that, at least under certain conditions, ZIFT may be beneficial as the method of transfer of ICSIderived embryos. The clinical circumstances under which ZIFT should be recommended following ICSI should be further investigated.

ZIFT for frozen–thawed embryo transfer For reasons that are not fully understood, cryopreservation has a detrimental effect on the implantation potential of frozen–thawed embryos.120 The insult to the embryos could occur during the actual freeze– thaw procedures, or result from suboptimal post-thaw culture conditions. It was therefore speculated that the tubal lumen environment may be more conducive for the health of frozen–thawed embryos and may improve their implantation potential. Although this approach has not been thoroughly evaluated, there are data suggesting that this indeed is the case. In a retrospective study, Frederick et al121 reported their experience with 54 tubal transfers of frozen– thawed embryos. A 41% clinical pregnancy rate and 24% live birth rate were achieved. In a retrospective analysis of a small group of patients, Abdalla et al reported comparable pregnancy rates after tubal or uterine transfers of frozen–thawed embryos resulting from donor oocytes.122 Finally, in a small prospective study reported by Van Voorhis et al,87 40 patients with patent fallopian tubes and at least three cryopreserved embryos were randomized to undergo either laparoscopic ZIFT or UET. Implantation rates, clinical pregnancy and ongoing pregnancy rates were compared. Tubal transfer of cryopreserved embryos resulted in significantly higher implantation (19% vs 10%), clinical (68% vs 24%), and ongoing pregnancy rates (58% vs 19%) when compared with UET. It was concluded that tubal transfer of cryopreserved embryos is highly effective and offers an improved pregnancy rate when compared with UET. Currently, there are no sufficient data to conclude whether ZIFT is advantageous in frozen–thawed embryo replacement cycles. This aspect of ZIFT should be also further explored.

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Clinical and technical issues related to ZIFT procedures Pronuclear-stage vs cleavage-stage tubal embryo transfer As mentioned above, ZIFT was originally described with pronuclear-stage embryo transfer into the fallopian tube.38 Subsequently, a variant in which day 2 embryos are replaced in the tubal lumen (TET) was introduced.123 Currently, both procedures are referred to as ZIFT. Whether the timing and embryonic stage at tubal transfer affect results has not been thoroughly evaluated. In a small retrospective study, Diedrich et al124 reported the results of 20 pronuclear-stage tubal transfers and 20 tubal transfers of embryos at the 2–8-cell stage. A mean of 2.9 embryos was transferred in both groups. Overall, there were 11 pregnancies (28%), six (30%) occurring with pronuclear stage and five (25%) occurring at the 2–8-cell stage. In a retrospective analysis, 176 patients who failed in 7.65 ± 3.7 previous IVF cycles, underwent 200 ZIFT and 73 EIFT procedures.125 Implantation and live birth rates were compared for both groups. Patients in both groups were found comparable for demographic and clinical parameters. Similar numbers of oocytes were retrieved and fertilized in both groups and 5.2 ± 1.2 zygotes/embryos were transferred. Implantation and live birth rates (10.5% and 26.5% vs 10.9% and 24.5% for ZIFT and EIFT, respectively) were comparable. It is concluded that tubal transfer of zygotes and day 2 cleavage-stage embryos are equally effective.

Multiple pregnancies with ZIFT It is well known that some pronuclear-stage embryos either arrest from cleaving or yield poor-quality embryos. It is therefore not unreasonable to transfer more pre-embryos in ZIFT procedures than embryos that have been selected based on morphologic criteria and cleavage rate on day 2 or 3 UET. In a retrospective study, Bollen et al reported results after transferring three oocytes, three zygotes, and three embryos in GIFT, ZIFT, and UET, respectively.82 Implantation and clinical pregnancy rates were significantly higher for ZIFT (18.2% and 38.5%) as compared to GIFT (8.4% and 19.4%) and UET (13.7% and 28.4%), respectively. Focusing on multiple pregnancy rates at 20 weeks’ gestation, 16% of GIFT pregnancies, 27% of ZIFT pregnancies, and 32% of UET pregnancies were multiple. Consequently, it was recommended to limit to three or fewer the number of zygotes/embryos transferred. In contrast, Toth et al86 and Tanbo et al85 obtained lower numbers of multiple pregnancies with the transfer of four zygotes (10% and 17%, respectively). By limiting the number of zygotes transferred from three to two, Devroey et al83 were able to reduce the multiple

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pregnancy rate from 23% (including 7.6% triplets) to zero, while maintaining a high implantation rate (28% and 24%, respectively) and a high clinical pregnancy rate (55% and 50%, respectively). Women with multiple failed UET cycles have been found to have an improved prognosis with transfer of six or more embryos, without a significant increase in the multiple pregnancy rate.126 For this reason, it has been our practice to replace five to six zygotes during ZIFT in patients with RIF. Furthermore, we normally offer ZIFT to RIF patients only if a minimum of three zygotes are available for transfer. Transferring a mean of 4.8 ± 1.6 zygotes resulted in a clinical pregnancy rate of 35.1% per transfer, and a multiple pregnancy rate of 19%, all of the multiples being twins.110 The favorable pregnancy rate obtained in RIF patients, with an acceptable multiple pregnancy rate and lack of high-order multiples, were all reassuring. Consequently, we have continued with the policy of transferring five to six zygotes. We found that the highest pregnancy rate is obtained with the transfer of five zygotes (63%), as compared to 38% with transfer of six embryos, and 26% with transfer of four embryos. We normally transfer five zygotes to RIF patients who are considered ‘good prognosis,’ i.e. young, have produced multiple eggs and zygotes, and have good-quality zygotes to select for transfer.95 This explains why the highest pregnancy rates were achieved in this group. On the other hand, patients who had four zygotes transferred, virtually had all their zygotes transferred without any pre-selection or zygotes left for cryopreservation. Similarly, patients who had six zygotes transferred did not have the favorable characteristics like those who had five zygotes replaced, which explains the lower pregnancy rate in this group. Analysis of our most recent data, using the above policy for selecting the number of zygotes to be transferred, has yielded a considerably higher multiple pregnancy rate than in our early report.93,110 Of 280 cycles, 96 pregnancies were achieved. Out of 72 ongoing pregnancies and live births, there were 38 singletons (52.8%), 31 twins (44.4%), and two triplets (2.8%). Out of 32 twin births, five were reduced from triplets, five reduced from quadruplets, and one reduced from quintuplets. The high-order multiple pregnancy rate was therefore 18% (13/72). Our efforts are currently focused on better selection of zygotes and patients in whom the number of zygotes transferred can be further reduced safely, without compromising the favorable results obtained with ZIFT in patients with RIF.

ZIFT in patients with a single patent tube Patients with a single patent tube represent a distinct subclass of tubal factor infertility. As early as 1989, Cittadini and Palermo advised the use of the ZIFT procedure ‘… if at least one healthy Fallopian tube is present.’127 However, the safety and efficacy of ZIFT in this specific group of patients has never been investigated.

It is almost impossible to determine the functional status of a single patent tube in the presence of a contralateral blocked or absent tube. There is always the fear that a single patent tube may be functionally damaged, and that transferring zygotes into such a tube may result in tubal pregnancy. Bollen et al reported their results with 223 ZIFT procedures in patients with at least one healthy tube.82 Although the proportion of patients with a single healthy tube was not reported, there were no ectopic pregnancies in the entire ZIFT group. In a nonrandomized study, Pool et al84 compared the results of ZIFT in patients with at least one normal tube to UET in patients with bilateral tubal disease. They have found significantly higher implantation (17% vs 8%) and ongoing pregnancy/delivery rates (34% vs 15.8%), with ZIFT vs UET. There were four ectopic pregnancies in 114 ZIFT procedures (3.5%) and no ectopic pregnancies following UET. Unfortunately, ZIFT outcome in patients with a single patent tube was not reported separately, so it is unclear whether ectopic pregnancies had occurred in this group. In our center, ZIFT is being offered to patients with a single patent tube who failed to conceive following multiple attempts of UET. We recently compared the outcome of ZIFT in patients with unilateral vs bilateral tubal patency.128 Overall, 112 tubal transfers were performed in 81 patients. Sixty-six patients with bilateral patent tubes underwent 97 ZIFT procedures, and 17 patients with a single patent tube underwent 15 tubal transfers. In two patients of the latter group, ZIFT could not be performed due to massive pelvic adhesions. A mean of 5.4 ± 1.3 and 5.6 ± 0.9 zygotes were transferred in the groups with bilateral and unilateral tubal patency, respectively. Implantation (11% vs 9.4%) and clinical pregnancy rates (37.1% vs 26.6%) were found comparable with bilateral and unilateral tubal patency, respectively. A single tubal pregnancy occurred in a patient with bilateral patent tubes. Despite the small numbers involved, our results are reassuring, indicating that the risk for extrauterine pregnancy may be similar, and certainly not increased with zygote transfer into a single patent tube, as compared to patients with bilateral tubal patency. Thus, whenever indicated, ZIFT may be offered to patients with a single patent tube.

ZIFT vs uterine transfer at the blastocyst stage With the recent availability of sequential media that allow extended culture to the blastocyst stage, the concept of blastocyst transfer has evolved mainly as a means of reducing the risk of multiple gestation. Favorable results have been obtained with uterine transfer at the blastocyst stage in good-prognosis patients.129,130 Menezo and Janny64 reported their results of a retrospective analysis comparing ZIFT with uterine transfer at the blastocyst stage after co-culture in an unselected population. A total of 137 ZIFT procedures were compared with 217 blastocyst transfers. While significantly more zygotes than

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blastocysts were transferred (2.6 ± 0.78 vs 2.06 ± 0.85, respectively; p = 0.001), the ongoing pregnancy rate per transfer was comparable at ~ 28%. Could uterine transfer at the blastocyst stage be beneficial for patients with RIF? Cruz et al reported a retrospective comparison of day 3 vs blastocyst-stage transfer in RIF patients.131 Twenty-two patients with a mean of 4.1 failed attempts underwent day 3 UET. These were compared to 15 patients with a mean of 3.8 failed attempts that underwent blastocyst transfer. Clinical pregnancy and implantation rates were significantly higher with blastocyst transfer (40% and 11.3% vs 9.1% and 3.4%, respectively). Since the concept of blastocyst transfer in patients with RIF is indeed attractive, we have conducted a randomized prospective study comparing ZIFT (n = 45; 7.7 ± 3.1 failed cycles) with blastocyst transfer (n = 40; 8.3 ± 3.9 failed cycles).132 Clinical pregnancy rates were 38% and 2.5% for the ZIFT and blastocyst transfer groups, respectively (p <0.0001). There was one pregnancy in the blastocyst transfer group that ended in a miscarriage. In summary, our experience shows that blastocyst transfer is not effective for the treatment of patients with multiple failed UET attempts. The different results obtained with blastocyst transfer in our study and by Cruz et al may be partially explained by the differences in patient characteristics: i.e. a mean of 4.1 and 8.1 previous failures by Cruz et al and ourselves, respectively. Nevertheless, the efficacy of ZIFT as a powerful treatment modality in RIF patients was once again substantiated.

Should a second ZIFT attempt be advised? To our knowledge, there are no studies in the literature which have directly addressed this issue. We have previously reported a high cumulative conception rate for two ZIFT cycles in patients with RIF.110 In the latter study, patients in the ZIFT group who failed to conceive with the first cycle were offered a second ZIFT attempt. Twenty-one patients completed a second ZIFT cycle. Thirty-two pregnancies were achieved following 91 ZIFT cycles in 70 patients, including six sets of twins. The overall pregnancy rate per patient was 45.7%, with a cumulative pregnancy rate for two ZIFT cycles of 59.3%. We recently extended the data from our own center, summarizing 112 completed ZIFT cycles in 79 patients. Twenty-four patients underwent more than one ZIFT attempt. Clinical pregnancy rate for a second ZIFT procedure was 74% (14/19 patients conceived). Two of four patients who underwent a third attempt conceived (50%) and one patient conceived at her fifth attempt. The cumulative pregnancy and live birth rates in this group were 49.4% and 44.4%, respectively. Our experience suggests that a second ZIFT attempt is beneficial and should be advised for patients with RIF after failure of a first ZIFT procedure.

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Summary and conclusions Despite the lack of convincing prospective data to support the tubal transfer of zygotes or embryos following IVF, there may be clinical conditions where the ZIFT procedure would be beneficial. Currently, the most valid indication for ZIFT appears to be RIF. This has been the consistent experience of our center as well as other centers in Israel performing ZIFT. The reasons for the high efficiency of ZIFT in RIF patients have not been fully elucidated. It is likely that the superiority of ZIFT over UET in RIF patients is related to the avoidance of some of the adverse events associated with transcervical UET, such as lack of endometrial trauma and junctional zone contractions, which may lead to embryo expulsion, as well as avoidance of inoculating the uterine cavity with cervical microorganisms. ZIFT may also be beneficial for patients with repeatedly difficult transcervical UET, although its efficacy for this indication has yet to be confirmed. There is evidence to suggest that ZIFT may be beneficial in patients with advanced maternal age, following ICSI and for frozen–thawed embryo transfers. All later indications should be evaluated by properly designed prospective studies. Results with ZIFT using either pronuclear-stage or cleavage-stage embryos are comparable, so that ZIFT appears to be equally effective on day 1 or 2 post-egg retrieval. ZIFT may be safely offered to patients with a single patent tube. Efforts should be made to limit the number of zygotes/embryos replaced in order to reduce the multiple pregnancy rates. This may be achieved by better selection of zygotes for transfer through more comprehensive evaluation of zygote quality, and by better characterization of patients who are likely to succeed with ZIFT. In summary, ZIFT appears to remain an effective treatment modality for selected infertile couples. More than two decades after its first successful application, there are only few data on ZIFT generated from controlled studies. Lack of statistical power and focus on specific infertility etiologies and conditions in previously published prospective studies mandates future research in order to clarify the role of ZIFT in ART.

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Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril 2002; 78: 918–31. Assisted reproductive technology in the United States: 1998 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril 2002; 77: 18–31. http://www.cdc.gov/reproductivehealth/ART00/ index.htm. Assisted reproductive technology in the United States: 2000 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril 2004; 81: 1207–20. Assisted reproductive technology in the United States: 2001 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril 2007; 87: 1253–66. Dickens CJ, Maguiness SD, Comer MT, et al. Human tubal fluid: formation and composition during vascular perfusion of the fallopian tube. Hum Reprod 1995; 10: 505–8. Tournaye H, Camus M, Ubaldi F, et al. Tubal transfer: a forgotten ART? Is there still an important role for tubal transfer procedures? Hum Reprod 1996; 11: 1815–18. Cohen J. Assisted hatching: indications and techniques. Acta Eur Fertil 1993; 24: 215–19. Bongso A, Ng SC, Fong CY, Ratnam S. Cocultures: a new lead in embryo quality improvement for assisted reproduction. Fertil Steril 1991; 56: 179–91. Gandolfi F, Moor RM. Stimulation of early embryonic development in the sheep by co-culture with oviduct epithelial cells. J Reprod Fertil 1987; 81: 23–8. Wiemer KE, Hoffman DI, Maxson WS, et al. Embryonic morphology and rate of implantation of human embryos following co-culture on bovine oviductal epithelial cells. Hum Reprod 1993; 8: 97–101. Yeung WS, Ho PC, Lau EY, Chan ST. Improved development of human embryos in vitro by a human oviductal cell co-culture system. Hum Reprod 1992; 7: 1144–9. Simon C, Mercader A, Garcia-Velasco J, et al. Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure. J Clin Endocrinol Metab 1999; 84: 2638–46. Menezo YJ, Guerin JF, Czyba JC. Improvement of human early embryo development in vitro by coculture on monolayers of Vero cells. Biol Reprod 1990; 42: 301–6. Menezo YJ, Janny L. Is there a rationale for tubal transfer in human ART? Hum Reprod 1996; 11: 1818–20. Fong CY, Bongso A. Comparison of human blastulation rates and total cell number in sequential culture media with and without co-culture. Hum Reprod 1999; 14: 774–81. Mansour RT, Aboulghar MA, Serour GI, Amin YM. Dummy embryo transfer using methylene blue dye. Hum Reprod 1994; 9: 1257–9.

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67. Knutzen V, Stratton CJ, Sher G, et al. Mock embryo transfer in early luteal phase, the cycle before in vitro fertilization and embryo transfer: a descriptive study. Fertil Steril 1992; 57: 156–62. 68. Poindexter AN 3rd, Thompson DJ, Gibbons WE, et al. Residual embryos in failed embryo transfer. Fertil Steril 1986; 46: 262–7. 69. Schulman JD. Delayed expulsion of transfer fluid after IVF/ET. Lancet 1986; 1: 44. 70. Bennett S, Waterstone J, Parsons J, Creighton S. Two cases of cervical pregnancy following in vitro fertilization and embryo transfer to the lower uterine cavity. J Assist Reprod Genet 1993; 10: 100–3. 71. Yovich JL, Turner SR, Murphy AJ. Embryo transfer technique as a cause of ectopic pregnancies in in vitro fertilization. Fertil Steril 1985; 44: 318–21. 72. Fanchin R, Righini C, Olivennes F, et al. Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization. Hum Reprod 1998; 13: 1968–74. 73. Lesny P, Killick SR, Tetlow RL, Robinson J, Maguiness SD. Embryo transfer – can we learn anything new from the observation of junctional zone contractions? Hum Reprod 1998; 13: 1540–6. 74. Ijland MM, Evers JL, Dunselman GA, Hoogland HJ. Subendometrial contractions in the nonpregnant uterus: an ultrasound study. Eur J Obstet Gynecol Reprod Biol 1996; 70: 23–4. 75. Fanchin R, Ayoubi JM, Righini C, et al. Uterine contractility decreases at the time of blastocyst transfers. Hum Reprod 2001; 16: 1115–19. 76. Levran D, Zahalka N, Malinger G, et al. Junctional zone contractions during zygote intrafallopian transfer. In: 59th Annual Meeting of the American Society for Reproductive Medicine, San Antonio, Texas, 2003: O–149. 77. Fanchin R, Harmas A, Benaoudia F, et al. Microbial flora of the cervix assessed at the time of embryo transfer adversely affects in vitro fertilization outcome. Fertil Steril 1998; 70: 866–70. 78. Egbase PE, Udo EE, Al-Sharhan M, Grudzinskas JG. Prophylactic antibiotics and endocervical microbial inoculation of the endometrium at embryo transfer. Lancet 1999; 354: 651–2. 79. Asch RH. Uterine versus tubal embryo transfer in the human. Comparative analysis of implantation, pregnancy, and live-birth rates. Ann N Y Acad Sci 1991; 626: 461–6. 80. Hammitt DG, Syrop CH, Hahn SJ, et al. Comparison of concurrent pregnancy rates for in-vitro fertilization–embryo transfer, pronuclear stage embryo transfer and gamete intra-fallopian transfer. Hum Reprod 1990; 5: 947–54. 81. Yovich JL, Yovich JM, Edirisinghe WR. The relative chance of pregnancy following tubal or uterine transfer procedures. Fertil Steril 1988; 49: 858–64. 82. Bollen N, Camus M, Staessen C, et al. The incidence of multiple pregnancy after in vitro fertilization and embryo transfer, gamete, or zygote intrafallopian transfer. Fertil Steril 1991; 55: 314–18. 83. Devroey P, Staessen C, Camus M, et al. Zygote intrafallopian transfer as a successful treatment for unexplained infertility. Fertil Steril 1989; 52: 246–9.

84. Pool TB, Ellsworth LR, Garza JR, et al. Zygote intrafallopian transfer as a treatment for nontubal infertility: a 2-year study. Fertil Steril 1990; 54: 482–8. 85. Tanbo T, Dale PO, Abyholm T. Assisted fertilization in infertile women with patent fallopian tubes. A comparison of in-vitro fertilization, gamete intrafallopian transfer and tubal embryo stage transfer. Hum Reprod 1990; 5: 266–70. 86. Toth TL, Oehninger S, Toner JP, et al. Embryo transfer to the uterus or the fallopian tube after in vitro fertilization yields similar results. Fertil Steril 1992; 57: 1110–13. 87. Van Voorhis BJ, Syrop CH, Vincent RD Jr, et al. Tubal versus uterine transfer of cryopreserved embryos: a prospective randomized trial. Fertil Steril 1995; 63: 578–83. 88. Preutthipan S, Amso N, Curtis P, Shaw RW. A prospective randomized crossover comparison of zygote intrafallopian transfer and in vitro fertilization–embryo transfer in unexplained infertility. J Med Assoc Thai 1994; 77: 599–604. 89. Tournaye H, Devroey P, Camus M, et al. Zygote intrafallopian transfer or in vitro fertilization and embryo transfer for the treatment of male-factor infertility: a prospective randomized trial. Fertil Steril 1992; 58: 344–50. 90. Fluker MR, Zouves CG, Bebbington MW. A prospective randomized comparison of zygote intrafallopian transfer and in vitro fertilization–embryo transfer for nontubal factor infertility. Fertil Steril 1993; 60: 515–19. 91. Balmaceda JP, Alam V, Roszjtein D, et al. Embryo implantation rates in oocyte donation: a prospective comparison of tubal versus uterine transfers. Fertil Steril 1992; 57: 362–5. 92. Clayton HB, Schieve LA, Peterson HB, et al. Ectopic pregnancy risk with assisted reproductive technology procedures. Obstet Gynecol 2006; 107: 595–604. 93. Weissman A, Eldar I, Farhi J, et al. Zygote intrafallopian transfer (ZIFT) in patients with repeated implantation failure: ten years experience of a single center. In: 61st Annual Meeting of the American Society for Reproductive Medicine, Montreal, Canada, 2005: S358–9. 94. Milki AA, Hardy RI, el Danasouri I, Giudice LC, Lamb EJ. Local anesthesia with conscious sedation for laparoscopic intrafallopian transfer. Fertil Steril 1992; 58: 1240–2. 95. Scott LA, Smith S. The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum Reprod 1998; 13: 1003–13. 96. Jansen RP, Anderson JC, Sutherland PD. Nonoperative embryo transfer to the fallopian tube. N Engl J Med 1988; 319: 288–91. 97. Jansen RP, Anderson JC. Catheterisation of the fallopian tubes from the vagina. Lancet 1987; 2: 309–10. 98. Ferraiolo A, Croce S, Anserini P, et al. ‘Blind’ transcervical transfer of gametes in the fallopian tube: a preliminary study. Hum Reprod 1991; 6: 537–40. 99. Diedrich K, Bauer O, Werner A, et al. Transvaginal intratubal embryo transfer: a new treatment of male infertility. Hum Reprod 1991; 6: 672–5. 100. Scholtes MC, Roozenburg BJ, Verhoeff A, Zeilmaker GH. A randomized study of transcervical intrafallopian

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transfer of pronucleate embryos controlled by ultrasound versus intrauterine transfer of four- to eight-cell embryos. Fertil Steril 1994; 61: 102–4. Jansen RP, Anderson JC. Transvaginal versus laparoscopic gamete intrafallopian transfer: a casecontrolled retrospective comparison. Fertil Steril 1993; 59: 836–40. Seracchioli R, Possati G, Bafaro G, et al. Hysteroscopic gamete intra-fallopian transfer: a good alternative, in selected cases, to laparoscopic intrafallopian transfer. Hum Reprod 1991; 6: 1388–90. Vorsselmans A, Platteau P, De Vos A, et al. Comparison of transfers to fallopian tubes or uterus after ICSI. Reprod Biomed Online 2003; 7: 82–5. Sahebkashaf H, Aleyassin A, Saidi H, et al. 4000 cases of intrafallopian transfer of ova immediately after intracytoplasmic sperm injection (RAPID ICSI-ZIFT) for treatment of severe male factor infertility. Fertil Steril 2001; 76 (Suppl 1): S2. Alleyassin A, Khademi A, Aghahosseini M, et al. Comparison of unilateral and bilateral transfer of injected oocytes into fallopian tubes: a prospective, randomized clinical trial. Fertil Steril 2006; 85: 96–100. Knickerbocker JJ, Thompson KA, Riley EA, et al. Hysteroscopic tubal transfer of ova after intracytoplasmic sperm injection (ICSI) as a treatment for severe male factor infertility. Fertil Steril 1997; 67: (Suppl 1): S209. Chen CD, Ho HN, Yang YS. Tubal embryo transfer improves pregnancy rate. Hum Reprod 1997; 12: 629–31. Tournaye H. Tubal embryo transfer improves pregnancy rate. Hum Reprod 1997; 12: 620–31. Templeton A, Morris JK. Reducing the risk of multiple births by transfer of two embryos after in vitro fertilization. N Engl J Med 1998; 339: 573–7. Levran D, Mashiach S, Dor J, Levron J, Farhi J. Zygote intrafallopian transfer may improve pregnancy rate in patients with repeated failure of implantation. Fertil Steril 1998; 69: 26–30. Aslan D, Elizur SE, Levron J, et al. Comparison of zygote intrafallopian tube transfer and transcervical uterine embryo transfer in patients with repeated implantation failure. Eur J Obstet Gynecol Reprod Biol 2005; 122: 191–4. Nabi A, Awonuga A, Birch H, Barlow S, Stewart B. Multiple attempts at embryo transfer: does this affect in-vitro fertilization treatment outcome? Hum Reprod 1997; 12: 1188–90. Thijssen RF, Hollanders JM, Willemsen WN, et al. Successful pregnancy after ZIFT in a patient with congenital cervical atresia. Obstet Gynecol 1990; 76: 902–4. Fluker MR, Bebbington MW, Munro MG. Successful pregnancy following zygote intrafallopian transfer for congenital cervical hypoplasia. Obstet Gynecol 1994; 84: 659–61. Diedrich K, Bauer O. Indications and outcomes of assisted reproduction. Baillières Clin Obstet Gynaecol 1992; 6: 373–88. Batzofin J, Tran C, Tan T, Nelson J, Serafini P. A comparison of clinical pregnancy and delivery rates between IVF and ZIFT in women over 40 years of age. In: Genet. JAR, ed. Ninth World

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Congress on In Vitro Fertilization and Assisted Reproduction, Vienna, Austria, 1995. Balmaceda JP, Gonzales J, Bernardini L. Gamete and zygote intrafallopian transfers and related techniques. Curr Opin Obstet Gynecol 1992; 4: 743–9. Boldt J, Schnarr P, Ajamie A, et al. Success rates following intracytoplasmic sperm injection are improved by using ZIFT vs IVF for embryo transfer. J Assist Reprod Genet 1996; 13: 782–5. La Sala GB, Campari C, Montanari R, et al. A retrospective comparison of 151 tubal versus 548 uterine embryo transfer cycles. Isr J Obstet Gynecol 1999; 109: 47–54. Levran D, Dor J, Rudak E, et al. Pregnancy potential of human oocytes – the effect of cryopreservation. N Engl J Med 1990; 323: 1153–6. Frederick JL, Ord T, Stone SC, Balmaceda JP, Asch RH. Frozen zygote intrafallopian transfer: a successful approach for transfer of cryopreserved embryos. Fertil Steril 1994; 61: 504–7. Abdalla HI, Baber RJ, Kirkland A, Leonard T, Studd JW. Pregnancy in women with premature ovarian failure using tubal and intrauterine transfer of cryopreserved zygotes. Br J Obstet Gynaecol 1989; 96: 1071–5. Asch RH, Balmaceda JP, Cittadini E, et al. Gamete intrafallopian transfer. International cooperative study of the first 800 cases. Ann NY Acad Sci 1988; 541: 722–7. Diedrich K, van der Ven H, al-Hasani S, Krebs D. Establishment of pregnancy related to embryo transfer techniques after in-vitro fertilization. Hum Reprod 1989; 4: 111–14. Weissman A, Eldar I, Ravhon A, et al. Timing intrafallopian transfer procedures. Reprod Biomed Online 2007; 15: 445–50. Azem F, Yaron Y, Amit A, et al. Transfer of six or more embryos improves success rates in patients with repeated in vitro fertilization failures. Fertil Steril 1995; 63: 1043–6. Cittadini E, Palermo R. Infertility in advanced reproductive age. Results of in vitro fertilization and embryo transfer according to the woman’s age. Acta Eur Fertil 1989; 20: 285–97. Farhi J, Weissman A, Nahum H, Levran D. Zygote intrafallopian transfer in patients with tubal factor infertility after repeated failure of implantation with in vitro fertilization–embryo transfer. Fertil Steril 2000; 74: 390–3. Schoolcraft WB, Gardner DK, Lane M, et al. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril 1999; 72: 604–9. Gardner DK, Schoolcraft WB, Wagley L, et al. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 1998; 13: 3434–40. Cruz JR, Dubey AK, Patel J, et al. Is blastocyst transfer useful as an alternative treatment for patients with multiple in vitro fertilization failures? Fertil Steril 1999; 72: 218–20. Levran D, Farhi J, Nahum H, et al. Prospective evaluation of blastocyst stage transfer vs zygote intrafallopian tube transfer in patients with repeated implantation failure. Fertil Steril 2002; 77: 971–7.

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49 Embryo transfer Leif Bungum, Mona Bungum

Introduction Embryo transfer (ET) is the final and most crucial step of the in vitro fertilization (IVF) procedure, characterized by a close collaboration between the clinician and the embryologist. Without healthy, good-quality embryos, embryo transfer is likely to fail. On the other hand, in case of a traumatic transfer the embryologists’ effort can be in vain, as well as all the effort invested in monitoring hormone stimulation and oocyte retrieval. Even with the transfer of high-quality embryos, the success rates in IVF remain relatively low: only 15– 20% of the transferred embryos will implant.1 Multiple factors have impact on the pregnancy outcome, including the technique of embryo transfer.2–6 Traditionally little attention has been paid to the embryo transfer technique. Myths, subjectivity, and little evidence-based knowledge have often been the basis for clinician’s choice of transfer technique.3,7 However, the growing tendency towards transferring fewer embryos, often a single embryo, has provided further incentives to improve implantation rates in IVF. Now, to an increasing extent, the embryo transfer technique is being recognized as a critical step in IVF. During the last few years several studies have shown improvements in clinical pregnancy rates resulting from a focus on different aspects of the gentle embryo transfer technique. Issues such as impact of physicians’ experience, trial transfer, catheter type, embryo deposition site, ultrasound-guided transfer, catheter loading technique, routine antibiotics, removing cervical mucus before embryo transfer, use of tenaculum, bed rest, acupuncture, and drugs to induce uterine relaxing, to mention the probably most important, have been studied. The aim of this chapter is to give a summary of the most recent reports of embryo transfer techniques and, based on these, give ideas on how to do a technically good embryo transfer.

The laboratory part of embryo transfer Embryo selection and timing of embryo transfer With the increasing trend towards replacement of few embryos or a single embryo in IVF,8,9 selection of the

most viable embryo for transfer is of outmost importance. In recent years, with the improved understanding of embryonic development and advances in the field of assisted reproduction, the developmental stage at which an embryo can be transferred has become more advanced, and embryo selection criteria have evolved accordingly. However, although new methods to assess embryo developmental competence are developing, the traditional morphological parameters are still the gold standard in embryo quality assessment. Whereas some groups practice blastocyst stage transfer, others prefer to do embryo transfer at the cleavage stage or at the pronuclei stage. Randomized studies have reported conflicting results;10,11 however, following a recent Cochrane report, embryo transfer day 5 was found to be significantly better than day 3 transfer in good-prognosis patients. In all other categories of patients no statistical difference in regard to pregnancy rates was seen.12

Loading of the catheter The dynamics involved in the procedure of loading the embryo transfer catheter is not well understood. In a recent study where an in vitro experimental model for ET simulations was used, Eytan and colleagues reported the importance of having a gas phase in the catheter load.13 Within the uterus the air bubbles were carried towards the fundus, thereby dragging behind them the medium containing the embryos. Eytan and colleagues also found that the speed of injection upon the catheter load into the uterus could influence the results: a low speed generated several air bubbles, which led to more of the transferred media being carried towards the fundal end of the uterus and thereby a likely enhanced implantation rate. This syringe–catheter complex has also been studied clinically2,4,14 as well as in a metaanalysis of Abou-Setta;15 however, there is no consensus whether air or fluid-filled catheters provide better results. So far, the speed of injection has not been studied clinically. Extended time between loading the catheter and deposition of the embryo(s) may have an effect on the pregnancy rate,16 possibly influenced by light,17

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temperature,18 and other environmental factors19 in which detrimental effects seem to increase during the time the embryos spend from the incubator to the uterus. Embryos may be retained in the catheter, which necessitates a retransfer. In order to minimize the risk of retained embryos, a slow withdrawal of the transfer catheter is recommended.13,20 Seen in light of the potential extended time for the embryos outside the incubator or uterus, it is understandable that Visser et al found a decreased pregnancy rate from 20% to 3% when embryos were retained and retransferred.21 However, their finding is controversial, as others have found no significant difference in pregnancy rate when retained embryos were retransferred.22,23 A systematic review of our own results, based on more than 5000 embryo transfers, demonstrated retained embryos in 1% of the transfers. In this group of patients the pregnancy result was similar to that of the other normal embryo transfers group (L Bungum, unpublished work). The importance of retransferring the embryos as fast as possible should, however, be stressed. Possible reasons for retained embryos are plugging of the tip with mucus or uterine tissue or the position of embryos in the catheter. Also, larger volumes of fluid transferred (60 µl) have been shown to result in retained embryos.24 Nevertheless, it is advisable to have a certain amount of media, as it will help to push out the embryo(s).25 Going back to the laboratory model of Eytan et al, they found evidence for using very small volumes of fluid (3 µl).13 However, their result is not clinically verified, and stands in contrast to findings of the previous mentioned studies.24 Which type of fluid or media the embryo should be transferred in has also been a matter of debate, ranging from the use of fibrin sealants26 and increased protein content,27 to supplement of hyaluronan.28 Enrichment of the embryo transfer medium with hyaluronan is probably the most physiological and now promising alternative; however, here also, the results of randomized controlled studies have been conflicting.29,30 Larger studies should be conducted. Our own practice is to load the Cook Sydney (Cook, Australia) inner catheter with a continuous column (20 µl) of the standard culture media (GIII-2, Vitrolife, Sweden) supplemented with 5% recombinant human albumin (GMM, Vitrolife) followed by the embryo(s) loaded towards the tip of the catheter with a small air column closest to the catheter opening. The syringe used is a sterile Hamilton Thorne glass syringe (Hamilton Thorne, UK). Other types of syringes should be tested before use to confirm that there is no embryo toxicity associated with them. During the transfer procedure, prior to loading of the catheter, the clinician always performs a trial transfer. While the embryologist is loading the embryo(s) into the inner catheter, the clinician keeps the outer sheath of the catheter in the cervical canal. Immediately after the transfer, the catheter is inspected under a light microscope for retained embryos.

The clinical part of embryo transfer Physician skills Any gynecologist can insert a transfer catheter through the cervical canal and dispose an embryo into the uterine cavity. One should, however, be aware of the possible variety in success rate bound to the individual performer in terms of obtaining a pregnancy. While some authors have not seen any difference in implantation rate between individual physicians,21 others state an important impact of ET providers’ skills in performing the procedure.31 Two papers reporting the result of more than 1500 cycles show implantation rates varying significantly between clinicians.25,31 In general, inter-individual differences in performance of the embryo transfer procedure is hard to prove. However, since the final outcome is influenced by blood/mucus, uterine contractions, and retained embryos, which in turn depends upon individual technical skills, it is likely that the clinician’s experience has an impact. Solid reports from programs involving a number of embryo transfer providers are likely to be trustworthy and state that important confounding parameters such as patient selection, stimulation protocols, embryo quality, and catheter type do not differ between the ET providers.25,31

Gentle atraumatic transfer This term refers to two important factors in optimizing the embryo transfer technique – namely, avoiding damage to the endometrium and unwanted uterine contractions – both of which appear more often in so-called difficult transfer and also have a clear impact on the clinical pregnancy rate.5,32 The term ‘gentle atraumatic transfer’ also covers important clinical features – i.e. a painless transfer with no problems passing the cervical canal and proper delivery of the embryo(s) within the uterine cavity – disposed at a level in accordance with the intention of the performer. A gentle atraumatic transfer is characterized by the lack of blood on the catheter tip, indicating bleeding from the endometrium. Embryo transfer may be performed blindly directly or after a trial transfer exploring eventual difficulties and measuring transfer depth, or it may be an ultrasoundguided procedure. Independent of the method chosen, a transfer may be classified as easy or difficult. Mansour et al33 estimated that up to 30% of the transfers could be classified as difficult. Whereas an easy transfer in most cases has no noxious impact on the anatomy and physiological processes within the uterus, a difficult transfer most probably does have such an impact.

Damage to the endometrium A frequent injury left by difficult transfers or by transfers where a suboptimal technique is used is damage to the endometrium with a subsequent negative impact on

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the receptivity of the endometrium and/or initiation of uterine contraction, which ultimately may displace or expel the embryos from the uterine cavity.24 Damage to the endometrium is associated with a diminished implantation rate after embryo transfer and may be the result of several factors such as bleeding, inflammation, and plugging of the catheter tip, which in turn may retain the embryos within the catheter.34,35 Even when the transfer appears easy and no blood is visible at the catheter tip, data from hysteroscopy performed after trial transfer show that significant harm can be done to the endometrium during a transfer procedure.36,37 However, it is important to bear in mind how difficult it is to emphasize the degree of ease or difficulty, because of the nature of its subjectivity.

Uterine contractions Uterine contractions are a physiological part of the activity in the uterine musculature.38 During the menstrual period the contractions form waves that can be directed towards the uterine cervix (anterograde). Successively, the direction of the waves turns towards a fundal direction (retrograde), which is the most prominent during ovulation and the early luteal phase when implantation occur.39 The quantity of these wavelike contractions has been found to negatively affect implantation rates after IVF.40 The practical impact of this observation is the fact that anterograde contractions may be provoked during the transfer procedure. In a study using radiopaque dye in trial transfer, only 58% of the dye stayed in the uterine cavity and the rest (immediately or delayed) expelled to the cervix and vagina.41 It is therefore logical to assume that transferred embryos may have the same fate, which in fact has also been observed and described previously.34,35 To prevent these unwanted uterine contractions, some precautions should be taken, such as using a soft transfer catheter,42,43 avoiding touching the uterine fundus,44 and manipulating gently while performing the transfer procedure.20 Moreover, as far as possible, one should avoid using a tenaculum. Such stimulation of the cervix may provoke uterine contractions,45 originating from a provoked release of oxytocin,46 which may have a negative impact on the final outcome of the treatment. In this context, using drugs to induce uterine relaxation to prevent potential harmful contractions at the time of embryo transfer is a logical approach to optimizing the final outcome. Thus far, vaginal progesterone, which resembles the hormonal sequence in a natural cycle, has been evaluated in regard to the initiation of the cycle.47 Vaginal progesterone is a commonly used luteal phase support, where the eventual additional beneficial relaxant effect of progesterone is already being implemented in the treatment.47 Recommending bed rest after embryo transfer or advising sexual abstinence relies upon the same assumptions concerning uterine contractions. Two published studies have revealed no statistically significant differences

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between bed rest or an immediate mobilization after embryo transfer.48,49 In one study sexual intercourse after embryo transfer was actually found to increase pregnancy rates.50

Tools to perform the gentle atraumatic transfer Trial of embryo transfer The value of a trial transfer in a nontreatment cycle has also been evaluated.33 Mansour and colleagues concluded that such a procedure would optimize the passage through the cervical canal, which in turn positively aids the implantation. It appears today, with improved ultrasound equipment, that this can be easily achieved by a careful examination of all levels of the uterus, including the optimal transfer depth.33,51 Performing a trial transfer at the time of oocyte retrieval, 2–5 days before embryo transfer, does not have a deleterious effect on the endometrium.52 On the other hand, the value of a trial transfer ahead of the embryo transfer has been questioned,53 due to the changed uterine anatomy between the trial and the real transfer.54 Miller and Frattarelli found that uterine depth significantly differed between the blind precycle trial transfer measurement and the ultrasound-guided ET measurement.54 Another variant practiced in our clinic is to perform the trial transfer within the same procedure as the real embryo transfer. The outer sheath of a soft catheter is inserted in the cervical canal, and the inner catheter is gently introduced through the internal os. Thereafter, the inner catheter is removed and left to the embryologist for loading of the catheter and completion of the procedure.

Choice of embryo transfer catheter Two recently published meta-analysis demonstrated an increased chance of clinical pregnancy in favor of soft transfer catheters.42,43 The rigid tight difficult transfer (TDT) catheter was compared with both soft catheters and other rigid catheters, showing a decreased chance of clinical pregnancy when the TDT catheter was used. It is likely that this finding comes down to the important feature of not harming the sensitive endometrium during the transfer procedure.

Embryo transfer with a full bladder The advantage of performing embryo transfer with a full bladder is a straightening of the uterocervical angle, which in a number of cases can simplify the introduction of the transfer catheter. The experience must be characterized as empirical, since no randomized trials have been published.

Depth of embryo replacement The site of embryo deposition has attracted a great deal of interest. A too-deep placement of the catheter

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tip increases the chance of fundal touch, jeopardizing the chance of pregnancy by causing damage to the endometrium and initiating uterine contractions. One report revealed that touching the fundus with the catheter tip stimulated harmful junctional zona contractions;35 another report stated that placing embryos 2 cm below the fundus was superior to a higher replacement.44 A study where ultrasonography was combined with ‘blind transfer’ for the performer, revealed a high rate of fundal touch or tubal ostia.34 One may conclude from the cited well-designed studies that paying attention to the depth of embryo deposition is an important issue in order to improve pregnancy rates.

Ultrasound-guided embryo transfer The value of ultrasound-guided embryo transfer has been debated for years. Ultrasound may be used in two ways: •

•

a careful examination of the cervical canal, with special emphasis on the cervical uterine angulations where glands may represent a possible starting point of a ‘via falsa’ calculating the embryo disposition depth prior to a subsequent ‘blind’ transfer.

Ultrasound used this way may represent a visualized guide immediately ahead of the transfer procedure. Embryo transfer may be guided by abdominal ultrasound through a filled bladder or vaginal ultrasound where the catheter has been introduced in the cervical canal before the ultrasound probe is applied to the vagina. A positive impact of this procedure on the pregnancy rate compared with clinical touch transfer has been shown in a meta-analysis of Buckett.55 In contrast, a recently published randomized trial including nearly 2300 patients could not confirm this finding.56

Cervical mucus Removing cervical mucus or flushing the cervical canal before embryo transfer has gained some focus in the literature. Hypothetically, two issues may be connected: first, the introduction of bacteria from the mucus into the uterine cavity during the transfer process; secondly, the possibility of the mucus covering the tip of the catheter causing interference with the deposition by retaining the embryos or dragging them into the cervix by the withdrawal of the catheter. The cervical mucus represents a mechanical as well as an immunological barrier between the sterile uterine cavity and the bacterial colonized vagina, and the mucal concentrations of immunoglobins vary through the menstrual cycle. However, the reported studies23,57 are not consistent with respect to the impact of removing

mucus. The findings can be interpreted in different ways. For instance, blood and mucus at the catheter tip associated with a negative effect on pregnancy rate because of mucus-associated problems, may instead be associated with traumatic transfer with damage to the endometrium.23 A randomized study by Visschers et al did not reveal any difference between the groups.57 Removing mucus is difficult, and may represent a noxious stimulus to the uterus by rigorous brushing or irrigations in the cervical canal, initiating unwanted contractions. Regarding the impact of bacterial contamination from cervical mucus, a recent randomized study could not justify routine use of antibiotics in connection with embryo transfer,58 whereas previous studies had shown a detrimental effect of bacterial contamination at the catheter tip.59

Acupuncture Although the evidence for the efficacy of acupuncture in relation to assisted reproductive technologies is limited, and the exact mechanisms of action are unknown, many IVF patients consult an acupuncturist during the treatment, in the hope of increasing their chance of obtaining a pregnancy. One paper reported an increased blood flow to the uterus;60 however, it is not known whether this is a positive feature or not. The few published randomized trials show marginal positive61 or no effect.62

Conclusion So far, the optimal embryo transfer protocol is not known. However, during the last few years an increasing number of publications have led to a more optimal transfer technique that contributes to better pregnancy results. In several of the issues thought to have impact on the implantation rates, data are conflicting or well-designed prospective, randomized trials are lacking. Still, we need to learn more. However, what is well accepted today is the importance of a gentle atraumatic transfer where no damage to the endometrium and minimal uterine contractions are induced. In order to succeed, it is advisable to perform a trial transfer, using a soft catheter and ultrasound to find the correct deposition site, which is 2 cm below the fundus. Moreover, the collaboration between the clinician and the embryologist appears to be important. The time from the embryo leaving the incubator until it is deposited in the uterine cavity is of utmost importance. How to load the catheter, with air or fluid, the amount of volume, or whether the transfer media should be enriched are more controversial issues. Following embryo transfer, no particular factor other than progesterone luteal phase support has been shown to have a positive effect on pregnancy outcome.

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Appendix: Protocol for embryo transfer • • •

• • •

• • •

Full bladder. Wash cervix with embryo culture media. Inspection of the cervical canal by ultrasound with special emphasis on the cervical uterine angulations where glands may represent a possible starting point of a ‘via falsa’ and to measure out the embryo disposition depth. Trial transfer to the internal os. Keep the outer sheath of the catheter in the cervical canal. Using a sterile Hamilton Thorne glass syringe (Hamilton Thorne, UK), load the Cook Sidney (Cook, Australia) or other preferred soft inner catheter with a continuous column (20 µl) of culture media followed by the embryo(s) loaded towards the tip of the catheter with a small air column closest to the catheter opening. Gentle insertion and deposition of embryo(s). Slow withdrawal of the transfer catheter. Immediate inspection of the catheter under a light microscope for retained embryos, blood, and mucus.

References 1. Edwards RG. Clinical approaches to increasing uterine receptivity during human implantation. Hum Reprod 1995; 10 (Suppl 2): 60–6. 2. Meldrum DR, Chetkowski R, Steingold KA, et al. Evolution of a highly successful in vitro fertilization–embryo transfer program. Fertil Steril 1987; 48: 86–93. 3. Kovacs GT. What factors are important for successful embryo transfer after in-vitro fertilization? Hum Reprod 1999; 14: 590–2. 4. Schoolcraft WB, Surrey ES, Gardner DK. Embryo transfer: techniques and variables affecting success. Fertil Steril 2001; 76: 863–70. 5. Sallam HN. Embryo transfer: factors involved in optimizing the success. Curr Opin Obstet Gynecol 2005; 17: 289–98. 6. Eytan O, Zaretsky U, Jaffa AJ, Elad D. In vitro simulations of embryo transfer in a laboratory model of the uterus. J Biomech 2007; 40: 1073–80. 7. Salha OH, Lamb VK, Balen AH. A postal survey of embryo transfer practice in the UK. Hum Reprod 2001; 16: 686–90. 8. Vilska S, Tiitinen A, Hyden-Granskog C, Hovatta O. Elective transfer of one embryo results in an acceptable pregnancy rate and eliminates the risk of multiple birth. Hum Reprod 1999; 14: 2392–5. 9. Hamberger L, Hardarson T, Nygren KG. Avoidance of multiple pregnancy by use of single embryo transfer. Minerva Ginecol 2005; 57: 15–19. 10. Bungum M, Bungum L, Humaidan P, Yding Andersen C. Day 3 versus day 5 embryo transfer: a prospective randomized study. Reprod Biomed Online 2003; 7: 98–104.

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11. Gardner DK, Vella P, Lane M, et al. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 1998; 69: 84–8. 12. Blake DA, Farquhar CM, Johnson N, Proctor M. Cleavage stage versus blastocyst stage embryo transfer in assisted conception. Cochrane Database Syst Rev 2007; 4: CD002118. 13. Eytan O, Elad D, Jaffa AJ. Evaluation of the embryo transfer protocol by a laboratory model of the uterus. Fertil Steril 2007; 88: 485–93. 14. Moreno V, Balasch J, Vidal E, et al. Air in the transfer catheter does not affect the success of embryo transfer. Fertil Steril 2004; 81: 1366–70. 15. Abou-Setta AM. Air fluid versus fluid-only models of embryo catheter loading: a systematic review and meta-analysis. Reprod Biomed Online 2007; 14: 80– 4. 16. Matorras R, Mendoza R, Exposito A, RodriguezEscudero FJ. Influence of the time interval between embryo catheter loading and discharging on the success of IVF. Hum Reprod 2004; 19: 2027–30. 17. Smith LC. Membrane and intracellular effects of ultraviolet irradiation with Hoechst 33342 on bovine secondary oocytes matured in vitro. J Reprod Fertil 1993; 99: 39–44. 18. Rocha A, Randel RD, Broussard JR, et al. High environmental temperature and humidity decrease oocyte quality in Bos taurus but not in Bos indicus cows. Theriogenology 1998; 49: 657–65. 19. Cohen J, Gilligan A, Esposito W, Schimmel T, Dale B. Ambient air and its potential effects on conception in vitro. Hum Reprod 1997; 12: 1742–9. 20. Mansour RT, Aboulghar MA. Optimizing the embryo transfer technique. Hum Reprod 2002; 17: 1149–53. 21. Visser DS, Fourie FL, Kruger HF. Multiple attempts at embryo transfer: effect on pregnancy outcome in an in vitro fertilization and embryo transfer program. J Assist Reprod Genet 1993; 10: 37–43. 22. Goudas VT, Hammitt DG, Damario MA, et al. Blood on the embryo transfer catheter is associated with decreased rates of embryo implantation and clinical pregnancy with the use of in vitro fertilization–embryo transfer. Fertil Steril 1998; 70: 878–82. 23. Nabi A, Awonuga A, Birch H, Barlow S, Stewart B. Multiple attempts at embryo transfer: does this affect in-vitro fertilization treatment outcome? Hum Reprod 1997; 12: 1188–90. 24. Poindexter AN 3rd, Thompson DJ, Gibbons WE, et al. Residual embryos in failed embryo transfer. Fertil Steril 1986; 46: 262–7. 25. Hearns-Stokes RM, Miller BT, Scott L, et al. Pregnancy rates after embryo transfer depend on the provider at embryo transfer. Fertil Steril 2000; 74: 80–6. 26. Bar-Hava I, Krissi H, Ashkenazi J, et al. Fibrin glue improves pregnancy rates in women of advanced reproductive age and in patients in whom in vitro fertilization attempts repeatedly fail. Fertil Steril 1999; 71: 821–4. 27. Khan I, Staessen C, Devroey P, Van Steirteghem AC. Human serum albumin versus serum: a comparative

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28.

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Textbook of Assisted Reproductive Technologies study on embryo transfer medium. Fertil Steril 1991; 56: 98–101. Valojerdi MR, Karimian L, Yazdi PE, et al. Efficacy of a human embryo transfer medium: a prospective, randomized clinical trial study. J Assist Reprod Genet 2006; 23: 207–12. Loutradi KE, Prassas I, Bili E, et al. Evaluation of a transfer medium containing high concentration of hyaluronan in human in vitro fertilization. Fertil Steril 2007; 87: 48–52. Urman B, Yakin K, Ata B, Isiklar A, Balaban B. Effect of hyaluronan-enriched transfer medium on implantation and pregnancy rates after day 3 and day 5 embryo transfers: a prospective randomized study. Fertil Steril 2007 Oct 11 [Epub ahead of print]. Karande VC, Morris R, Chapman C, Rinehart J, Gleicher N. Impact of the “physician factor” on pregnancy rates in a large assisted reproductive technology program: do too many cooks spoil the broth? Fertil Steril 1999; 71: 1001–9. Abusheikha N, Lass A, Akagbosu F, Brinsden P. How useful is cervical dilatation in patients with cervical stenosis who are participating in an in vitro fertilization–embryo transfer program? The Bourn Hall experience. Fertil Steril 1999; 72: 610–12. Mansour R, Aboulghar M, Serour G. Dummy embryo transfer: a technique that minimizes the problems of embryo transfer and improves the pregnancy rate in human in vitro fertilization. Fertil Steril 1990; 54: 678–81. Woolcott R, Stanger J. Potentially important variables identified by transvaginal ultrasound-guided embryo transfer. Hum Reprod 1997; 12: 963–6. Lesny P, Killick SR, Tetlow RL, Robinson J, Maguiness SD. Embryo transfer – can we learn anything new from the observation of junctional zone contractions? Hum Reprod 1998; 13: 1540–6. Cevrioglu AS, Esinler I, Bozdag G, Yarali H. Assessment of endocervical and endometrial damage inflicted by embryo transfer trial: a hysteroscopic evaluation. Reprod Biomed Online 2006; 13: 523–7. Murray AS, Healy DL, Rombauts L. Embryo transfer: hysteroscopic assessment of transfer catheter effects on the endometrium. Reprod Biomed Online 2003; 7: 583–6. Abramowicz JS, Archer DF. Uterine endometrial peristalsis – a transvaginal ultrasound study. Fertil Steril 1990; 54: 451–4. Lyons EA, Taylor PJ, Zheng XH, et al. Characterization of subendometrial myometrial contractions throughout the menstrual cycle in normal fertile women. Fertil Steril 1991; 55: 771–4. Fanchin R, Righini C, Olivennes F, et al. Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization. Hum Reprod 1998; 13: 1968–74. Mansour RT, Aboulghar MA, Serour GI, Amin YM. Dummy embryo transfer using methylene blue dye. Hum Reprod 1994; 9: 1257–9. Buckett WM. A review and meta-analysis of prospective trials comparing different catheters used for embryo transfer. Fertil Steril 2006; 85: 728–34.

43. Abou-Setta AM, Al-Inany HG, Mansour RT, Serour GI, Aboulghar MA. Soft versus firm embryo transfer catheters for assisted reproduction: a systematic review and meta-analysis. Hum Reprod 2005; 20: 3114–21. 44. Frankfurter D, Trimarchi JB, Silva CP, Keefe DL. Middle to lower uterine segment embryo transfer improves implantation and pregnancy rates compared with fundal embryo transfer. Fertil Steril 2004; 81: 1273–7. 45. Lesny P, Killick SR, Robinson J, Raven G, Maguiness SD. Junctional zone contractions and embryo transfer: is it safe to use a tenaculum? Hum Reprod 1999; 14: 2367–70. 46. Dorn C, Reinsberg J, Schlebusch H, et al. Serum oxytocin concentration during embryo transfer procedure. Eur J Obstet Gynecol Reprod Biol 1999; 87: 77–80. 47. Fanchin R, Righini C, de Ziegler D, et al. Effects of vaginal progesterone administration on uterine contractility at the time of embryo transfer. Fertil Steril 2001; 75: 1136–40. 48. Sharif K, Afnan M, Lashen H, et al. Is bed rest following embryo transfer necessary? Fertil Steril 1998; 69: 478–81. 49. Botta G, Grudzinskas G. Is a prolonged bed rest following embryo transfer useful? Hum Reprod 1997; 12: 2489–92. 50. Tremellen KP, Valbuena D, Landeras J, et al. The effect of intercourse on pregnancy rates during assisted human reproduction. Hum Reprod 2000; 15: 2653–8. 51. Knutzen V, Stratton CJ, Sher G, et al. Mock embryo transfer in early luteal phase, the cycle before in vitro fertilization and embryo transfer: a descriptive study. Fertil Steril 1992; 57: 156–62. 52. Katariya KO, Bates GW, Robinson RD, Arthur NJ, Propst AM. Does the timing of mock embryo transfer affect in vitro fertilization implantation and pregnancy rates? Fertil Steril 2007; 88: 1462–4. 53. Stafford-Bell MA. Which factors are important for successful embryo transfer after in-vitro fertilization? Hum Reprod 1999; 14: 2678–9. 54. Miller KL, Frattarelli JL. The pre-cycle blind mock embryo transfer is an inaccurate predictor of anticipated embryo transfer depth. J Assist Reprod Genet 2007; 24: 77–82. 55. Buckett WM. A meta-analysis of ultrasound-guided versus clinical touch embryo transfer. Fertil Steril 2003; 80: 1037–41. 56. Drakeley AJ, Jorgensen A, Sklavounos J, et al. A randomized controlled clinical trial of 2295 ultrasound-guided embryo transfers. Hum Reprod 2008; 23: 1101–6. 57. Visschers BA, Bots RS, Peeters MF, Mol BW, van Dessel HJ. Removal of cervical mucus: effect on pregnancy rates in IVF/ICSI. Reprod Biomed Online 2007; 15: 310–15. 58. Brook N, Khalaf Y, Coomarasamy A, Edgeworth J, Braude P. A randomized controlled trial of prophylactic antibiotics (co-amoxiclav) prior to embryo transfer. Hum Reprod 2006; 21: 2911–15. 59. Egbase PE, Udo EE, Al-Sharhan M, Grudzinskas JG. Prophylactic antibiotics and endocervical microbial inoculation of the endometrium at embryo transfer. Lancet 1999; 354: 651–2.

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Embryo transfer 60. Stener-Victorin E, Waldenstrom U, Andersson SA, Wikland M. Reduction of blood flow impedance in the uterine arteries of infertile women with electroacupuncture. Hum Reprod 1996; 11: 1314–17. 61. Paulus WE, Zhang M, Strehler E, El-Danasouri I, Sterzik K. Influence of acupuncture on the

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pregnancy rate in patients who undergo assisted reproduction therapy. Fertil Steril 2002; 77: 721–4. 62. Domar AD, Meshay I, Kelliher J, Alper M, Powers RD. The impact of acupuncture on in vitro fertilization outcome. Fertil Steril 2008; in press.

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50 Anesthesia and in vitro fertilization Ethan E Harow

Introduction In 1978 the first child was born of an externally fertilized human egg.1 Since then the techniques for in vitro fertilization (IVF) have been widely applied. In the early stages, laparoscopic retrieval of oocytes was commonly performed. Nowadays, transvaginal ultrasound-guided oocyte retrieval (TUGOR) is the technique of choice. It is one of the most stressful components of assisted reproductive treatment.2,3 Patients are often anxious following hormonal manipulation. Anesthesia technique is important in a successful program. Patient comfort and safety are important considerations. There are many options available to the anesthesiologist. General anesthesia, monitored sedation with or without local anesthesia, and regional technique have all been used and studied. The predominant procedure requiring anesthesia is the TUGOR. There is a wide variation in technique for this procedure. In a survey of practice in the UK there was no uniformity of technique. There were 60 centers that responded to the survey. Sedation was used by 46%, while 28% used general anesthesia, and 12% used regional with sedation; the remaining 14% used a mixed technique or regional alone.4 Thus, the method of anesthesia delivery varies among practices. There are few reports to define the current standard of care. In addition, there appears to be a lack of consensus among practitioners performing assisted reproduction procedures in regard to anesthetic medications employed. This was confirmed by a recent Cochrane review5 that included 12 trials. There was no single method or delivery system that appeared superior for pregancy rates and pain relief. Pregnancy rates with in vitro fertilization improve as the number of high-quality embryos available for transfer increases. The technique used needs to focus on this goal. The various options will be explored as well as current information on some of the commonly used anesthetic agents.

Anesthetic technique used in TUGOR Sedative technique Conscious sedation is an acceptable and desirable way to produce amnesia and analgesia. It offers many

advantages to the women undergoing assisted reproduction. It is relatively easy to deliver, the drugs are well tolerated, and few immediate- or long-term side effects occur with its proper use.6 Women found oocyte aspiration to be less painful than expected.7 This lends itself to a sedative technique. Ultrasoundguided transvaginal retrieval is usually performed under parenteral sedation.8 In the USA, 95% of the programs use conscious sedation.6 In a reported study, 84% of the centers in the UK used sedation.9 It has been reported that there is a greater pregnancy rate with sedative technique (28.2%) than with general anesthesia (16.3%).10 The potential for life-threatening complications such as cardiac, respiratory, anaphylaxis, and drug interactions are present with sedative technique. Therefore, safe guidelines regarding patient preparation, sedationist equipment, and monitoring need to be established.9 The two major considerations when choosing the desired agents are whether the substances enter the follicular fluid, and if they are toxic. Fentanyl has only minimal penetration into follicular fluid.11 Meperidine reveals no influence on embryo in the mouse model.12 Midazolam fails to reveal any detrimental effects and is not found in measurable quantities in follicular fluid.13–15 Propofol has distinct advantages, with its favorable recovery and antiemetic property. The effects of propofol on fertilization, embryo cleavage, and pregnancy rates have been studied extensively. Early studies showed that increased exposure adversely affected cleavage without interfering with fertilization.16 In the mouse model, Dupypere et al17 showed an adverse affect on fertilization but not on cleavage. Propofol exhibited a lower ongoing pregnancy rate when compared with isoflurane in laparoscopic pronuclear stage transfer (PROST).18 In addition, concentrations of propofol increase in follicular fluid during oocyte retrieval.19 In a later study, it was observed that although propofol follicular concentration increased with time, there was no difference in the ratio of mature to immature oocytes. In addition, there were no differences found in fertilization, cleavage, and embryo cell number with the use of propofol.20 When compared with paracervical block, there were also no differences in fertilization rate, embryo cleavage rate, and implantation.21 When the following sedative combinations were

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compared – propofol + alfentanil, midazolam + fentanyl, and propofol + fentanyl – they did not differ significantly in terms of hemodynamic variables and recovery characteristics.2 The combination of midazolam and alfentanil did produce a prolonged recovery. Wilhelm et al,10 in a nonrandomized study, showed that a monitored anesthesia technique with remifentanil resulted in a higher pregnancy rate then a general anesthetic technique with alfentanil, propofol, and either isoflurane or propofol maintenance. Patient-controlled analgesia with propofol and alfentanil was found to be inferior to physician-controlled analgesia with diazepam and meperidine, although both methods were acceptable to the patients.3 Midazolam and ketamine sedative combination were found to be a favorable alternative to general anesthesia with fentanyl, propofol, and isoflurane.22

General anesthetic technique General anesthesia can be induced intravenously (most common) or with an inhalatory agent. Maintenance of general anesthesia can also be with a volatile agent or with intravenous anesthetics. One gas nitrous oxide (N2O) and the vapors of three volatile liquids (isoflurane, desflurane, sevoflurane) represent the commonly used inhaled anesthetics. Intravenous maintenance can be with a variety of agents or combinations thereof. This may include, but is not limited to, narcotics, sedatives, or rapid induction agents. In the earlier stages of assisted reproduction, general anesthesia was the technique of choice.21,22 At the present time, general anesthesia is the second most common technique for TUGOR, and the most common technique for laparoscopic zygote intrafallopian transfer (ZIFT) or gamete intrafallopian transfer (GIFT).8 Anesthetic agents used for either general or local anesthesia have been detected in follicular fluid.11 Propofol, midazolam, fentanyl, and alfentanil have been found in follicular fluid during transvaginal oocyte retrieval. As these general anesthetics were detected in follicular fluid, concern arose that these drugs may be potentially harmful to the oocyte and thus might interfere with IVF success. Early studies reported that general anesthesia adversely affected fertilization rates and possibly cleavage rates.24–26 Thiopental and thiamylal concentrations in follicular fluid are greater than plasma concentrations up to 50 minutes post-intravenous administration.11 The vast majority of studies showed that halogenated fluorocarbons and N2O were detrimental, causing decreased cleavage rate and increased spontaneous abortion.8 A decreased fertilization rate of oocytes collected after prolonged exposure to 50% N2O and 1% isoflurane or enflurane anesthesia was reported by Jensen et al.27 Propofol with N2O was associated with a lower clinical and ongoing pregnancy rate compared with isoflurane for laparoscopic PROST.18 However, balanced anesthesia with N2O and an opioid is a most appealing option of several authors.8

Hammedah et al28 showed a higher retrieval of oocytes with remifentanil + propofol or isoflurane-based general anesthesia than with sedation with midazolam, diazepam, or propofol. This was attributed to improved comfort for both the patient and the gynecologist during the transvaginal puncture procedure. The higher number of retrieved oocytes had a lower fertilization rate, resulting in an almost identical mean number of fertilized oocytes per patient. Oocytes obtained from smaller follicles show a lower rate of fertilization.29 With the increased comfort of general anesthesia, more of the smaller follicles are aspirated. Isoflurane has been shown to inhibit mouse embryo development, which was not seen with a balanced anesthesia of nitrous oxide and narcotic.3 Fertilization and cleavage of mature oocytes collected during TUGOR were analyzed following general anesthesia or intravenous sedation. There were no significant differences between the first and last collected oocyte, except for a trend towards lower fertilization rate with longer exposure to anesthetic drugs.27 The key is to aim for a pharmacologic exposure of the shortest duration.30 It has been our experience in over 1000 cases that a balanced anesthetic technique gives a very satisfactory result. Patients are given midazolam 1 mg or a small dose of propofol (20 mg) intravenously while being prepared for general anesthesia. This is then followed by a 50 µg dose of fentanyl, and propofol (1.5–2 mg/kg). Spontaneous ventilation via face mask is maintained with 50/50% of O2/N2O. Incremental doses of propofol 20–40 mg are given as necessary to maintain optimal operating conditions. Postoperative pain is controlled with nonsteroidal anti-inflammatory drugs (NSAIDs) and dipyrone. Patients are discharged to home within 1–2 hours, with negligible nausea and vomiting.

Regional anesthesia technique Paracervical block using lidocaine 1% 50–200 mg with sedation has been used for egg retrieval. Doses of 150 mg were found to be equally effective as 200 mg.31 Locoregional when compared with general anesthesia in a meta-analysis showed similar cleavage and pregnancy rates.32 Spinal anesthesia is a viable alternative to general or sedation for oocyte retrieval. In an early study, spinal anesthesia was used successfully for laparoscopic egg retrieval.33 Martin et al used a combination of low-dose fentanyl (10 µg) and low-dose hyperbaric 1.5% lidocaine (45 mg) spinal for TUGOR.34 This combination left the patients comfortable during the procedure and in the post-anesthetic care unit. Tsen et al compared low-dose spinal bupivacaine (3.75 mg) with fentanyl (25 µg) to lidocaine (30 mg) and fentanyl (25 µg).35 Other than taking approximately 30 minutes longer to micturation and to discharge, the bupivacaine compared favorably to the lidocaine in all aspects. Epidural anesthesia is also an effective method for transvaginal oocyte retrieval. However, the use of epidural did not demonstate any advantage over intravenous sedation using propofol and

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nitrous oxide.36 Epidural has also been found to be a safe anesthetic for GIFT.37

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of this agent are found in follicular fluid, it fails to show any detrimental effects.13,14 It is recommended for use to induce sedation in human IVF.45 A combination of midazolam and fentanyl was found to be a good selection for transvaginal oocyte retrieval.2

Propofol

Narcotics

Propofol is a useful agent in assisted reproduction and is chemically unrelated to earlier anesthetic drugs. It is a highly lipophilic agent with a fast onset and a short, predictable duration of action due to its rapid penetration of the blood–brain barrier and distribution to the central nervous system.38 An increased amount of propofol is recovered in follicular fluid with increased dose and time.19,30 When compared with thiopental, there were no significant differences for fertilization rate, cleavage rate, pregnancy rate, implantation, and abortion rate.39 Data reveal no harmful effect on the oocyte, fertilization, cleavage, or early embryo development.20 It was found to be a favorable agent when used in combination with fentanyl or with alfentanil.9

Remifentanil-based general anesthesia without nitrous oxide is a suitable alternative to sedation and may be recommended for oocyte retrieval.28 Fentanyl and meperidine did not show adverse effects on mouse embryo development.12 Fentanyl has only minimal penetration into follicular fluid.46 The alfentanil follicular fluid level is 10-fold lower than the serum concentrations at the same time points.47 Fentanyl or alfentanil were found to be favorable agents when used in combination with propofol.2,20

Nitrous oxide The use of nitrous oxide (N2O) in TUGOR in either general or sedative technique is controversial. N2O inactivates methionine synthetase, thereby diminishing the amount of thymidine available for DNA synthesis in dividing cells; this effect lasts 24–72 hours. Nitrous oxide has been found to be toxic to two-cell mouse embryos.40 Gonen et al,41 in a nonrandomized study, concluded that N2O has a deleterious effect on IVF outcome, which manifests during post-embryo transfer. This effect leads to a lower clinical pregnancy and delivery rate. However, the inactivation of methionine synthetase proceeds slowly in the human liver, and thus the effect of N2O is minimal. In addition, the low solubility of N2O exposes the oocyte to this gas for only a short period, as rapid transfer to an O2–CO2 media occurs after oocyte retrieval. Nutrientrich media also afford protection to the oocyte from the effects of N2O on methionine synthetase. N2O may actually increase the rate of in vitro fertilization by reducing the concentration of other potentially toxic and less diffusible anesthetics.42 Nitrous oxide was found not to inhibit mouse embryo development.12 There was no significant difference between rate of fertilization or pregnancy when comparing isoflurane and nitrous oxide vs isoflurane and oxygen.42 These results were also confirmed by Matt et al.43

Midazolam Midazolam is probably the most commonly used benzodiazepine for conscious sedation. Midazolam has no adverse effect on fertilization and on mouse embryo development.44 Although minimal amounts

Neuroleptanesthesia Neuroleptanesthesia can be produced by a combination of droperidol and fentanyl. Droperidol produces hypnosis, sedation, and antiemetic effects, while fentanyl produces analgesia. This combination has been shown to cause extreme endocrinological adverse effects.8 Higher plasma prolactin levels and lower plasma progesterone levels were observed in the neuroleptanesthesia group than in the halothane group during and after TUGOR. 48

Inhalatory agents Isoflurane inhibits early mouse embryo development in vitro.12 Combinations of halogenated fluorocarbons and nitrous oxide were found to be detrimental in the majority of studies.8 They caused decreased cleavage rates and increased spontaneous abortions. Matt et al43 did not find a significant effect of nitrous oxide and isoflurane anesthesia on human IVF pregnancy rates.

Ketamine In a randomized prospective study, the combination of midazolam and ketamine was found to be a good sedative alternative to general anesthesia. A comparable number of oocytes were found, with no difference in rate of embryo transfers and pregnancies between the two groups. Patient satisfaction was similar. 22

Alternative therapy for female infertility Acupuncture Acupuncture is an element of traditional Chinese medicine traced back at least 4000 years. It has both

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physiologic and psychologic benefits.49 Among its many uses are the relief of nausea and vomiting, dental pain, addiction, headache, menstrual cramps, tennis elbow, fibromyalgia, myofascial pain, osteoarthritis, asthma, and carpal tunnel syndrome.50 However, there are few quality clinical acupuncture studies with properly designed and conducted randomized clinical trials.51 Acupuncture affects the hypothalamic–pituitary– ovarian axis and the pelvic organs, and reduces anxiety and stress. Acupuncture restores patterns of energy flow disrupted during disease states. It increases β-endorphin levels up to 24 hours’ post treatment. β-Endorphin precursors are found in the hypothalamus, pituitary, medulla, and peripheral tissues, including intestines and ovaries. Aleem et al52,53 demonstrated the presence of immunoreactive β-endorphins in follicular fluids of both normal and polycystic ovaries. β-Endorphin levels are impacted by acupuncture treatment, which in turn affects gonadotropin-releasing hormone secretion and the menstrual cycle. Therefore, it may influence ovulation and fertility. Ovulatory cycles occur significantly more often in acupuncture groups compared with the control group, however, and with equal pregnancy rates. Acupuncture has a central sympathoinhibitory effect and may therefore contribute to a decreased uterine artery impedance and, in turn, increase uterine artery blood flow. This can have the salutary effect of increasing endometrial thickness, and increasing the success rate of implantation. Paules et al,50 in a randomized prospective study, compared pregnancy rates in 160 patients undergoing IVF. Acupuncture was performed in 80 patients at 25 minutes before and after embryo transfer (ET). The clinical pregnancy rate for the acupuncture group was greater than for the control group (42.5% vs 26.3%; p = 0.03). Fertility can be influenced by stress, and this can increase during the IVF process. Stress hormones influence the ovulatory menstrual cycle through the hypothalamic–pituitary–ovarian axis. Acupuncture induces in many individuals an increased sense of well-being, calmness, and improved sleep. It appears almost as effective as antidepressant drugs in the treatment of some patients with anxiety and depression. 49 Electroacupuncture and auricular electroacupuncture have been used and proved to be effective in reducing analgesia requirements during IVF.54,55 However, electroacupuncture cannot be recommended as the sole pain-relieving method at oocyte aspiration. It might be an alternative for women desiring a nonpharmacologic method. Another advantage is less postoperative tiredness and confusion.56 Thus, acupuncture is a nontoxic, relatively affordable therapy with possible indications as an adjunct in assisted reproduction. It offers an alternative for women who are intolerant, ineligible, or contraindicated for conventional anesthesia and analgesia (see Table 50.1).

Table 50.1

Beneficial effects of acupuncture in infertility

1. 2. 3. 4.

Sympathoinhibitory Increased β-endorphin levels Antidepressant, antianxiolytic Neuroendocrine effect on hypothalamic–pituitary–ovarian axis 5. Increased uterine blood flow

Conclusion For nearly three decades there has been tremendous progress in the field of assisted reproduction. The techniques employed in aspiration of the oocyte and the laboratory manipulations have all been modified and updated. The anesthetic, which is important to the comfort of the patient and for the gynecologist to maximize the harvesting of oocytes, plays an important role in a successful outcome. The anesthetic agents must be short acting, with minimal side effects. They should have little penetration into the follicle, and the oocyte should not be harmed by their presence. The key is short exposure to the least toxic agent. Neuroleptanesthesia is not recommended, and there remains some controversy over the use of nitrous oxide and the inhalatory agents. The majority of anesthetic agents have been deemed safe for use.

References 1. Steptoe PC, Edwards RG. Birth after the reimplantation of a human embryo. Lancet 1978; 2: 336. 2. Hadimioglu N, Titiz T, Dosemeci L, Erman M. Comparison of various sedation regimens for transvaginal oocyte retrieval. Fertil Steril 2002; 78(3): 648–9. 3. Lok I, Chan M, Chan D, et al. A prospective randomized trial comparing patient-controlled sedation using propofol and alfentanil and physician-administered sedation using diazepam and pethidine during transvaginal ultrasound-guided oocyte retrieval. Hum Reprod 2002; 17(8): 2101–6. 4. Bokhari A, Pollard B. Anaesthesia for assisted conception; a survey of UK practice. Eur J Anaesthesiol 1999; 16(4): 225–30. 5. Kwan I, Bhattacharya S, Knox F, McNeil A. Conscious sedation and analgesia for oocyte retrieval during in vitro fertilization procedures. Cochrane Database Syst Rev 2005; 3: CD004829. 6. Ditkoff E, Plumb J, Selick A, Sauer M. Anesthesia practices in the United States common to in vitro fertilization (IVF) centers. J Assist Reprod Genet 1997; 14(3): 145–7. 7. Gejermall AL, Sener-Victorin E, Cerne A, et al. Pain aspects in oocyte aspiration for IVF. Reprod Biomed Online 2007; 14(2): 184–90. 8. Jennings J, Moreland K, Peterson CM. In vitro fertilisation: a review of drug therapy and clinical management. Drugs 1996; 52(3): 313–43. 9. Elkington N, Kehoe J, Acharya U. Recommendations for good practice for sedation in assisted conception. Hum Fertil (Camb) 2003; 6: 77–80.

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Anesthesia and in vitro fertilization 10. Wilhelm W, Hammadeh M, White P, et al. General anesthesia versus monitored anesthesia care with remifentanil for assisted reproductive technologies: effect on pregnancy rate. J Clin Anesth 2002; 14: 1–5. 11. Endler GC, Stout M, Magyar DM, et al. Follicular fluid concentrations of thiopental and thiamylal during laparoscopy for oocyte retrieval. Fertil Steril 1987; 48: 828–33. 12. Chetkowski R, Nass T. Isoflurane inhibits early mouse embryo development in vitro. Fertil Steril 1988; 49: 171–3. 13. Schnell VL, Ataya K, Sacco A, et al. Midazolam at physiological levels does not adversely affect mouse in vitro fertilization, embryo development, and cleavage rate [abstract]. Proc 45th Meeting of the American Fertility Society, November 11–16, 1989, San Francisco, Birmingham, AL: American Fertility Society, 1989: 109. 14. Chapineau J, Bazin J-E, Terrisse M-P, et al. Assay for midazolam in liquor folliculi during in vitro fertilization under anesthesia. Clin Pharm 1993; 12: 770–3. 15. Soussis I, Boyd O, Paraschos T, et al. Follicular fluid levels of midazolam, fentanyl, and alfentanil during transvaginal oocyte retrieval. Fertil Steril 1995; 64(5): 1003–7. 16. Palot M, Harika G, Visseaux H, et al. [Use of nitrous oxide in general anesthesia for oocyte retrieval]. Ann Fr Anesth Reanim 1989; 8: R147 [in French]. 17. Dupypere HT, Dhont M, De Sutter P, et al. The influence of propofol on in vitro fertilization in mice. Program of the 7th World Congress on IVF and Assisted Procreations June 30 to July 3, 1991. Paris: World Congress on IVF and Assisted Procreations, 1991: 151. 18. Vincent R, Syrop C, Van Voorhis B, et al. An evaluation of the effect of anesthetic technique on reproductive success after laparoscopic pronuclear stage transfer. Anesthesiology 1995; 82: 352–8. 19. Christiaens F, Janssenswillen C, Verbough C, et al. Propofol concentration in follicular fluid during general anaesthesia for transvaginal oocyte retrieval. Hum Reprod 1999; 14(2): 345–8. 20. Ben-Shlomo I, Moskovich R, Golan J, et al. The effect of propofol anaesthesia on oocyte fertilization and early embryo quality. Hum Reprod 2000; 15(10): 2197–9. 21. Christiaens F, Janssenswillen C, Van Steirteghem AC, et al. Comparison of assisted reproductive technology performance after oocyte retrieval under general anaesthesia (propofol) versus paracervical local anaesthesia block: a case-controlled study. Hum Reprod 1998; 13: 2456–60. 22. Ben-Shlomo I, Moskovich R, Katz Y, Shalev E. Midazolam/ketamine sedative combination compared with fentanyl/propofol/isoflurane anaesthesia for oocyte retrieval. Hum Reprod 1999; 14(7): 1757–9. 23. Ben-Shlomo I, Etchin A, Perl A, et al. Midazolam fentanyl sedation in conjunction with local anesthesia during oocyte retrieval for in vitro fertilitzation. J Assist Reprod Genet 1992; 9: 85–7. 24. van der Ven H, Dietrich K, Al-Hasani S, et al. The effect of general anaesthesia on the success of

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embryo transfer following human in-vitro fertilization. Hum Reprod 1988; 3(Suppl): 81–3. Boyers SP, Lavy G, Russell JB, et al. A paired analysis of in vitro fertilization and cleavage rate of firstversus last-recovered preovulatory human oocytes exposed to varying intervals of 100% CO2 pneumoperitoneum and general anesthesia. Fertil Steril 1987; 48: 975–81. Hayes MF, Sacco AG, Savoy-Moore RT, et al. Effect of general anesthesia on fertilization and cleavage of human oocytes in vitro. Fertil Steril 1987; 48: 828–33. Jensen JT, Boyers SP, Grunfeld LH, et al. Anesthesia exposure may affect fertilization rates in human oocytes collected by ultrasound aspiration. Presented at the Fifth World Congress on In Vitro Fertilization and Embryo Transfer, Norfolk, VA. 1987: 48. Hammadeh M, Wilhelm W, Huppert A, Rosenbaum P, Schmidt W. Effects of general anaesthesia vs. sedation on fertilization cleavage and pregnancy rates in an IVF program. Arch Gynecol Obstet 1999; 263: 56–9. Quiglry M, Wolf D, Maklad N, et al. Follicular size and number in human in vitro fertilization. Fertil Steril 1982; 38: 678–81. Coetsier T, Dhont M, De Sutter P, et al. Propofol anaesthesia for ultrasound-guided oocyte retrieval: accumulation of the anaesthetic agent in follicular fluid. Hum Reprod 1992; 7: 1422–4. Ng EHY, Tang OS, Chui DKC, Ho PC. Comparison of two different doses of lignocaine used in paracervical block during oocyte collection in an IVF programme. Hum Reprod 2000; 15(10): 2148–51. Kim WO, Kil HK, Koh SO, Kim JI. Effects of general and locoregional anesthesia on reproduction outcome for in vitro fertilization: a meta-analysis. J Korean Med Sci 2000; 15(1): 68–72. Endler G, Magyar D, Hayes M, Moghissi K. Use of spinal anesthesia in laparoscopy for in vitro fertilization. Fertil Steril 1985; 43(5): 809–10. Martin R, Tsen L, Tzeng G, et al. Anesthesia for in vitro fertilization: the addition of fentanyl to 1.5% lidocaine. Anesth Analg 1999; 88(3): 523–6. Tsen L, Schultz R, Martin R, et al. Intrathecal lowdose bupivacaine versus lidocaine for in vitro fertilization procedures. Reg Anesth Pain Med 2000; 26(1): 52–6. Botta G, D’Angelo A, Giovanni D, et al. Epidural anesthesia in an in vitro fertilization and embryo transfer program. J Assist Reprod Genet 1995; 12(3): 187–90. Chung P, Timothy Y, Mayer J, et al. Gamete intrafallopian transfer; comparison of epidural vs. general anesthesia. J Reprod Med 1998; 43(8): 681–6. Kanto J, Gepts E. Pharmacokinetic implications for the clinical use of propofol. Clin Pharmacokinet 1989; 17(5): 308–26. Huang HW, Huang FJ, Kung F, et al. Effects of induction anesthetic agents on outcome of assisted reproductive technology: a comparison of propofol and thiopental sodium. Changgeng Yi Xue Za Zhi 2000; 23(9): 513–19. Warren JR, Shaw B, Steinkampf MP. Effects of nitrous oxide on preimplantation mouse embryo cleavage and development. Biol Reprod 1990; 43: 158–61.

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41. Gonen O, Shulman A, Ghetler Y, et al. The impact of different types of anesthesia on in vitro fertilizationembryo transfer treatment outcome. J Assist Reprod Genet 1995; 12(10): 678–82. 42. Rosen M, Roizen M, Eger E, et al. The effect of nitrous oxide on in vitro fertilization success rate. Anesthesiology 1987; 67: 42–4. 43. Matt DW, Steingold KA, Dastvan CM, et al. Effects of sera from patients given various anesthetics on preimplantation mouse embryo development in vitro. J In Vitro Fert Embryo Transf 1991; 8(4): 191–7. 44. Swanson R, Leavitt M. Fertilization and mouse embryo development in the presence of midazolam. Anesth Analg 1992; 74(4): 549–54. 45. Trout S, Vallerand A, Kemmann E. Conscious sedation for in vitro fertilization. Fertil Steril 1998; 69(5): 799–808. 46. Schoeffler PF, Levron JC, Hany L, et al. Follicular concentration of fentanyl during laparoscopy for oocyte retrieval – correlation with in vitro fertilization results. Anesthesiology 1988; 69: A663. 47. Shapira S, Chrubasik S, Hoffman A, et al. Use of alfentanil for in vitro fertilization oocyte retrieval. J Clin Anesth 1996; 8(4): 282–5. 48. Naito Y, Tamai S, Fukata J, et al. Comparison of endocrinological stress response associated with transvaginal ultrasound-guided oocyte pick-up under halothane anaesthesia and neuroleptanaesthesia. Can J Anesth 1989; 36: 633–6. 49. Andersson S, Lundeberg T. Acupuncture – from empiricism to science: functional background to

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acupuncture effects in pain and disease. Medical Hypotheses 1995; 45: 271–81. Paulus W, Zhang M, Strehler E, et al. Influence of acupuncture on the pregnancy rate in patients who undergo assisted reproduction therapy. Fertil Steril 2002; 77(4): 721–4. Stener-Victorin E, Wasdenström U, Nilsson L, et al. A prospective randomized study of electroacupuncture versus alfentanil as anaesthesia during oocyte aspiration in in-vitro fertilization. Hum Reprod 1999; 14(10): 2480–4. Aleem F, Eltabbakh G, Omar R, et al. Ovarian follicular fluid beta-endorphin levels in normal and polycystic ovaries. Am J Obstet Gynecol 1987; 156: 1197–200. Aleem F, Omar R, Eltabbakh G, et al. Immunoreactive beta-endorphin in human ovaries. Fertil Steril 1986; 45: 507–11. Humaidan P, Brock K, Bungum L, Stener-Victoria E. Pain relief during oocyte retrieval – exploring the role of different frequencies of electro-acupuncture. Reprod Biomed Online 2006; 13(1): 120–5. Sator-Katzenschlager SM, Wölfler MM, KozekLangenecker SA, et al. Auricular electro-acupuncture as an additional perioperative analgesic method during oocyte aspiration in IVF treatment. Hum Reprod 2006; 21(8): 2114–20. Gejervall AL, Stener-Victorin E, Moller A, et al. Electro-acupuncture versus conventional analgesia: a comparison of pain levels during oocyte aspiration and patients’ experience of well-being after surgery. Hum Reprod 2005; 20(3): 728–35.

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51 Medical considerations of single embryo transfer Outi Hovatta

The health of children born after in vitro fertilization The health of children born after in vitro fertilization (IVF) has been followed up since the first of them were born. In the beginning, the numbers of children were small, but then national and international registers were established. Accumulated data showed that children born as a result of IVF had a higher risk of abnormalities when compared to conventionally born children, as described in a review article by Edwards and Ludwig.1 The risk ratio of having some abnormality was 1.2–1.4 in the reviewed literature. There are many possible causes for such abnormalities: parental factors are likely,1 but multiple pregnancies and their consequences appear to be the most common one.

Risks of multiple pregnancies for the children The increased risk of premature birth even among twin pregnancies, not to mention triplets and higherorder multiple pregnancies, was the main cause of morbidity and mortality among IVF children. This became very clear in a Swedish nationwide analysis of all 5680 IVF children born in 1982–1995 in this country.2 Data regarding all the IVF children were compared to the children born in the general population during the same time (1 505 724 children), using Swedish Medical Birth Registry and the Registry of Congenital Malformations. There were 27% multiple births after IVF, but only 1% in the control population. The rate of preterm births was 30.3% among the IVF infants, while it was only 6.3% in the control population. The percentage of low birth weight (<2500 g) infants after IVF was 27.4% while it was 4.6% among the control infants. The perinatal mortality was 1.9% in the IVF group, but 1.1% in the controls. The high frequency of multiple births and maternal characteristics were regarded as the main factors for adverse outcome, and not the IVF technique itself.

A closer analysis of the abnormalities among the IVF children was very alarming. Strömberg et al,3 studied the same population of Swedish IVF children and found that among the 2060 twins out of the 5680 IVF children, the risk of a neurological diagnosis, in particular, was significantly higher than that amongst the control children. It did not differ from the risk of control twins. The most common neurological diagnosis was cerebral palsy, for which IVF children had an increased risk ratio of 3.7 (2.0–6.6, 95% confidence intervals). The risk ratio for IVF singletons was 2.8 (1.3–5.8, 95% confidence intervals). Also the risk of developmental delay was four-fold among the IVF children. As a consequence of these results, the Swedish IVF clinics have been allowed to transfer only one embryo at a time: in exceptional cases, two. In Denmark, a large register study including all 8602 infants born after IVF or intracytoplasmic sperm injection (ICSI) between 1995 and 2000 was carried out.4 The cohort included 3438 twins and 5164 singletons. A significantly increased risk of preterm delivery was found between twins and singletons. The risk of prematurity was 10-fold increased amongst all births before 37 completed weeks, and 7.4-fold increased in births before 32 completed weeks. The stillbirth rate was doubled in twins (13.1/1000), when compared to that in singletons (6.6/1000). In addition to premature births, congenital anomalies are also more common in twin pregnancies.5 A 2.3-fold higher prevalence of major malformation (9.3%) was found among IVF infants when compared with control infants. Of the IVF infants with malformations, 70% were born from twin or triplet pregnancies.6 Congenital heart disease is more common among twins than singletons.6–8 In the large Danish register study, the total malformation rate (minor + major, 73.7/1000) in twins was significantly higher than that in singletons (55.0/1000).4 Patent ductus arteriosus, typical of preterm birth, was very common among twins. BenAmi et al9 found an increased risk of anencephaly among twin infants born after assisted reproduction. Other perinatal complications typical of twin pregnancies also occur as well in IVF twin pregnancies.

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One of the causes why IVF singleton pregnancies also have more growth retardation and other perinatal problems is the vanishing twin syndrome, which is actually a consequence of an IVF twin pregnancy.10,11 The risk of premature birth is further enhanced in other conditions predisposing to prematurity, such as uterine malformations. Twin pregnancies should not be induced among individuals who have any known increased risk factor of premature delivery.

Risks of multiple pregnancies for the mothers Multiple pregnancies are not risk-free for the mother, either. The risk of pre-eclampsia, gestational hypertension, placental abruption and placenta previa is higher among twin pregnancies than it is among singletons.12–14 All these conditions bear consequences for the mother’s health. Impaired glucose tolerance and pregnancy-induced diabetes15 are more common during multiple pregnancies. Pregnancy of a woman who has diabetes always bears high risks for both the mother and the fetus, and all possible actions should be taken to diminish such risks.16 Obesity further increases the risks of gestational diabetes and hypertension.17 Increasing the risk of diabetic complications with a twin pregnancy by transferring more than one embryo is not acceptable. For a diabetic woman, elective single embryo transfer (SET) is always indicated, irrespective of the embryo quality. Turner’s syndrome is a medical condition in which the majority of the affected women need donated oocytes.18,19 These women often have cardiac anomalies and hypertension, and it is not justified to make these pregnancies even more risky by transferring more than one embryo at a time. The most dangerous complication among Turner’s syndrome women is aortic dissection, and several cases have occurred during pregnancy.20 In Turner’s syndrome, elective SET only can be accepted. Oocyte donation is a procedure that gives relatively high pregnancy rates. Söderström-Anttila et al21 and Söderström-Anttila and Vilska22 showed that elective SET gives excellent pregnancy rates in oocyte donation. Elective SET is further motivated in this group of women because they have an increased risk of hypertension in pregnancy.23 The likelihood of operative delivery, with possible early and late complications, is higher in multiple pregnancies,4 which is another maternal indication not to transfer more than one embryo at a time.

Elective single embryo transfer as a method of avoiding multiple pregnancies During the early days of IVF, the evolving embryo culture techniques did not facilitate high pregnancy rates. In early statistics, the pregnancy rate per SET

was clearly lower than that after transfer of more than one embryo. This urged clinicians to try to improve the treatment results by transfer of multiple embryos. The consequence was to increase the number of multiple pregnancies. However, pediatricians who saw the increase in complications caused by this new epidemic of multiple births alarmed the IVF community relatively early. They were particularly active in Northern Europe. In Finland, we started elective SETs in the mid 1990s. We then analyzed the pregnancy rates after SET during a 1-year period in two IVF units in Helsinki,24 and demonstrated very clearly the difference between nonelective and elective SET. If only one embryo was available for transfer, the pregnancy rate was 20% per transfer, but if an elective SET was carried out in this nonselected patient population, a pregnancy rate of 29.7 % was achieved. It was similar to that in two-embryo transfer (29.4%). That elective SETs result in much better pregnancy rates than nonelective SETs, and that such pregnancy rates are similar to those achieved in transfer of two embryos when top- or good-quality embryos are available, were soon demonstrated in two prospective randomized trials. Gerris et al25 randomized 53 couples with a female partner <34 years old and who had at least two top-quality embryos for elective SET or two embryo transfer (double ET). The pregnancy rate after elective SET was 42.3%, and that among the couples with double ET was 48.1%, with 30% twins. We carried out a multicenter study in Finland26 in which 144 couples with good-quality embryos were randomized to elective SET or double ET. The pregnancy rate per transfer was 32.4% in the SET group and 47.1% in the double ET group. The difference was not statistically significant. The cumulative pregnancy rates after frozen embryo transfers were 47.3% in the SET and 58.6% in the double ET groups. After double ET, there were 39% twins. A large North European multicenter study was then carried out.27 Couples in which the female partner was <36 years old and who had at least two goodquality embryos were randomized for receiving either two embryos or first a single fresh embryo and then a frozen–thawed single embryo. A pregnancy resulting in at least one live birth was encountered in 142 of the 331 women (42.9%) who received two fresh embryos, and in 128 of the women (38.8%) who received first one fresh and then one frozen embryo. The pregnancy rate after SET was not substantially lower than that after double ET. But the twinning rate after SET (0.8%) was significantly (p <0.001) lower when compared to that after ET (33.1%). Pregnancy outcome was not affected when blastocyststage embryos were transferred; as shown in an analysis of couples undergoing a single blastocyst transfer (n = 52, live birth rate 53.8%) or a double blastocyst transfer (n = 187, live birth rate 54.4%). In the single blastocyst transfer, the twin rate was 3.1%, while it was 51% in the double blastocyst transfer.28

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An effective and active cryopreservation policy, particularly in connection with SET, increases the pregnancy rates per oocyte retrieval.29 Elective SET also reduces multiple pregnancies in frozen embryo transfers.30 Elective SET has proved to be a method by which multiple pregnancies can be reduced without decreasing the overall pregnancy rates.31 In real life, it has not only been equally effective but also, economically, substantially cheaper than double embryo transfer with all its complications.32,33 In order to be as effective as double ET, there have to be some criteria to select the couples for elective SET.34 In a nonselected population, SET resulted in a lower pregnancy rate. There are now data from Sweden and Belgium – which have a law and guideline for regular SET – a that the pregnancy rates have remained similar in these countries, while the rate of multiple pregnancies has dropped significantly.35,36

Optimizing criteria for single embryo transfer In order to achieve equal pregnancy rates between single and double embryo transfers, some kind of selection has to be made to balance the likelihood of pregnancy and minimize the risk of twins.34 The criteria presented in all the above-cited articles include the age of the female partner, the number of earlier unsuccessful cycles, and embryo quality. In Sweden, these criteria have in most clinics become stricter during the period in which SET has been practiced. The National Board of Health and Welfare in Sweden has collected statistics from all IVF clinics,36 and the units by themselves have agreed criteria using which a twin rate of <5% and an unchanged pregnancy rate could be achieved. The present recommended criteria for elective SET are: the age of the woman <40 years old; not more than three failed IVF/ICSI cycles; and at least two good-quality embryos. Regarding cleavagestage embryos; the criteria are: less than 20% fragmentation; the embryo fills an even zona pellucida; no multinuclear blastomers; four cells on day 2; and eight cells on day 3. An exception of one of these parameters still counts a good-quality embryo. Early cleavage is an additional sign urging SET. At blastocyst stage, an expanded blastocyst on day 5 indicates good quality. In frozen embryo transfers, an intact embryo after thawing is indicative for SET.

Conclusions Multiple pregnancies, including twins, cause high risks for the health of the infants and the mothers. The risks are largely caused by the high incidence of premature birth, but also the incidence of congenital anomalies is increased among twins. Twin pregnancies can be almost completely avoided by carrying out elective SET. In order to obtain equal pregnancy

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rates between single and double embryo transfers, certain criteria have to be used for selection of the couple to SET. A twin rate of <5% can be achieved if a single good-quality embryo is transferred to a woman under <40 years old during her three first IVF/ICSI cycles, and she has good-quality embryos available. Two embryos can be transferred to women who do not fulfill these criteria. If the embryo is intact after thawing, only a single frozen–thawed embryo should be transferred. Irrespective of the woman’s age, the number of earlier cycles, or the embryo quality, a SET should be carried out in situations in which the woman has a known high risk of premature birth, such as uterine abnormalities. The same is true as regards maternal contraindications for multiple pregnancies, such as diabetes, hypertension, or Turner’s syndrome.

References 1. Edwards RG, Ludwig M. Are major defects in children conceived in vitro due to innate problems in patients or to induced genetic damage? Reprod Biomed Online 2003; 7: 131–82. 2. Bergh T, Ericson A, Hillensjö T, Nygren KG, Wennerholm UB. Deliveries and children born after in-vitro fertilisation in Sweden 1982–95: a retrospective cohort study. Lancet. 1999; 354: 1579–85. 3. Strömberg B, Dahlquist G, Ericson A, et al. Neurological sequelae in children born after in-vitro fertilisation: a population-based study. Lancet 2002; 359: 461–5. 4. Pinborg A, Loft A, Nyboe Andersen A. Neonatal outcome in a Danish national cohort of 8602 children born after in vitro fertilization or intracytoplasmic sperm injection: the role of twin pregnancy. Acta Obstet Gynecol Scand 2004; 83: 1071–8. 5. Sperling L, Kiil C, Larsen LU, et al. Detection of chromosomal abnormalities, congenital abnormalities and transfusion syndrome in twins. Ultrasound Obstet Gynecol 2007; 29: 517–26. 6. Merlob P, Sapir O, Sulkes J, Fisch B. The prevalence of major congenital malformations during two periods of time, 1986–1994 and 1995–2002 in newborns conceived by assisted reproduction technology. Eur J Med Genet 2005; 48: 5–11. 7. Caputo S, Russo MG, Capozzi G, et al. Congenital heart disease in a population of dizygotic twins: an echocardiographic study. Int J Cardiol 2005; 10: 293–6. 8. Hajdu J, Beke A, Marton T, et al. Congenital heart diseases in twin pregnancies. Fetal Diagn Ther 2006; 21: 198–203. 9. Ben-Ami I, Vaknin Z, Reish O, et al. Is there an increased rate of anencephaly in twins? Prenat Diagn 2005; 25: 1007–10. 10. Spellacy WN. Antepartum complications in twin pregnancies. Clin Perinatol 1988; 15: 79–86. 11. Pinborg A, Lidegaard O, Freiesleben NC, Andersen AN. Vanishing twins: a predictor of smallfor-gestational age in IVF singletons. Hum Reprod 2007; 22: 2707–14.

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12. Duckitt K, Harrington D. Risk factors for pre-eclampsia at antenatal booking: systematic review of controlled studies. BMJ 2005; 330: 565. 13. Erez O, Vardi IS, Hallak M, et al. Preeclampsia in twin gestations: association with IVF treatments, parity and maternal age. J Matern Fetal Neonatal Med 2006; 19: 141–6. 14. Allen VM, Wilson RD, Cheung A; Genetics Committee of the Society of Obstetricians and Gynaecologists of Canada (SOGC); Reproductive Endocrinology Infertility Committee of the Society of Obstetricians and Gynaecologists of Canada (SOGC). Pregnancy outcomes after assisted reproductive technology. J Obstet Gynaecol Can 2006; 28: 220–50. 15. Adler-Levy Y, Lunenfeld E, Levy A. Obstetric outcome of twin pregnancies conceived by in vitro fertilization and ovulation induction compared with those conceived spontaneously. Eur J Obstet Gynecol Reprod Biol 2007; 133: 173–8. 16. Hod M, Jovanovic L. Improving outcomes in pregnant women with type 1 diabetes. Diabetes Care 2007; 30: e62. 17. Dokras A, Baredziak L, Blaine J, et al. Obstetric outcomes after in vitro fertilization in obese and morbidly obese women. Obstet Gynecol 2006; 108: 61–9. 18. Foudila T, Söderström-Anttila V, Hovatta O. Turner’s syndrome and pregnancies after oocyte donation. Hum Reprod 1999; 14: 532–5. 19. Hovatta O. Pregnancies in women with Turner’s syndrome. Ann Med 1999; 31: 106–10. 20. Bondy CA. Turner Syndrome Study Group. Care of girls and women with Turner syndrome: a guideline of the Turner Syndrome Study Group. J Clin Endocrinol Metab 2007; 92: 10–25. 21. Söderström-Anttila V, Vilska S, Mäkinen S, Foudila T, Suikkari AM. Elective single embryo transfer yields good delivery rates in oocyte donation. Hum Reprod 2003; 18: 1858–63. 22. Söderström-Anttila V, Vilska S. Five years of single embryo transfer with anonymous and non-anonymous oocyte donation. Reprod Biomed Online 2007; 15: 428–33. 23. Söderström-Anttila V, Tiitinen A, Foudila T, Hovatta O. Obstetric and perinatal outcome after oocyte donation: comparison with in-vitro fertilization pregnancies. Hum Reprod 1998; 13: 483–90. 24. Vilska S, Tiitinen A, Hydén-Granskog C, Hovatta O. Elective transfer of one embryo results in an acceptable pregnancy rate and eliminates the risk of multiple birth. Hum Reprod 1999; 14: 2392–5.

25. Gerris J, De Neubourg D, Mangelschots K, et al. Prevention of twin pregnancy after in-vitro fertilization or intracytoplasmic sperm injection based on strict embryo criteria: a prospective randomized clinical trial. Hum Reprod 1999; 14: 2581–7. 26. Martikainen H, Tiitinen A, Tomás C, et al; Finnish ET Study Group. One versus two embryo transfer after IVF and ICSI: a randomized study. Hum Reprod 2001; 16(9): 1900–3. 27. Thurin A, Hausken J, Hillensjö T, et al. Elective single-embryo transfer versus double-embryo transfer in in vitro fertilization. N Engl J Med 2004; 351: 2392–402. 28. Styer AK, Wright DL, Wolkovich AM, Veiga C, Toth TL. Single-blastocyst transfer decreases twin gestation without affecting pregnancy outcome. Fertil Steril 2008; 89: 1702–8. 29. Tiitinen A, Hydén-Granskog C, Gissler M. What is the most relevant standard of success in assisted reproduction? The value of cryopreservation on cumulative pregnancy rates per single oocyte retrieval should not be forgotten. Hum Reprod 2004; 19: 2439–41. 30. Hydén-Granskog C, Unkila-Kallio L, Halttunen M, Tiitinen A. Single embryo transfer is an option in frozen embryo transfer. Hum Reprod 2005; 20: 2935–8. 31. Tiitinen A, Unkila-Kallio L, Halttunen M, HydenGranskog C. Impact of elective single embryo transfer on the twin pregnancy rate. Hum Reprod 2003; 18: 1449–53. 32. Gerris J, De Sutter P, De Neubourg D, et al. A real-life prospective health economic study of elective single embryo transfer versus two-embryo transfer in first IVF/ICSI cycles. Hum Reprod 2004; 19: 917–23. 33. Fiddelers AA, Severens JL, Dirksen CD, et al. Economic evaluations of single- versus doubleembryo transfer in IVF. Hum Reprod Update 2007; 13: 5–13. 34. van Montfoort AP, Fiddelers AA, Land JA, et al. eSET-irrespective of the availability of a good-quality embryo in the first cycle only is not effective in reducing overall twin pregnancy rates. Hum Reprod 2007; 22: 1669–74. 35. Van Landuyt L, Verheyen G, Tournaye H, et al. New Belgian embryo transfer policy leads to sharp decrease in multiple pregnancy rate. Reprod Biomed Online 2006; 13: 765–71. 36. Karlström PO, Bergh C. Reducing the number of embryos transferred in Sweden – impact on delivery and multiple birth rates. Hum Reprod 2007; 22: 2202–7.

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52 Endometriosis and ART Andy Huang, Mark Hunter, Alan H DeCherney

Introduction Endometriosis, as a clinical entity, has been recognized and intensely investigated for well over 100 years. Despite the accumulation of an enormous amount of information, uncertainty still exists regarding etiologies, clinical consequences, and treatment efficacy. The two most common complaints leading to a diagnosis of endometriosis are pelvic pain and infertility. The advancement of innovative medical and surgical approaches, such as gonadotropin-releasing hormone (GnRH) agonists and laparoscopically guided laser ablation, have proven quite effective in improving many of the symptoms associated with endometriosis. It does appear that assisted reproductive technology (ART) is becoming an indispensable asset in providing affected couples with viable pregnancies, and with the accumulation of randomized trials, the role of both long and short GnRH protocols is becoming clear.

Endometriosis and infertility There is little debate that the extensive anatomical distortion and tubal obstruction, frequently attributed to severe endometriosis, does impair fertility. Less clear is the reported association between minimal or mild endometriosis and infertility, in the absence of any mechanical disruption. Although there is no conclusive evidence that minimal to moderate endometriosis actually causes infertility, several studies dating back to the 1930s have suggested that there is at least an association between the two.1 In the 1970s, three studies retrospectively compared the incidence of endometriosis in women undergoing laparoscopy for infertility or voluntary sterilization.2–4 The incidences of endometriosis ranged from 21% to 48% in the infertile women, while endometriosis was noted in only 1.3–5% of fertile women undergoing tubal ligation. More recent studies,5,6 including one prospective investigation,7 have demonstrated that among women undergoing insemination with donor sperm due to severe male-factor infertility, those with coexisting endometriosis had markedly fewer conceptions per exposure than women who did not have the disease.

Although the above studies were methodologically imperfect and far from conclusive, virtually every area within the reproductive process has been intensely investigated, in an attempt to describe a causal relationship between endometriosis and infertility. The results of several tangential lines of investigation have added to the confusion, as studies are frequently in direct contradiction to one another. Investigators have suggested that women with mild to moderate endometriosis have a higher incidence of endocrine abnormalities,8 anovulation,9 corpus luteum insufficiency,10 hyperprolactinemia,11 luteinized unruptured follicle syndrome,12 and spontaneous abortions.13 However, other well-organized, prospective studies have found most of these factors to be either normal or lacking in clinical significance.14–19 Immune dysfunction in endometriosis has become the focus of more recent efforts, as it is hypothesized that immunity plays a role in the pathogenesis of the disease. Several immunologic abnormalities, which could potentially impair fertility, have been identified. Researchers have reported increased B-cell activity, with the production of specific antibodies against endometrial antigens, T-cell and macrophage dysfunction, and non-specific polyclonal B-cell activation. In addition, some have reported increased production of cytokines and eicosanoids in the peritoneal fluid and sera, which may affect sperm motility and velocity, acrosome reactivity, sperm penetration, embryo implantation, and early development.14,20 As with other factors, many conflicting reports have emerged. Furthermore, it is not at all clear which is the cause and which the effect, or what role each abnormality actually plays in the pathogenesis of endometriosis-associated infertility. As stated, one argument that has been proposed against a causal relationship between endometriosis and infertility is in the outright failure of medical or surgical treatment to significantly improve pregnancy success in these patients. The use of medical treatments, otherwise successful in alleviating the nonreproductive symptoms of endometriosis, has failed to demonstrate a reasonable improvement in fertility.21 Most studies investigating the effect of surgical ablation of endometriotic lesions, by any one of a number of techniques, have failed to show

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an increased fecundity. One recent randomized study, however, did show an improved rate of pregnancy for women with minimal/mild endometriosis treated with ablation of endometriotic lesions, when compared with a control group receiving diagnostic laparoscopy alone.22 However, this study has been criticized for having a lower fecundity rate among untreated patients than would normally be expected, for notifying patients of their treatment status, and for following pregnancies to only 20 weeks. Subsequently, another randomized study, which looked at actual birth rates, failed to demonstrate a reproductive benefit for patients whose lesions were ablated, but had lower power than the first study.23 When the results are combined, there was no significant statistical heterogeneity and the increased chance of achieving pregnancy after surgery was only 8.6% (95% confidence interval [CI] 2.1,15).24 Thus, surgically ablating visible endometriosis lesions only potentially benefits pregnancy outcomes minimally.

Ovulation induction and insemination Controlled ovarian hyperstimulation (COH), in combination with intrauterine insemination (IUI), has proven to be a cost-effective and appropriate first-line treatment for many infertility diagnoses.25 However, it is not entirely clear that this approach is as effective for patients with endometriosis. Deaton et al26 demonstrated increased fecundity in patients treated with clomiphene citrate and IUI. However, Fedele and collegues27 reported that the increased conception rate with COH and IUI did not follow with a significantly different pregnancy rate at 6 months. Furthermore, a retrospective comparison of COH and IUI reported per-cycle pregnancy rates of 6.5, 11.8, and 15.3% for endometriosis, male-factor, and unexplained infertility, respectively.28 Similarly, although with more optimistic results, a prospective, observational study reported pregnancy rates of 16.3 and 33.6% following COH/IUI in patients with endometriosis and unexplained infertility, respectively.29 In a meta-analysis, Hughes30 reported that a diagnosis of endometriosis decreased the per-cycle COH/IUI conception rate by half. Also, a recent prospective, randomized study reported live birth rates of 11 and 2% for endometriosis patients undergoing COH/IUI and no treatment, respectively.31 While this demonstrates a live birth odds ratio (OR) of 5.5 for the treatment group, the actual percentage of live births after treatment remains relatively low. Failure of COH/IUI has recently been correlated with advanced endometriosis. A retrospective study of 92 patients found that more than one-third of patients failing to conceive after four ovulatory cycles of clomiphene citrate had stage III or IV disease, an endometrioma, pelvic adhesions, and/or tubal disease.32 Most recently, however, a retrospective, controlled cohort study of 259 COH/IUI cycles found no difference in cycle pregnancy rate and cumulative live-birth rate between

women with surgically treated minimal to mild endometriosis and women with unexplained infertility, indicating potentially improved outcomes after surgical treatment.33 The advent of aromatase inhibitors has added to the armamentarium of therapeutic modalities for the treatment of endometriosis. With its efficacy in treating the endometriosis-associated pain, more formally established34,35 studies are underway to evaluate its utility in ovulation induction. Most recently, Wu et al36 found that a third-generation aromatase inhibitor was able to achieve a reasonable pregnancy rate, with a thicker endometrium but fewer ovulatory follicles, when randomized and compared with clomiphene citrate.

Endometriosis and assisted reproductive technology Treatment strategies for the infertile couple must be based on the specific situation. For the young women with only minimal or mild endometriosis, expectant management may be the most appropriate course. However, for women approaching the end of their reproductive age, the chances of conceiving drop precipitously. In these women, intervention, in the form of COH/IUI, or in vitro fertilization (IVF) may be warranted more expeditiously.37 The lower cost and low complication rate of ovulation induction and IUI make the combination an attractive first step. However, for women with severe endometriosis or tubal disease, or when male-factor or a combination of etiologies are involved, assisted reproduction such as IVF may be pursued sooner. In addition, IVF offers the added benefit of being able to directly observe key events in the conception process, such as the assessment of gamete quality, the observation of fertilization, and the evaluation of early embryo development. As a result, the increasing use of ART in the treatment of endometriosis-associated infertility may help to answer some of the questions regarding this elusive association. It is thought that the use of IVF–ET (embryo transfer) in the infertile patient with endometriosis removes critical steps in reproduction, such as fertilization and early embryo development, from an in vivo environment that some have suggested is hostile to these processes. Thus, it has been anticipated that endometriosis patients will have IVF outcomes approaching those of other infertility etiologies. Recent studies, however, confirm that endometriosis patients, particularly those with moderate to severe disease, had lower pregnancy rates.38,39 Certainly, the development of GnRH agonists and transvaginal oocyte retrieval has been associated with increased success in the use of IVF for endometriosis-associated infertility. However, the value of reported ART results must be considered along with the understanding that there is great clinical and laboratory variability among centers, leading to a

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Study Mahadevan et al, 198381 Wardle et al, 198562 Matson et al, 198643 Frydman et al, 1987 Inoue et al, 199252 Mills et al, 1992 Simon et al, 199469 Dmowski et al, 199549 Gerber et al, 199551 Olivennes et al, 199554 Tanbo et al, 199565 Arici et al, 199667 Padigas et al, 199675 Huang et al, 1997

1.51 (0.32,7.10) 0.52 (0.06,4.85) 0.36 (0.13,1.01) 0.83 (0.39,1.80) 1.21 (0.87,1.69) 0.94 (0.47,1.86) 0.26 (0.12,0.54) 1.31 (0.72,2.39) 0.84 (0.58,1.20) 0.93 (0.61,1.41) 0.89 (0.60,1.32) 0.49 (0.24,1.02) 1.64 (0.82,3.30) 0.68 (0.32,1.46)

Overall (95% Cl)

0.81 (0.72,0.91)

0.1

1 Odds ratio

7

wide range of reported pregnancy rates. Furthermore, most studies are retrospective and observational and are therefore of limited value in reaching definitive conclusions regarding therapy efficacy. Barnhart et al40 performed a meta-analysis on the studies evaluating the effects of endometriosis on the outcomes of assisted reproductive technologies. They evaluated a total of 22 articles and concluded that, overall, patients with endometriosis had lower pregnancy rates, decreased fertilization and implantation rates, and a decreased number of oocytes retrieved compared to controls of tubal factor infertility (Fig 52.1).

Controlled ovarian hyperstimulation and oocyte retrieval As the practice of assisted reproduction has evolved over the past two decades, so has the efficacy of IVF in the treatment of endometriosis. With regards to the effect of endometriosis on COH and oocyte retrieval, an obvious divide exists between earlier studies, using clomiphene citrate with laparoscopic oocyte retrieval, and more recent investigations benefiting from the development of GnRH agonists and ultrasound-guided transvaginal retrieval. Earlier studies did in fact report a reduced oocyte yield in patients with endometriosis undergoing IVF. In one small study, Chillik and colleagues41 compared patients with either no endometriosis, mild to moderate endometriosis, or severe disease, and reported that oocyte yield was reduced in those patients of advanced stage. Oehninger et al42 reported a similar effect on oocyte retrieval for patients with stage III or IV endometriosis. Both studies suggested that oocyte yield was impaired in this group of patients due to technical difficulties at the time of laparoscopic oocyte retrieval. Alternatively, other researchers have reported decreased folliculogenesis in

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Percent weight 0.6 0.7 2.8 4.0 16.9 4.6 8.0 5.0 18.1 12.0 14.0 6.0 3.1 4.3

Fig 52.1 Unadjusted meta-analysis of odds of pregnancy in endometriosis patients vs tubal factor controls. (Reproduced from Barnhart et al,40 with permission.)

patients with endometriosis.43–46 Furthermore, Dlugi and colleagues47 and more recently Somigliana and colleagues48 reported a significantly lower number of preovulatory follicles in patients with endometriomas, when compared to patients with hydrosalpinges. Several contemporary studies utilizing GnRH agonists and transvaginal retrieval have not confirmed that endometriosis has a significant effect on oocyte yield. Dmowski et al49 retrospectively analyzed 237 IVF cycles and found no difference in either folliculogenesis or in the number of oocytes obtained for women with or without endometriosis. In a casecontrol study comparing 65 cycles of IVF for women with endometriosis, to 98 cycles of IVF in patients with tubal infertility, Bergendal et al50 found no difference in folliculogenesis or oocyte retrieval. Several recent studies have further concluded that there is no difference in the number of oocytes obtained in patients with mild to moderate endometriosis, when compared to patients with more severe disease.51–54 Barnhart et al demonstrated a lower number of oocytes retrieved (OR = 0.82, 95% CI 0.75–0.90) for patients with endometriosis when compared to patients with tubal factor.40 The improvement in IVF outcomes, brought about by the development of GnRH agonists, is largely undisputed. Olivennes and colleagues54 reported a significantly improved clinical pregnancy rate for patients treated with GnRH agonists, when compared with standard, gonadotropin-only, ovarian-stimulation protocols (Fig 52.1). Other investigations have reported similar results.55 Long-term GnRH agonist suppression has been thought to repress further endometriotic lesions and improve IVF outcome for patients with endometriosis. Dicker and associates,56 as well as Rickes et al57 reported a significantly higher clinical pregnancy rate after 6 months of GnRH agonist therapy, compared with

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Table 52.1

Comparison of IVF–ET outcomes for women with and without endometriosis

Study

Group

Number of cycles

Clinical pregnancies (%/cycle)

Mahadevan81

I–IV Tubal I–IV Tubal

14 261 17 47

14 10 6 11

I/II III/IV I II III IV Tubal I/II III/IV Tubal I/II III/IV I/II III/IV Tubal

10 14 24 37 36 57 40 135 141 994 191 35 61 93 49

60 7 13 14 6 2 18 16 8 13 24 20 13 3 14

Wardle62

Chillik41 43

Matson

82

Sharma

42

Oehninger Yovich44

ovarian stimulation with gonadotropins alone. Chedid et al58 also investigated the use of a 3-month and a 3-week GnRH agonist down-regulation protocol and reported a significantly increased oocyte yield, when compared with controls receiving only gonadotropins. Although they noted an improved pregnancy rate, it did not reach statistical significance. Nakamura and colleagues59 compared GnRH agonist suppression for 60 days with a shorter, midluteal down-regulation, prior to ovulation induction. They reported pregnancy rates of 67 and 27%, for longer and shorter protocols, respectively. Marcus et al60 also reported a significantly higher pregnancy rate for patients treated with longer GnRH agonist protocols (Table 52.1), although they used different GnRH agonists for the two groups and assigned patients based on their refusal to accept the longer regimen. Surrey et al61 investigated a 3-month course of GnRH agonist therapy prior to IVF–ET, and found the agonist therapy to be associated with a significantly higher ongoing pregnancy rate. Conversely, Chedid and colleagues58 found no difference between long and short GnRH agonist administrations. For now, it appears that endometriosis patients respond to ovarian stimulation in a manner that is similar to other infertility etiologies. Although standard gonadotropin stimulation protocols work reasonably well, the addition of longer GnRH agonist down-regulation may increase IVF success and should be considered on a case-by-case basis.

Study

Group

Number of cycles

Clinical pregnancies (%/cycle)

Inoue52

I II III IV Other I–IV Tubal

111 78 51 69 372 360 160

40 42 47 42 44 29 36

I/II III/IV Tubal I/II III/IV Other I/II III/IV Tubal I–IV Tubal I/II III/IV

100 29 1139 89 30 118 43 46 147 65 98 45 39

29 52 41 25 30 21 12 15 24 28 30 44 33

Olivennes54 Geber51

Dmowski49

Arici67

Bergendal50 Pal53

Fertilization and early embryo development It is unclear as to the degree to which endometriosis is a detriment to the process of fertilizing oocytes in vitro, as several investigations have now reported significantly impaired fertilization rates for these patients. One early study noted fertilization rates per oocyte of 33, 63, and 68% for patients with endometriosis, unexplained infertility, and tubal infertility, respectively,62 while another reported a marked impairment in fertilization with the presence of an endometrioma.47 More recently, Bergendal et al50 reported fertilization rates of 60 and 78% for patients with endometriosis and tubal factor, respectively (p <0.0001). Other investigators have reported significantly lower fertilization success for stage III or IV endometriosis, when compared with stage I or II.53,54 With regard to early embryo development, researchers have reported fewer embryos reaching the 4-cell stage at 48 h,63 a reduced number of blastomeres at 72 h,64 and lower cleavage rates when endometriosis is compared with tubal-factor or unexplained infertility.65 Furthermore, Brizek and colleagues66 retrospectively analyzed video records of 235 embryos and found a statistically significant increase in the incidence of aberrant nuclear and cytoplasmic morphology within embryos from patients with endometriosis. Conversely, there have been several large studies that have failed to detect an impairment in fertilization. Dmowski et al48 analyzed 237 cycles and found

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no difference in either the fertilization rate or the early cleavage rate among patients with endometriosis or tubal-factor infertility. Another case-control study, also comparing endometriosis with tubal factor, found no evidence of either impaired fertilization or a decrease in embryo quality.67 In comparing the effect of progressive endometriosis stages on fertilization and embryo development, Inoue and colleagues52 found no difference in either the fertilization rate or the embryo transfer rate for 309 patients with stage I– IV endometriosis. Furthermore, Bergendal et al,50 although reporting impaired fertilization for women with endometriosis, noted no difference in either the cleavage rate or the morphologic embryo score, when compared with tubal infertility. As it remains, the question of a significant effect by endometriosis on fertilization and in vitro embryo development has yet to be answered. Barnhart et al40 showed an overall decrease in fertilization rate when all endometriosis patients were compared to patients with tubal infertility, but when stratified by stage of disease, patients with severe endometriosis actually had an increase in fertilization rates. However, more recent studies have shown that any impaired fertilization has little or no effect on the ultimate outcome of IVF, as pregnancy rates for patients with endometriosis are comparable with other etiologies. Most recently, Suzuki et al68 found that endometriosis affects oocyte number but not embryo quality or pregnancy outcome, irrespective of the presence of an ovarian endometrioma. Perhaps the clinical insignificance of impaired fertilization is due to the fact that improved ovarian stimulation and oocyte-recovery techniques have led to a surplus of available oocytes for fertilization. An increased oocyte yield can readily sustain a slight decrease in fertilization capacity to produce enough embryos for implantation. It is unclear what role, if any, intracytoplasmic sperm injection (ICSI) may play in the fertilization of oocytes from women with endometriosis.

Implantation, pregnancy, and loss Assuming a minimum number of good-quality embryos are available for transfer, a successful live birth is dependent on adequate implantation and a low rate of spontaneous abortion. However, as a result of the transfer of multiple embryos, a lower rate of implantation has not necessarily translated into a low pregnancy rate. Although a few contemporary studies have in fact reported reduced implantation rates, most have failed to demonstrate a correspondingly low pregnancy rate for patients with endometriosis (Table 52.1). Some early studies have shown a decrease in the implantation rate with a subsequent decrease in the pregnancy rate.42,43,61 In a small study, Chillik and colleagues41 reported a significantly lower implantation and pregnancy rate for patients with stage III or IV endometriosis when compared to

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patients with tubal factor or endometriosis of a lesser severity. Matson and Yovich43 demonstrated pregnancy rates of 18, 13, 14, 6, and 2%, for patients with tubal factor, and stage I–IV endometriosis, respectively. Arici et al,67 in a case-control study of 284 IVF cycles, reported a significantly lower implantation rate of 3.9% for patients with endometriosis, compared with 8.1 and 7.2% for tubal infertility and unexplained infertility, respectively. They also demonstrated a trend towards a lower pregnancy rate in patients with endometriosis, although this did not reach significance. More recent studies have taken this finding and added live-birth and cumulative pregnancy rates. Omland et al39 found the live birth rate after transfer of two embryos to be 66.0% compared with 78.8% for unexplained etiology of infertility. Kuivasaari et al38 found a significantly lower cumulative pregnancy rate after 1–4 IVF/ICSI treatments in women with stage III/IV endometriosis compared to women with stage I/II endometriosis and a control of women with tubal infertility. While Simon and colleagues69 also reported lower implantation and pregnancy rates for patients with endometriosis vs tubal infertility, they added a dimension to the data by analyzing the outcomes of oocyte donation from donors with and without endometriosis. They reported comparable implantation and pregnancy rates for women with and without endometriosis, who received oocytes from donors without endometriosis. However, patients who received oocytes from endometriotic ovaries had significantly lower implantation rates. Another study reported on 239 oocyte donor cycles and found that the presence of endometriosis in the recipient had no effect on implantation or pregnancy rates, regardless of the disease stage.70 From this, it has been suggested that an endometriosis-associated impairment of implantation results from a compromise to the potential of the oocyte or early embryo, and not to the endometrium itself. Several large investigations have failed to demonstrate either an impaired implantation rate or a lower pregnancy rate for patients with endometriosis, comparing stage by stage or with other infertility etiologies.50–54 Geber and colleagues51 reported pregnancy rates in 140 cycles of 40 and 45% for patients with endometriosis or tubal infertility, respectively. Olivennes et al noted similar pregnancy rates of 29% for endometriosis and 36% for tubal factor,54 while another study reported 28 and 30%, respectively.50 Inoue et al52 in a study of 681 women with and without endometriosis, found no difference in the IVF conception rate between the two groups. Several comparisons within endometriosis stages have reported similar pregnancy rates despite increasing disease severity.42,49,51,67 Pal and colleagues53 analyzed 85 IVF cycles in endometriosis patients with either stage I–II or stage III–IV disease. Although they reported a lower fertilization rate for patients with stage III or IV

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Table 52.2 Comparing stage III–IV disease with stage I–II disease: results of bivariate analysis and multiple logistic regression comparing endometriosis (Endo) patients with stage III–IV disease with patients with stage I–II disease

Outcome Pregnancy rate Fertilization rate Implantation rate Mean oocyte count Peak E2

Endo III–IV

Endo I–II

p

Crude OR (95% CI)

Adjusted ORa (95% CI)

13.84 74.47 10.23 6.70 1447.74

21.12 58.38 11.31 8.19 5813.38

<0.001 <0.001 0.003 <0.001 <0.001

0.60 (0.42–0.87) 1.11 (1.09–1.13) 0.93 (0.89–0.98) 0.83 (0.78–0.87) N/A

0.64 (0.35–1.17) Not interpretable 0.21 (0.15–0.32) 0.31 (0.24–0.39) N/A

Note: Total number of observations: 699; N/A, not applicable. a Adjusted for publication date and age. Reproduced from Barnhart et al,40 with permission.

endometriosis, clinical pregnancy rates did not differ significantly between the two groups. In his metaanalysis, Barnhart et al40 calculated that the adjusted OR of achieving pregnancy compared with the group of controls was 0.56, 0.79, and 0.46, respectively, for overall patients, stage I/II, and stage III/IV patients respectively (Table 52.2). A few studies have associated endometriosis with increased pregnancy loss during IVF cycles. Oehninger and colleagues42 noted a higher miscarriage rate following IVF among patients with stage III or IV endometriosis, when compared to those with less severe disease. Yanushpolsky et al63 reported, along with a diminished oocyte yield and poor embryo quality, a significantly higher early pregnancy loss when endometriomas were aspirated at the time of oocyte retrieval. However, another large study comparing patients with aspirated endometriomas to others with endometriosis found no difference in oocyte yield, embryo quality, pregnancy rate, or miscarriage.71 Furthermore, most studies have not reported a significant endometriosis-associated increase in pregnancy loss.50,51

Endometriosis and GIFT There are limited data concerning the effect of endometriosis on gamete intrafallopian transfer (GIFT). Guzick and colleagues,72 in a retrospective, casecontrol study, reported significantly different pregnancy rates of 32 and 47% for patients with or without endometriosis, respectively. Another study analyzed GIFT outcomes in patients with endometriosis and found decreased folliculogenesis and a lower oocyte yield with more severe disease, although the clinical pregnancy rate did not differ between patients with different stages of the disease or with other infertility etiologies.46 In an early observational study, Yovich and Matson73 reported a significantly higher pregnancy rate for patients with severe endometriosis treated with GIFT than for those undergoing IVF. Another study, however, failed to find a difference between the two.74 As with much of the ART data concerning

endometriosis, no prospective, randomized studies exist comparing GIFT with IVF. With a lack of compelling evidence and an impressive success rate for IVF, it is difficult to assess the role of the more invasive GIFT procedure in initial attempts at assisted reproduction for patients with endometriosis.

Surgery and ART As stated earlier, the data are conflicting regarding the effect of surgery on fertility in patients with endometriosis. Unfortunately, there have been no prospective, randomized studies investigating the effect of surgery for endometriosis on ART outcome. One retrospective study compared IVF with repeat surgery for patients with stage III or IV endometriosis.75 Recently, however, a Cochrane Review of two randomized trials comparing the effectiveness of laparoscopic surgery in the treatment of subfertility associated with endometriosis vs other treatment modalities or placebo found that use of laparoscopic surgery may improve the chance of pregnancy by an OR of 1.6.76 Pregnancy rates were reported as 70% over two cycles of IVF compared with 24% for the 9 months following surgery. There are no similar randomized studies evaluating the effects of surgery on severe disease. A nonrandomized study24 demonstrated that the cumulative probability of pregnancy in 216 infertile patients with severe disease 2 years after surgery was significantly increased. In another study, Garcia-Velasco et al reported no difference in fertilization, implantation, or pregnancy rates for patients who had undergone removal of an endometrioma, as compared to patients with suspected endometriomas that were not removed.77 In another recent randomized study comparing minimal and severe endometriosis with treatment modality, there was a significant difference in the cumulative probability of pregnancy rates between operative laparoscopy and expectant of GnRH analog treatment in patients with minimal disease. With severe disease, however, severity of disease, the number of endometriomata, their size and unilateral or bilateral existence did not significantly affect the estimated cumulative

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pregnancy rates.78 Until better data are available, however, no definitive conclusions can be drawn regarding the role of surgery for endometriosis prior to ART. In fact, one recent study79 finds that in the absence of tubal occlusion or severe male-factor infertility, laparoscopy may still be considered for the treatment of endometriosis even after multiple failed IVF cycles.

Future directions Some researchers have suggested that endometriosis is associated with an impaired folliculogenesis and a decreased oocyte yield. Although the data are conflicting, it is possible that the introduction of GnRH antagonists and aromatase inhibitors may represent another large step forward in improving ovarian stimulation protocols and increasing IVF success. Furthermore, the use of donor oocytes has been suggested to improve efficacy in patients with endometriosis. As ovarian hyperstimulation protocols become more tolerable, and as oocyte cryopreservation becomes efficacious and efficient, it is possible that an increased number of women with endometriosis who have failed standard IVF will benefit from donation. There is evidence for and against an endometriosisassociated impairment of oocyte fertilization in vitro. One of the tremendous benefits of fertilizing an oocyte in vitro is the ability to assess the process on a case-bycase basis. For patients with endometriosis who are experiencing fertilization difficulty, it is likely that ICSI will prove to be a valuable addition to the technology of assisted reproduction for this disease. Indeed, ICSI has proven to be of tremendous worth in achieving pregnancy in couples with male-factor infertility. Minguez et al78 analyzed 980 cycles of ICSI for couples with male-factor infertility, of which 101 cycles were also complicated by endometriosis. They found no significant difference in fertilization, implantation, or pregnancy rates with coexisting endometriosis. Finally, there is an increasing interest in the prolongation of in vitro embryo maturation, with many investigators studying the efficacy of blastocyst development and implantation. An endometriosis-associated detriment to implantation may be responsible for some IVF failures. Although reports are conflicting, some have suggested an impaired early embryo development in patients with endometriosis. It is possible that the practice of in vitro maturation to the blastocyst stage in these patients may allow for the transfer of a more selected group of healthier embryos, thus improving the implantation rate. It is anticipated that the improvements from this approach will eventually raise the implantation rate to a point at which it will become routine to transfer no more than one or two embryos at a time, thereby significantly lowering the incidence of multiple pregnancies. Furthermore, the adoption of various techniques in embryo manipulation, such as assisted hatching, may also have a positive effect on the implantation rate for these patients.

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Conclusion It is important to stress the heterogeneous nature of the data that has been reviewed. Laboratory and clinical practices vary greatly from center to center, as do the corresponding IVF success rates. Randomized, prospective studies designed to answer key questions about the optimum algorithmic approach to the treatment of endometriosis-associated infertility simply do not exist. With the evidence evaluated as a whole, it does appear that IVF outcomes have improved significantly for endometriosis patients with the adoption of GnRH agonists and transvaginal oocyte retrieval. Although ART procedure alterations are site-specific, the vast majority of endometriosis patients undergo the same treatment protocol as for those patients with tubalfactor or unexplained infertility. There is, to date, no compelling evidence that endometriosis patients benefit from significant alterations from standard ART protocols or procedures, with the notable exception of prolonged GnRH agonist down-regulation. Until large, randomized, prospective studies have answered questions regarding the optimum length of down-regulation, the use of in vitro maturation or manipulation, the role of autoantibodies and immunosuppression, and other controversies, it is likely that patients with endometriosis will continue to undergo the same treatment protocol as everyone else. At the very least, it can be said that ART represents a tremendous advancement for women who, for whatever reason, have been unable to achieve pregnancy. For the patient with endometriosis, evolving options in pharmacotherapy and assisted reproduction may finally offer the blessing of a pain-free and reproductive life.

References 1. Counseller VS. Endometriosis: a clinical and surgical review. Am J Obstet Gynecol 1938; 36: 877–88. 2. Hasson HM. Incidence of endometriosis in diagnostic laparoscopy. J Reprod Med 1976; 16: 135–8. 3. Strathy JH, Molgaard CA, Coulam CB, Melton LJ. Endometriosis and infertility: a laparoscopic study of endometriosis among fertile and infertile women. Fertil Steril 1982; 38: 667–72. 4. Drake TS, Grunert GM. The unsuspected pelvic factor in the infertility investigation. Fertil Steril 1980; 34: 27–31. 5. Hammond MG, Jordan S, Sloan CS. Factors affecting pregnancy rates in a donor insemination program using frozen semen. Am J Obstet Gynecol 1986; 155: 480–5. 6. Yeh J, Seibel MM. Artificial insemination with donor sperm: a review of 108 patients. Obstet Gynecol 1987; 70: 313–16. 7. Jansen RPS. Minimal endometriosis and reduced fecundibility: prospective evidence from an artificial insemination by a donor program. Fertil Steril 1986; 46: 141–3. 8. Bancroft K, Vaughan-Williams CA, Elstein M. Pituitary–ovarian function in women with minimal

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Textbook of Assisted Reproductive Technologies or mild endometriosis and otherwise unexplained infertility. Clin Endocrinol 1992; 36: 177–81. Matorras R, Rodriguez F, Perez C, et al. Infertile women with and without endometriosis: a case control study of luteal phase and other infertility conditions. Acta Obstet Gynecol Scand 1996; 75: 826–31. Pittaway DE, Maxson W, Daniell J, et al. Luteal phase defects in infertility patients with endometriosis. Fertil Steril 1983; 39: 712–13. Hirschowitz JS, Soler NG, Wortsman J. The galactorrhea-endometriosis syndrome. Lancet 1978; 1: 896–8. Mio Y, Toda T, Harada T, Terakawa N. Luteinized unruptured follicle in the early stages of endometriosis as a cause of unexplained infertility. Am J Obstet Gynecol 1992; 167: 271–3. Wheeler JM, Johnston BM, Malinak LR. The relationship of endometriosis to spontaneous abortion. Fertil Steril 1983; 39: 656–60. Burns WN, Schenken RS. Pathophysiology of endometriosis-associated infertility. Clin Obstet Gynecol 1999; 42: 586–610. Thomas EJ, Lenton EA, Cooke ID. Follicle growth patterns and endocrinological abnormalities in infertile women with minor degrees of endometriosis. Br J Obstet Gynaecol 1986; 93: 852–8. Kusuhara K. Luteal function in infertile patients with endometriosis. Am J Obstet Gynecol 1992; 167: 274–7. Matalliotakis I, Panidis D, Vlassis G, et al. PRL, TSH and their response to the TRH test in patients with endometriosis before, during, and after treatment with danazol. Gynecol Obstet Invest 1996; 42: 183–6. Pittaway DE, Vernon C, Fayez JA. Spontaneous abortions in women with endometriosis. Fertil Steril 1988; 50: 711–15. Matorras R, Rodriguez F, Gutierrez de Teran G, et al. Endometriosis and spontaneous abortion rate: a cohort study in infertile women. Eur J Obstet Gynecol Reprod Biol 1998; 77: 101–5. Senturk LM, Arici A. Immunology of endometriosis. J Reprod Immunol 1999; 43: 67–83. Hughes E, Fedorkow DM, Collins J, Vandekerckhove P. Ovulation suppression versus placebo in the treatment of endometriosis. In: Lilford R, Hughes E, Vandekerckehove P, eds, Subfertility Module of the Cochrane Database of Systematic Reviews. Oxford: The Cochrane Collection, 1997. Marcoux S, Maheux R, Berube S. The Canadian Collaborative Group on Endometriosis. Laparoscopic surgery in infertile women with minimal or mild endometriosis. N Engl J Med 1997; 336: 217–22. Gruppo Italiano per lo Studio dell’Endometriosi. Ablation of lesions or no treatment in minimal–mild endometriosis in infertile women: a randomized trial. Hum Reprod 1999; 14: 1332–4. Al-Inany HG, Crosignani PG, Vercellini P. Evidence may change with more trials: concepts to be kept in mind [letters]. Hum Reprod 2000; 15: 2447–8. Guzick DS, Carson SA, Coutifaris C, et al. Efficacy of superovulation and intrauterine insemination in the treatment of infertility. N Engl J Med 1999; 340: 177–83.

26. Deaton JL, Gibson M, Blackmer KM, et al. A randomized, controlled trial of clomiphene citrate and intrauterine insemination in couples with unexplained infertility or surgically corrected endometriosis. Fertil Steril 1990; 54: 1083–8. 27. Fedele L, Parazzini F, Radici E, et al. Buserelin acetate versus expectant management in the treatment of infertility associated with mild endometriosis: a randomized clinical trial. Fertil Steril 1992; 58: 28–31. 28. Nuojua-Huttunen S, Tomas C, Bloigu R, et al. Intrauterine insemination treatment in subfertility: an analysis of factors affecting outcome. Hum Reprod 1999; 14: 698–703. 29. Omland A, Tanbo T, Dale PO, Abyholm T. Artificial insemination by husband in unexplained infertility compared with infertility associated with peritoneal endometriosis. Hum Reprod 1998; 13: 2602–5. 30. Hughes EG. The effectiveness of ovulation induction and intrauterine insemination in the treatment of persistent infertility: a meta-analysis. Hum Reprod 1997; 12: 1865–72. 31. Tummon IS, Asher LJ, Martin JS, Tulandi T. Randomized controlled trial of superovulation and insemination for infertility associated with minimal or mild endometriosis. Fertil Steril 1997; 68: 8–12. 32. Capelo F, Kumar A, Steinkampf M, et al. Laparoscopic evaluation following failure to achieve pregnancy after ovulation induction with clomiphene citrate. Fertil Steril 2003; 80: 1450–3. 33. Werbrouck E, Spiessens C, Meuleman C, D’Hooghe T. No difference in cycle pregnancy rate and in cumulative live-birth rate between women with surgically treated minimal to mild endometriosis and women with unexplained infertility after controlled ovarian hyperstimulation and intrauterine insemination. Fertil Steril 2005; 86: 566–71. 34. Karaer O, Oruc S, Koyuncu FM. Aromatase inhibitors: possible future applications. Acta Obstet Gynecol Scand 2004; 83: 699–706. 35. Attar E, Bulun S. Aromatase inhibitors: the next generation of therapeutics for endometriosis? Fertil Steril 2006; 85: 1307–18. 36. Wu HH, Wang NM, Cheng ML, Hsieh JN. A randomized comparison of ovulation induction and hormone profile between the aromatase inhibitor anastrozole and clomiphene citrate in women with infertility. Gynecol Endocrinol 2007; 23: 76–81. 37. The Practice Committee of the American Society of Reproductive Medicine. Endometriosis and infertiity. Fertil Steril 2006; 86: S156–60. 38. Kuivasaari P, Hippelainen M, Anttila M, Heinonen S. Effect of endometriosis on IVF/ICSI outcome: stage III/IV endometriosis worsens cumulative pregnancy and live-born rates. Hum Reprod 2005; 20: 3130–5. 39. Omland AK, Abyholm T, Fedorcsak P, et al. Pregnancy outcome after IVF and ICSI in unexplained, endometriosis-associated and tubal factor infertility. Hum Reprod 2005; 20: 722–7. 40. Barnhart K, Dunsmoor-Su R, Coutifaris C. Effect of endometriosis on in vitro fertilization. Fertil Steril 2002; 77: 1148–55.

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Endometriosis and ART 41. Chillik CF, Acosta AA, Garcia JE, et al. The role of in vitro fertilization in infertile patients with endometriosis. Fertil Steril 1985; 44: 56–61. 42. Oehninger S, Acosta AA, Kreiner D, et al. In vitro fertilization and embryo transfer (IVF/ET): an established and successful therapy for endometriosis. J In Vitro Fert Embryo Transf 1988; 5: 249–56. 43. Matson PL, Yovich JL. The treatment of infertility associated with endometriosis by in vitro fertilization. Fertil Steril 1986; 46: 432–4. 44. Yovich JL, Matson PL, Richardson PA, Hilliard C. Hormonal profiles and embryo quality in women with severe endometriosis treated by in vitro fertilization and embryo transfer. Fertil Steril 1988; 50: 308–13. 45. Yovich JL, Matson PL. The influence of infertility etiology on the outcome of IVF–ET and GIFT treatments. Int J Fertil 1990; 35: 26–33. 46. Chang MY, Chiang CH, Hsieh TT, et al. The influence of endometriosis on the success of gamete intrafallopian transfer (GIFT). J Assist Reprod Genet 1997; 14: 76–82. 47. Dlugi AM, Loy RA, Dieterle S, et al. The effect of endometriomas on in vitro fertilization outcome. J In Vitro Fert Embryo Transf 1989; 6: 338–41. 48. Somigliana E, Infantino M, Benedetti F, et al. The presence of ovarian endometriomas is associated with a reduced responsiveness to gonadotropins. Fertil Steril 2006; 86: 192–6. 49. Dmowski WP, Rana N, Michalowska J, et al. The effect of endometriosis, its stage and activity, and of autoantibodies on in vitro fertilization and embryo transfer success rates. Fertil Steril 1995; 63: 555–62. 50. Bergendal A, Naffah S, Nagy C, et al. Outcome of IVF in patients with endometriosis in comparison with tubal-factor infertility. J Assist Reprod Genet 1998; 15: 530–4. 51. Geber S, Paraschos T, Atkinson G, et al. Results of IVF in patients with endometriosis: the severity of the disease does not affect outcome, or the incidence of miscarriage. Hum Reprod 1995; 10: 1507–11. 52. Inoue M, Kobayashi Y, Honda I, et al. The impact of endometriosis on the reproductive outcome of infertile patients. Am J Obstet Gynecol 1992; 167: 278–82. 53. Pal L, Shifren JL, Isaacson KB, et al. Impact of varying stages of endometriosis on the outcome of in vitro fertilization–embryo transfer. J Assist Reprod Genet 1998; 15: 27–31. 54. Olivennes F, Feldberg D, Liu HC, et al. Endometriosis: a stage by stage analysis – the role of in vitro fertilization. Fertil Steril 1995; 64: 392–8. 55. Oehninger S, Brzyski RG, Muasher SJ, et al. In vitro fertilization and embryo transfer in patients with endometriosis: impact of a gonadotrophin releasing hormone agonist. Hum Reprod 1989; 4: 541–4. 56. Dicker D, Goldman JA, Levy T, et al. The impact of long-term gonadotropin-releasing hormone analogue treatment on preclinical abortions in patients with severe endometriosis undergoing in vitro fertilization–embryo transfer. Fertil Steril 1992; 57: 597–600. 57. Rickes D, Nickel I, Kropf S, Kleinstein J. Increased pregnancy rates after ultralong postoperative therapy

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with gonadotropin-releasing hormone analogs in patients with endometriosis. Fertil Steril 2002; 78: 757–62. Chedid S, Camus M, Smitz J, et al. Comparison among different ovarian stimulation regimens for assisted procreation procedures in patients with endometriosis. Hum Reprod 1995; 10: 2406–11. Nakamura K, Oosawa M, Kondou I, et al. Metrodin stimulation after prolonged gonadotropin releasing hormone agonist pretreatment for in vitro fertilization in patients with endometriosis. J Assist Reprod Genet 1992; 9: 113–17. Marcus SF, Edwards RG. High rates of pregnancy after long-term down-regulation of women with severe endometriosis. Am J Obstet Gynecol 1994; 171: 812–17. Surrey ES, Silverberg KM, Surrey MW, Schoolcraft WB. Effect of prolonged gonadotropin-releasing hormone agonist therapy on the outcome of in vitro fertilization–embryo transfer in patients with endometriosis. Fertil Steril 2002; 78: 699–704. Wardle PG, Mitchell JD, McLaughlin EA, et al. Endometriosis and ovulatory disorder: reduced fertilisation in vitro compared with tubal and unexplained infertility. Lancet 1985; 2: 236–9. Yanushpolsky EH, Best CL, Jackson KV, et al. Effects of endometriomas on oocyte quality, embryo quality, and pregnancy rates in in vitro fertilization cycles: a prospective, case-controlled study. J Assist Reprod Genet 1998; 15: 193–7. Pellicer A, Oliveira N, Ruiz A, et al. Exploring the mechanism(s) of endometriosis-related infertility: an analysis of embryo development and implantation in assisted reproduction. Hum Reprod 1995; 10 (Suppl 2): 91–7. Tanbo T, Omland A, Dale PO, Abyholm T. In vitro fertilization/embryo transfer in unexplained infertility and minimal peritoneal endometriosis. Acta Obstet Gynecol Scand 1995; 74: 539–43. Brizek CL, Schlaff S, Pellegrini VA, et al. Increased incidence of aberrant morphological phenotypes in human embryogenesis – an association with endometriosis. J Assist Reprod Genet 1995; 12: 106–12. Arici A, Oral E, Bukulmez O, et al. The effect of endometriosis on implantation: results from the Yale University in vitro fertilization and embryo transfer program. Fertil Steril 1996; 65: 603–7. Suzuki T, Izumi SI, Matsubayashi H, Awaji H, et al. Impact of ovarian endometrioma on oocyte and pregnancy outcome in in vitro fertilization. Fertil Steril 2005; 83: 908–13. Simon C, Gutierrez A, Vidal A, et al. Outcome of patients with endometriosis in assisted reproduction: results from in vitro fertilization and oocyte donation. Hum Reprod 1994; 9: 725–9. Sung L, Mukherjee T, Takeshige T, et al. Endometriosis is not detrimental to embryo implantation in oocyte recipients. J Assist Reprod Genet 1997; 14: 152–6. Isaacs JD Jr, Hines RS, Sopelak VM, Cowan BD. Ovarian endometriomas do not adversely affect pregnancy success following treatment with in vitro fertilization. J Assist Reprod Genet 1997; 14: 551–3.

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72. Guzick DS, Yao YA, Berga SL, et al. Endometriosis impairs the efficacy of gamete intrafallopian transfer: results of a case-control study. Fertil Steril 1994; 62: 1186–91. 73. Yovich JL, Matson PL. The influence of infertility etiology on the outcome of IVF–ET and GIFT treatments. Int J Fertil 1986; 35: 26–33. 74. Tanbo T, Dale PO, Abyholm T. Assisted fertilization in infertile women with patent fallopian tubes. A comparison of in vitro fertilization, gamete intrafallopian transfer, and tubal embryo stage transfer. Hum Reprod 1990; 5: 266–70. 75. Pagidas K, Falcone T, Hemmings R, Miron P. Comparison of reoperation for moderate (stage III) and severe (stage IV) endometriosis-related infertility with in vitro fertilization–embryo transfer. Fertil Steril 1996; 65: 791–5. 76. Jacobson T, Barlow D, Koninckx P, et al. Laparoscopic surgery for subfertility associated with endometriosis (Cochrane Review). In: The Cochrane Library, Issue 1, 2004. Chichester, UK: John Wiley and Sons, Ltd.

77. Garcia-Velasco JA, Mahutte NG, Corona J, et al. Removal of endometriomas before in vitro fertilization does not improve fertility outcomes: a matched, casecontrol study. Fertil Steril 2004; 81: 1194–7. 78. Minguez Y, Rubio C, Bernal A, et al. The impact of endometriosis in couples undergoing intracytoplasmic sperm injection because of male infertility. Hum Reprod 1997; 12: 2282–5. 79. Littman E, Giudice L, Lathi R, Berker B, et al. Role of laparoscopic treatment of endometriosis in patients with failed in vitro fertilization cycles. Fertil Steril 2005; 84: 1574–8. 80. Mahadevan MM, Trounson AO, Leeton JF. The relationship of tubal blockage, infertility of unknown cause, suspected male infertility, and endometriosis to the success of in vitro fertilization and embryo transfer. Fertil Steril 1983; 40: 755–62. 81. Sharma V, Pampiglione J, Riddle A, et al. An analysis of factors influencing the establishment of a clinical pregnancy in an ultrasound-based ambulatory in vitro fertilization program. Fertil Steril 1988; 49: 468–78.

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53 Polycystic ovaries and ART Thomas H Tang, Adam H Balen

Introduction Polycystic ovary syndrome (PCOS) is a common endocrine disorder that affects women and accounts for 87% and 26% of patients attending a gynecological endocrine clinic presenting with history of oligomenorrhea and amenorrhea respectively.1–6 Polycystic ovary syndrome is characterized by hyperandrogenism, menstrual disturbance, anovulatory infertility, and obesity. Obesity often magnifies the clinical features of PCOS.7,8 It is also a heterogeneous disorder, ranging from a classic presentation described by Stein and Leventhal,9 with features of obesity, amenorrhea, and hirsutism, to women with a normal cyclicity and yet with ultrasound evidence of polycystic ovarian morphology.10,11 In the last 15 years, a large body of evidence has indicated that increased insulin resistance and compensatory hyperinsulinemia play a key role in the pathogenesis of PCOS.2,3,5,6,12 The current first-line therapy is weight loss through lifestyle modification in the obese group of women and then ovulation induction treatment with clomi phene.13,14 Approximately 80% of women respond to clomiphene treatment and the cumulative pregnancy rate after 6 months of treatment is between 40 and 50%.15,16 Gonadotropin ovulation induction therapy is usually offered to those patients who have failed to respond to clomiphene. In addition, ovarian diathermy has been advocated by some as an effective alternative option.17,18 Assisted conception treatment is an effective therapy for women with PCOS who are refractory to standard ovulation induction therapies or who have coexisting infertility factors.19,20

Prevalence and diagnosis The prevalence of PCOS is determined by the diagnostic criteria being used,11,21–23 hospital- or community-based studies,4,24–26 and the ethnic background of the population.21,27–29 Knochenhauer et al21 reported that the prevalence of PCOS in an unselected population of 195 black

and 174 white women of the southeastern United States was 4.7% and 3.4%, respectively. The subjects were recruited at the time of their pre-employment physical examination. However, the diagnostic criteria were based on the definition of PCOS that arose from National Institutes of Health (NIH) consensus (1990): namely, ovulatory dysfunction and clinical/biochemical evidence of hyperandrogenism. However, the ultrasound (US) morphology of polycystic ovary (PCO) was not included. A similar prevalence rate (6.7%) was also observed in a Greek population by using the same diagnostic criteria. 29 In contrast, a study by Michelmore et al 11 which included US morphology of PCO and menstrual cycle disturbance, revealed the prevalence of PCOS was 26% in 230 volunteers between 18 and 25 years old. These subjects were recruited from two universities and two general practice surgeries in Oxford. Furthermore, other population-based studies, by Farquhar et al30 (n = 183), Clayton et al31 (n = 190), and Polson et al 10 (n = 257) demonstrated that the prevalence of US PCO morphology is common, at between 21 and 23%. However, a significant proportion of the subjects (25%) were without any clinical features of PCOS. 10 Nevertheless, the nonoral contraceptive PCO users had a higher incidence of hirsutism (Ferriman and Gallwey score >7) than controls (Clayton et al31). Similar findings were reported by Farquhar et al30 with 57% of the studied population with PCO being found to have an irregular menstrual cycle and/or hyperandrogenism. PCOS encompasses a broad spectrum of signs and symptoms of ovarian dysfunction that have been highlighted in a recent 2003 Rotterdam consensus workshop.32 The panel of experts concluded that PCOS remains as a syndrome of ovarian dysfunction, with the cardinal features of menstrual disturbance, hyperandrogenism, and PCO morphology. No single diagnostic criterion (such as hyperandrogenism or PCO) is sufficient for the clinical diagnosis. The revised diagnostic criteria of PCOS is as follows, with two of the following being required:

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Oligo- and/or anovulation, i.e. menstrual disturbance. Clinical and/or biochemical signs of hyperandrogenism. PCO on US. Exclusion of other etiologies (such as congenital adrenal hyperplasia, androgen-secreting tumor, Cushing’s syndrome) by appropriate investigation of menstrual disturbance and hyperandrogenism.

With the improvement of the resolution of US scanning and increasingly acceptable transvaginal scanning procedures, more detail of ovarian morphology can be studied compared with the transabdominal technique (Fig 53.1). The 2003 Rotterdam consensus also defined the diagnostic criteria for US PCO morphology as either 12 or more follicles measuring 2–9 mm in diameter or increased ovarian volume over 10 cm3, by using transvaginal US scan. The distribution of follicles and a description of the stroma are not required in the diagnosis.33 The threshold of 12 follicles of 2–9 mm diameter per ovary offered the best specificity (99%) and sensitivity (75%) for the diagnosis of PCOS.34 Jonard et al34 also reported that there was a significantly increased number of follicles of 2–5 mm in diameter compared with controls, whereas the number of follicles of 6–9 mm in diameter was not different. It was postulated that the hyperandrogenic microenvironment within the PCO results in an increased recruitment of growing follicles of 2–5 mm, followed by the arrest at 6–9 mm.34

Response of the polycystic ovary to stimulation for IVF The response of the polycystic ovary to ovulation induction aimed at the development of unifollicular ovulation is well documented and differs significantly from that of normal ovaries. The response tends to be slow, with a significant risk of ovarian hyperstimulation and/or cyst formation.35–37 Conventional in vitro fertilization (IVF) nowadays depends on inducing multifol-

Fig 53.1

A transvaginal ultrasound image of a polycystic ovary.

licular recruitment. It is thus to be expected that the response of the polycystic ovary within the context of an IVF program should also differ from the normal. Dor et al38 showed that significantly more oocytes were recovered per cycle in the PCOS group than in women with tubal factor infertility (19.4 vs 5.4, p <0.005), but this was associated with lower fertilization rates (40.4% vs 67.6%, p <0.001) (Table 53.1). Similar findings were observed in subsequent reports of women with PCOS.19,39,40 MacDougall et al39 reported the outcome of (IVF) in 76 patients diagnosed as having PCO on their pretreatment ultrasound scan, and compared it with that in 76 control patients who had ultrasonically normal ovaries (see Table 53.1). The subjects were matched for age, cause of infertility, and stimulation regimen. Despite receiving significantly less human menopausal gonadotropin (hMG), patients with PCO diagnosed by ultrasound had significantly higher serum estradiol concentrations on the day of human chorionic gonadotropin (hCG) administration (5940 ± 255 vs 4370 ± 240 pmol/l, p <0.001), developed more follicles (14.9 ± 0.7 vs 9.8 ± 0.6, p <0.001), and produced more oocytes (9.3 ± 0.6 vs 6.8 ± 0.5, p = 0.003). Fertilization rates were, however, reduced in PCO patients (52.8 ± 3.4% vs 66.1 ± 3.4%, p = 0.007). There was no significant difference in cleavage rates. The pregnancy rate per embryo transfer was 25.4% in the PCO group and 23.0% in the group with normal ovaries. Of the PCO patients, 10.5% developed moderate/severe ovarian hyperstimulation syndrome (OHSS), compared with none in the controls (p = 0.006). Patients with and without PCO undergoing IVF had similar pregnancy and live birth rates, as each had similar numbers of goodquality embryos for transfer. Despite the fact that they often require a lower total dose of gonadotropin during stimulation compared with women with normal ovaries, women with PCOS are at a greater risk of developing moderate to severe OHSS, quoted at 10–18% vs 0.3–5% et al39–42 Kodama40 also demonstrated a significantly higher incidence of cancellation of embryo transfer in the PCOS group due to failure of fertilization and the risk of OHSS. These findings are also supported by the data from a recent meta-analysis which demonstrated significantly more oocytes retrieved from women with PCOS, with lower fertilization rates compared with controls.43 However, Heijnen et al43 did not confirm that women with PCOS had lower clinical pregnancy rates and live birth rates than non-PCOS women. On the other hand, caution is required to interpret the meta-analysis, because most of the included studies are retrospective case-controlled studies with a significant heterogeneity with regard to the subject demographic data and treatment regimens. Although most retrospective data indicated that the pregnancy rates per transfer were comparable to controls,19,39,40 the miscarriage rates following IVF treatment were increased in women with PCOS (35.8% vs 23.6% in women with normal ovaries, p = 0.0038).42,44 The

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Table 53.1

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The outcome of IVF treatment between women with normal ovaries and polycystic ovaries Dor et al38

No. of patients No. of oocytes per cycle Fertilization rates (%) Pregnancy rates per aspiration (%) Pregnancy rates per embryo transfer (%)

MacDougall et al39

Homburg et al19

PCOS

Normal

p-value

PCOS

Normal

p-value

PCOS

Normal

p-value

16 19.4 40.4 30.7 –

37 5.4 67.6 29.7 –

0.004 < 0.001 N.S. –

76 9.3 52.8 21 25.4

76 6.8 66.1 18 23

0.003 0.007 N.S. N.S.

68 14.2 57.3 – 22.6

68 10.5 65.7 – 26.5

0.002 < 0.002 – N.S.

unfavorable outcomes are related to their high body mass index (BMI),45–48 the increased waist–hip ratio49 and insulin resistance.50 Fedorcsak et al45 reported that women with a BMI >25 kg/m2 undergoing IVF treatment had a relative risk of 1.77 (95% confidence interval [CI] 1.05–2.97) in miscarriage before 6 weeks’ gestation compared with those with a BMI <25 kg/m2. Likewise, women with a waist–hip ratio between 0.70 and 0.79 had a pregnancy rate of 29.9%, compared with 15.9% in women with a waist–hip ratio >0.80 (odds ratio [OR] = 0.42, 95% CI 0.2–0.9; p = 0.03).49 This finding is still significant after adjustment for age, smoking, BMI, parity, and number of embryos transferred. A consequence of obesity among women with PCOS is an increased requirement for follicle-stimulating hormone (FSH) stimulation.45,50–53 Therefore, they may not respond to a low-dose stimulation regimen. However, once the dose of FSH is increased and the threshold reached, the subsequent response can be explosive, with an increasing risk of OHSS. The mechanism of poor response to gonadotropins is uncertain but it is likely to be related to hyperinsulinemia and insulin resistance.54 Hyperandrogenism and hyperinsulinemia are the cardinal features of PCOS.6 Teissier et al55 suggested that the follicular endocrine microenvironment is related to oocyte quality in women undergoing IVF. This study showed that testosterone levels in the follicular fluid were significantly elevated in women with PCOS follicles compared with women with normal ovaries. They also demonstrated that there were significantly higher levels of follicular testosterone in those follicles with meiotically incompetent oocytes compared with follicles with meiotically competent oocytes in PCOS patients. It was concluded that the excess follicular androgen concentration could affect oocyte maturation and quality. Hence, high androgen levels may contribute to a lower fertilization rate among the oocytes retrieved from women with PCOS compared to those without. Additionally, insulinresistant women with PCOS require a higher dose of exogenous gonadotropins during stimulation and are at greater risk of overstimulation compared with the noninsulin-resistant women with PCOS.45,47,50,56,57 There are several possible explanations for the excessive response of the PCO to ovarian stimulation. Women with PCOS have an increased number of antral

follicles.58–60 These follicles are not atretic despite the fact that they have a high androgen:estrogen ratio similar to the follicles undergoing atresia.61 In other words, there is an increased cohort of selectable antral follicles which are sensitive to exogenous gonadotropins. An increased number of antral follicles is also reflected by elevation of anti-Müllerian hormone (AMH) levels in women with PCOS compared with women with normal ovaries.62 An increased stockpile of antral follicles is contributed by an increase in recruitment of primordial follicles from the resting pool.60,63 This process partly results from increased insulin/insulin-like growth factor (IGF)64 and androgen concentrations.65– 70

Jonard and Dewailly58 demonstrated a positive correlation between serum testosterone concentrations and androstenedione levels in PCOS patients and the number of antral follicles (2–5 mm in diameter). A pretreatment of aromatase inhibitor increases the concentration of intraovarian androgen levels, resulting in an improved ovarian response in the low-responder women undergoing IVF cycles.71 Taken together, increased androgen levels result in an increase in primordial follicles’ recruitment from the resting pool. It has been consistently reported that patients with PCOS have higher estradiol levels on the day of hCG administration than women with normal ovaries.38– 40,72,73 These observations may be explained by an increase in levels of androgen substrates6 and in aromatase activity.73,74 Under in vitro conditions, the granulosa cells obtained from PCO exhibit a higher response to FSH stimulation than those size-matched follicles from normal ovaries.75–79 This may result from a higher number of FSH receptors attributed to the stimulatory effects of androgen on FSH receptors’ synthesis.69 Androgen also enhances the production of steroids from the granulosa cells in response to the stimulatory effect of gonadotropins. Furthermore, androgen promotes insulin-like growth factor-I (IGF-I) and IGF-I receptor gene expression in the follicular cells in the growing follicles up to the small antral stage.65,80 In company with the effects of insulin, IGF-I increases in FSH responsiveness of gonadotropindependent stages of the follicular development.73,81,82 Many women with PCOS, particularly those who are obese, have compensatory hypersecretion of insulin in

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response to the insulin resistance that is specifically related to the polycystic ovary syndrome83 and to that caused by obesity. The ovary is stimulated by insulin, which acts, as it were, as a co-gonadotropin. Insulin augments theca cell production of androgens in response to stimulation by luteinizing hormone (LH)84 and granulosa cell production of estrogen in response to stimulation by FSH.85 Ovarian hyperstimulation syndrome is the most serious iatrogenic complication of IVF treatment.86 In severe cases, although uncommon, patients develop life-threatening conditions, such as hypovolemia, thromboembolism, hemoconcentration, oligouria, electrolyte imbalance, ascites, hydrothorax (Fig 53.2), pericardial effusion, or even adult respiratory distress syndrome. The management of OHSS is discussed in Chapter 56. It has long been recognized that patients with PCOS undergoing IVF treatments are at a greater risk of developing OHSS.87 However, the actual incidence is difficult to quantify, with the reported rates varying between 10% and 18%.39–41,88 A recent metaanalysis indicated that the incidence of OHSS was rarely reported among the studies on the outcomes of IVF in women with PCOS.43 Patients who developed OHSS had higher estradiol concentrations and more oocytes retrieved than those without OHSS.39,89–91 The cardinal feature of the pathogenesis of OHSS is an increase in capillary permeability.92 Vascular endothelial growth factor (VGEF) is an endothelial cell mitogen with potent angiogenic properties93 and is thought to be a key mediator of OHSS.89–91 Serum and follicular VEGF levels on the day of egg retrieval are elevated in patients with PCOS compared with controls, and are also increased in women who develop OHSS.94 Although there is a positive correlation between the serum VEGF and estradiol level on the day of hCG administration, VEGF is a better predictor for OHSS.90,95 Furthermore, OHSS is also known to be more common in a pregnancy cycle or in an IVF cycle using hCG for luteal support.86 An in vitro study revealed that the expression of VEGF mRNA in human luteinized granulosa cells was dose and time dependently enhanced by hCG.96 A simi-

Fig 53.2 A chest X-ray demonstrates a left-side pleural effusion with compression of the lower lobe in a case of severe OHSS.

lar finding of the direct effect of hCG on the expression of VEGF was reported in a recent study.97 The expression was also higher in the OHSS group than controls.97a Additionally, Miele et al98 demonstrated that both insulin and IGF-I increased VEGF mRNA expression. Agrawal et al99 also showed that insulin augmented both gonadotropin and hCG in the production of VEGF from the human luteinized granulosa cells. This is in agreement with a recent in vitro study on the stimulating effects of insulin and IGF on VEGF production by human luteinized granulosa cells in a comparison between PCOS and non-PCOS women.100

Superovulation strategies for women with PCO and/or PCOS Pituitary desensitization with a gonadotropin-releasing hormone (GnRH) agonist has become almost universal in assisted conception clinics. The reversible hypogonadotropic hypogonadism so produced permits enhanced control of follicular development and improved pregnancy rates in IVF programs.101,102 Suppression of endogenous LH by GnRH agonists may have a particular advantage to the woman with the PCOS, so that oocyte-containing follicles can develop in the sensitive polycystic ovary free from the adverse environment of high-tonic LH concentrations.103,104 These oocytes appear to fertilize better than those obtained in cycles without pituitary desensitization, suggesting that it is indeed the abnormal hormonal milieu, rather than the polycystic ovary itself, that is the problem for women with PCOS.103–105 There are few studies that specifically compare different treatment regimens for women with and without PCOS, and those that do vary in their definition and diagnosis of the syndrome.106,107 The two particular aims of treatment in this group of women are the correction of the abnormal hormone milieu, by suppressing elevated LH and androgens, and the avoidance of ovarian hyperstimulation. Prolonged pituitary desensitization avoids the initial surge of gonadotropins with the resultant ovarian steroid release that occurs in the short GnRH protocol. While the long protocol theoretically provides controlled stimulation, the polycystic ovary is still more likely than the normal ovary to become hyperstimulated.108 With both long and short protocols, significantly more eggs are collected from women with polycystic than normal ovaries and, interestingly, the total dose of exogenous gonadotropins is the same for either regimen. It was also proposed that a longer period of desensitization (30 instead of 15 days) is of benefit by reducing androgen levels;106 in the latter study, the longer duration of treatment did not improve pregnancy rates but did apparently decrease the incidence of hyperstimulation. In general, when GnRH agonists are used, the evidence is that the long protocol has advantages over other schedules109 and there is every

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reason to suppose this conclusion will hold true for patients with PCOS as well as for those with normal ovaries. Nonetheless, it has to be accepted that only a few randomized controlled comparisons of the various schedules have been carried out specifically in PCOS cases (see below). The other debate in ovarian stimulation for women with the PCOS is whether using FSH alone confers any benefit over HMG. Preparations of purified urinary FSH contain some LH activity, usually less than 1%, and an early study suggested that ovulation induction can be achieved without exogenous LH.110 In patients with hypogonadotropic hypogonadism, follicular maturation is, however, often incomplete and inconsistent because LH, by its action on thecal cells, is required for full ovarian steroidogenesis.111,112 Thus, the presence of some LH in the hypogonadotropic condition is facilitatory to normal follicular development. Most studies have found no benefit from the use of FSH alone in ovulation induction for anovulatory infertility.113,114 So far as IVF is concerned, we have reported the results of a meta-analysis of randomized controlled comparisons of urinary-derived FSH and hMG.115 In contrast to the earlier study of Daya et al,116 we assessed the outcome in relation to the preceding schedule of treatment with GnRH analog, on the principle that the action of these hormones on the ovary would be determined in part at least by the endocrine milieu in which they were administered. Our analysis showed that, in studies in which the long protocol of GnRH desensitization was used, we could detect no difference in outcome between ovarian stimulation with urinary-derived FSH or with hMG preparations. In recent years, recombinant follicle-stimulating hormone (rFSH) has increasingly been used in ovulation induction and IVF treatments. Although there is pituitary suppression by GnRH agonists during the IVF treatment, a low endogenous LH level is sufficient to permit an adequate steroidogenesis in the mature follicles. Recombinant follicle-stimulating hormone is synthesized by transfecting Chinese hamster ovary cell lines with both FSH subunit genes. It has a higher bioactivity than urinary FSH,117 and results in a higher number of oocytes retrieved in IVF treatment, and a shorter duration of treatment in clomiphene-resistant anovulatory patients.118 The same group also reported that the initial dose of rFSH can be reduced to 100 IU, achieving a similar response as the usual starting dose of 150–225 IU.119 Marci et al 120 demonstrated that a low-dose stimulation protocol with rFSH can lead to high pregnancy rates in IVF patients with polycystic ovaries who are at risk of a high ovarian response to gonadotropins. This protocol may potentially reduce the risk of OHSS. Teissier et al121 demonstrated that women with PCOS undergoing IVF cycles using hMG had higher testosterone and estradiol levels compared with those using rFSH, due to higher serum LH levels. Van Wely et al122 reported a metaanalysis and systematic review on the effectiveness and

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the outcomes of IVF cycles with a long protocol using GnRH agonist, between ovarian stimulation with rFSH and urinary FSH. This review concluded that there was no evidence of a difference between hMG and rFSH in respect to the number of oocytes retrieved and the ongoing pregnancy/live birth rates (OR = 1.27; 95% CI 0.98–1.64). No significant differences were detected in the rates of miscarriage, multiple pregnancy, and OHSS. Andersen et al123 also revealed that there was no difference in the pregnancy outcomes following stimulation with highly purified hMG or rFSH. However, these results will of course need to be updated when data specifically derived from women with PCOS become available from randomized controlled trials (RCTs) using recombinant gonadotropin preparations, GnRH antagonists rather than superactive agonists. The recent introduction of schedules of gonadotropin stimulation that incorporate treatment with GnRH antagonists holds promise for patients with PCO and PCOS. GnRH antagonists do not activate the GnRH receptors and produce a rapid suppression of gonadotropin secrection within hours. The new IVF protocol using GnRH antagonists can offer a shorter and simpler treatment in comparison with the long protocol using GnRH agonists.124,125 A Cochrane Database of Systematic Reviews, including 27 RCTs comparing the GnRH antagonist to the long protocol of GnRH agonist, has been published recently. This showed a significant reduction in the incidence of severe OHSS with the antagonist protocol, with a relative risk of 0.61 (95% CI 0.42–0.89).126 However, this review also demonstrated that clinical pregnancy and live-birth rates were significantly lower, with ORs of 0.84 (95% CI 0.72–0.97) and 0.81 (95% CI 0.69–0.98), respectively. Patients with PCOS undergoing IVF using the GnRH antagonist protocol were found to have an earlier follicular growth and higher estradiol concentrations during the initial stimulation phase with rFSH compared with long agonist protocol.127 Despite a shorter stimulation period with antagonist protocol, the number of oocytes retrieved, overall fertilization, and clinical and ongoing pregnancy rates were not different.127 Furthermore, a significantly lower OHSS rate was also observed in the antagonist group. These findings are consistent with previous studies128,129 and a recent meta-analysis by Griesinger et al130 Nevertheless, among OHSS patients with female factor infertility undergoing an antagonist cycle, a significantly higher proportion was caused by PCOS than in the non-OHSS group (65.2% vs 8.1%; p <0.05).131 Another advantage of using GnRH antagonists is that the native GnRH or GnRH agonist can displace the antagonist from the GnRH receptors at the pituitary level. Therefore, in a GnRH antagonist IVF cycle, GnRH agonist can be administered to induce an LH surge and to trigger the final oocyte maturation and ovulation.132,133 Itskovitz-Eldor et al134 demonstrated a rapid rise of LH concentrations after administration of GnRH

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agonist, and a peak in LH levels at 4 hours after the injection. The pattern of the induced LH surge was similar to those observed in the natural cycle. Fauser et al135 showed that the outcomes of IVF treatment in terms of the number of oocytes retrieved, the proportion of metaphase II oocytes, the fertilization rates, the number of good-quality of embryos, the implantation rates, and the pregnancy rates were comparable to using hCGtriggering ovulation. The findings were confirmed by a recent RCT by Acevedo et al.136 Triggering of ovulation with GnRH agonist is potentially more physiological and can reduce the risk of OHSS compared with using hCG, due to a shorter half-life of LH (60 minutes vs 32– 34 hours). A few small recently published studies were able to support this view.133,136,137 However, a metaanalysis conducted by Griesinger et al138 demonstrated that GnRH agonist administration is associated with a significantly reduced likelihood of achieving a clinical pregnancy (0.21, 95% CI 0.05–0.84; p = 0.03). Therefore, a large RCT is required to evaluate these findings. A recent multicenter, double-blind study revealed that new recombinant human luteinizing hormone can be as effective as hCG in inducing the final follicular maturation in IVF treatment,125 with a lower incidence of OHSS. This clinical effect can be beneficial for women with polycystic ovaries undergoing either GnRH antagonist or agonist IVF cycles. However, its clinical application in ART has not yet been clearly established. At present, therefore, we recommend the long protocol of pituitary desensitization for women identified as having PCO or PCOS. When using urinary-derived gonadotropin preparations, we employ a dose of 75–150 units of an FSH-containing preparation for women under the age of 35 years old and 150 units for older women. This is intentionally lower than our usual starting dose of 225 units for women under the age of 35 years old. When using recombinant gonadotropin preparations, we recommend reducing the dose by at least 25– 30%. The dose should be further modified if the patient has had either an exuberant or a poor response in a previous cycle of treatment. Follicular development is then monitored principally by daily ultrasonography from day 8 of stimulation, with additional measurements of serum estradiol being helpful in some cases. Because of the lower pregnancy rates associated with the antagonist protocols, it is not recommended as a first-line IVF treatment regimen. However, for women with PCOS who have previously hyperresponded to FSH stimulation, using an antagonist protocol may be a safer option.

The impact of obesity and insulin resistance on the outcome of the IVF treatment Women with PCOS undergoing IVF treatment are associated with higher miscarriage rates.40,48,139 Patients

with android obesity, which is a common feature of PCOS, and a high BMI (>25 kg/m2), were found to have low pregnancy rates after IVF.49,54,57 These observations were consistent with the early studies on pregnancy outcomes after ovulation induction with gonadotropin in obese PCOS women.50 Fedorcsak et al47,57 concluded that obesity (>25 kg/m2), independent of insulin resistance, is associated with the gonadotropin resistance as well as the risk of miscarriage. Fewer oocytes were also retrieved from obese women. The number of oocytes collected and the quality of transferred embryos were positively correlated. In other words, embryo qualities declined along with the number of oocytes recovered. A recent retrospective study of over 1000 women undergoing IVF treatment demonstrated that morbidly obese women (BMI >40 kg/m2) had a 25.3% IVF cycle cancellation rate compared with 10.9% in normal-weight women (OR = 2.73, 95% CI 1.49–5.0).140 Tian et al141 also revealed that increased insulin resistance is an independent risk factor of miscarriage after adjustment of BMI and age of women undergoing fertility treatment. Hence, obese women with PCOS should lose weight before commencing IVF treatment in order to improve the outcome. Furthermore, women with PCOS are known to be at a greater risk of developing gestational diabetes, pre-eclampsia, and preterm birth.142 Therefore, an improvement of preconceptual lifestyle would decrease the pregnancy related morbidities. The serum androgen levels rise during the ovarian stimulation in IVF cycles143 and the levels tend to be higher in patients with PCOS.40 Check et al,144 Takeuchi et al,145 and Kodaman et al146 suggested that high androgen levels negatively affect the pregnancy outcome. Okon et al147 showed that there was a negative correlation between androgen concentrations and uterine placental protein 14 (PP14, also known as glycodelin) levels in women with PCOS with history of recurrent miscarriage. Glycodelin is an important secretory protein from the endometrium and is a marker of endometrial receptivity.148,149 Serum glycodelin levels were found to be increased in the conception compared with the nonconception IVF cycles.150,151 An in vitro study on the human endometrium by Tuckerman et al152 showed that androstenedione caused a dose-dependent decrease in the production of glycodelin and endometrial cell proliferation. These effects were inhibited when co-cultured with cyproterone (antiandrogen). Maliqueo et al153 also reported that the protein expression of sex hormone-binding globulin (SHBG) in the endometrial stroma was significantly lower in women with PCOS compared with controls. This implies a greater bioavailability of androgen in the endometrium of women with PCOS. In addition, women with PCOS exhibited a greater endometrial androgen receptor expression compared with normal fertile controls.149 This expression is mainly at the glandular and luminal epithelium. High levels of estrogen and androgen up-regulate the expression of androgen receptors,

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whereas progesterone poses an opposite effect.154,155 Androgen also suppresses the endometrial expression of αvβ3 integrin, a cell adhesion molecule that normally appears on the apical surface of glandular and luminal epithelium at the time those embryos implant. Therefore, its level reflects the endometrial receptivity.146,156 On the basis of the above evidence, high androgen levels may have detrimental effects on endometrial receptivity145 and, consequently, affect the outcomes of pregnancy.

The use of metformin in IVF treatment Insulin resistance and compensatory hyperinsulinemia contribute to the pathogenesis of PCOS.3 A number of studies investigated the beneficial effects of using insulin-sensitizing agents, mainly metformin, on women with PCOS. Although many of these studies were small, collectively, a majority of them demonstrated improvements in menstrual regularity, spontaneous ovulation rates, serum androgens, and insulin levels.157–160 There was also some evidence suggesting that metformin can improve response to clomiphene and gonadotropin ovulation induction therapy.159–162 However, more recent large RCTs were unable to substantiate these benefits,163–165 especially among women who are overweight. Hyperinsulinemia is often associated with hyperandrogenism. Teissier et al55 suggested that the follicular endocrine microenvironment is related to oocyte quality in women undergoing IVF. As mentioned previously, high androgen levels may contribute to a lower fertilization rate among the oocytes retrieved from women with PCOS compared to those women without PCOS. Therefore, co-treatment with metformin in IVF treatment may also improve the response to exogenous gonadotropins. A recent publication by Stadtmauer et al166 reported that the use of metformin in patients with PCOS undergoing IVF treatment improved the number of mature oocytes retrieved and the overall fertilization and pregnancy rates. However, caution is required to interpret the retrospective observational data. Since then, only a few RCTs, an open-label study by Fedorcsak et al46 with 17 patients and the first RCT by Kjotrod et al167 with 73 subjects, have been conducted to ascertain the benefit of using metformin for women with PCOS undergoing IVF cycles. Recently, our group published the largest RCT, with 94 subjects with PCOS undergoing 101 consecutive cycles.168 All the subjects underwent a conventional long protocol with GnRH agonist employing a lowdose step-up stimulation regimen with a starting dose of 100 IU of rFSH. All the patients commenced metformin or placebo at the start of down-regulation until the day of egg retrieval. The mean duration of medication was 28 days. The mean age was 31 years old, and the mean BMI was 27 kg/m2. Our study demonstrated that a short course of metformin during the

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IVF cycle resulted in an improved clinical pregnancy rate per cycle (38.5% vs 16.3%, p = 0.023) and live birth rate per cycle (32.7% vs 12.2%, p = 0.027) compared with a placebo group. Furthermore, metformin also reduced the risks of OHSS (3.8% vs 20.4%, p = 0.023), despite the fact that it neither enhanced the response to the stimulation nor improved the fertilization rate. Additionally, metformin also reduced the serum estradiol, androgen, fasting insulin, and VEGF concentrations on the day of administration of hCG. The regimen was also well tolerated, with a low rate of withdrawal. Since all the women received a similar number and quality of embryos, the favorable pregnancy outcomes in the metformin group may be the result of an improvement of negative factor(s) unrelated to oocyte quality in women with PCOS. Similar observations were also reported by Ludwig et al.42 It is possible that metformin improves the pregnancy outcome, maybe through its ability to reduce androgen production. As mentioned previously, hyperandrogenism may have a detrimental effect on endometrial receptivity. Recently, increased insulin resistance has been found to be associated with an increase of miscarriage after IVF treatment.141 Hence, a reduction of insulin levels in the metformin group may also explain the improved pregnancy outcome. A reduction of estradiol concentration can be partly explained by a decrease in androgen substrates induced by metformin. Furthermore, Nestler et al169 revealed that decreasing serum insulin with metformin reduces ovarian cytochrome P450c17α activity, and hence ameliorates hyperandrogenism. There is also evidence to suggest that metformin has direct effects on human ovarian steroidogenesis. Mansfield et al170 and Attia et al171 demonstrated a reduction in androstenedione production from the theca cells when they were co-cultured with metformin. In addition, metformin was also found to reduce aromatase activity directly.74 These findings may also explain the rapid biochemical improvements in the serum testosterone and estradiol concentrations observed in our study after a short course of metformin therapy. Metformin appears to reduce estradiol levels and the risk of overstimulation by lowering VEGF concentrations. A recent meta-analysis conducted by Costello et al172 which also included our current data, reported that metformin reduces the incidence of OHSS (OR= 0.21; 95% CI 0.11–0.41). However, this meta-analysis did not include our study analyzing the effect of metformin on estradiol concentrations in IVF treatment. Therefore, a repeat analysis was carried out and it demonstrated the beneficial effect of metformin on the reduction of estradiol levels (WMD −2.60 nmol/l, 95% CI −3.87 to −1.33) (Fig 53.3). Our study was unable to show metformin improved the response to stimulation, the number of oocytes retrieved, the fertilization rates, the cleavage rates or the embryo quality.168 These findings were consistent with the other prospective studies,46,167 but contradicted the

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Study or subcategory

N

Metformin Mean (SD)

N

Control Mean (SD)

Visnova 2003 Kjotrod 2001 Onalan 2005 Tang

71 31 53 52

12.56 (6.21) 6.80 (3.96) 14.56 (7.04) 7.45 (4.72)

66 30 55 49

22.69 (11.59) 7.60 (5.36) 14.51 (5.19) 10.32 (7.77)

Total

207

WMD (fixed) 65% CI

Weight %

200

WMD (fixed) 95% CI

16.33 28.77 29.55 25.35

–10.13 [–13.28, –6.98] –0.00 [–0.17, 1.57] 0.05 [–2.29, 2.39] –2.87 [–5.40, –0.34]

100.00

–2.60 [–3.87, –1.33]

Test for heterogeneity: χ2 = 29.18, df = 3 (P < 0.00001), I2 = 89.7% Test for overall effect: Z = 4.00 (P < 0.0001) –10

–5 0 Favors treatment

5 Favors control

10

Figure 53.3. Comparison of metformin vs placebo or no treatment in IVF with the outcome of maximum serum estradiol concentrations (nmol/l) during stimulation.

findings of Stadtmauer et al166 despite similar BMI in the study populations. This may be partly explained by the fact that Stadtmauer et al166 compared the outcomes to their historic data. Artini et al94 demonstrated that more immature oocytes were retrieved from women with PCOS than from women with normal ovaries undergoing IVF treatment. In addition, Tarin and Pellicer173 reported that high responders (>11 oocytes retrieved) in IVF treatment had a lower estradiol level per follicle, with a higher incidence of diploid oocytes and cytoplasmic immaturity, than those with less than 10 oocytes retrieved. Therefore, the main factor to improve the fertilization rates and to reduce the risk of developing OHSS in women with PCOS undergoing IVF treatment is to reduce the antral follicle counts before stimulation. As mentioned previously, hyperinsulinemia and hyperandrogenism play a major role in the initial recruitment and growth of the primordial follicles. Therefore, a more prolonged treatment with metformin before starting the IVF cycle might be expected to improve oocyte quality, since the duration of maturation from primary follicles to the antral follicles stage is more than 4 months. Fleming et al62 also demonstrated that a protracted treatment with metformin (over 4 months) decreased the follicle counts and AMH levels compared with a very rapid reduction of androstenedione concentrations by metformin (within weeks). These findings suggest that a reduction of insulin and androgen levels reduces the recruitment of primordial follicles. Since the duration of folliculogenesis in humans is >4 months, a reduction of antral follicle counts and AMH levels would not be evident until a prolonged treatment with metformin. However, Kjotrod et al167 did not find that pretreatment with metformin for at least 4 months improved the response, the number of oocytes retrieved, and the fertilization rates. A potential advantage of using a short course of metformin, as in the current study, is that it improves the compliance and reduces the withdrawal rate. Currently, a low-dose step-up regimen should be continuously employed during the stimulation for the high responders undergoing IVF treatment. Regarding the safety of using metformin, recent retrospective174 and prospective data175,176 have been reassuring without any effects on birth defects or motor-social development.

In vitro maturation In recent years, in vitro maturation (IVM) has attracted much interest as a new assisted reproductive technique.177,178 The immature oocytes are retrieved from the antral follicles within the unstimulated ovaries via the transvaginal approach.178 The oocytes are subsequently matured in vitro in a special formulated culture medium for 24–48 hours. The mature oocytes are fertilized with or without the ICSI technique, and the selected embryos are transferred to the uterus 2–3 days later. Although IVM is labor-intensive compared with conventional IVF treatment, there are a number of clinical advantages in IVM cycles without using exogenous gonadotropin. Patients, in general, require less monitoring, are free from any side effects associated with exogenous gonadotropin and, most importantly, they can avoid the risk of OHSS. Since patients with PCOS have more antral follicles and a higher risk of developing OHSS compared with those without, IVM may be a promising alternative to conventional IVF.179 Some studies reported that the maturation rate of immature oocytes recovered from patients with PCOS were lower than those from women with normal regular menstrual cycles.180 However, Chian et al181 demonstrated that priming with hCG before the retrieval of immature oocytes from unstimulated women with PCOS improved the maturation rates. In a prospective observational study of 180 cycles, Child et al178 demonstrated that significantly more immature oocytes were retrieved from PCO (10 ± 5.1) and PCOS (11.3 ± 9.0) groups than from women with normal ovaries (5.1 ± 3.7), p <0.05. The overall oocyte maturation and fertilization rates were similar among the three groups. The subsequent pregnancy and live birth rates per transfer were significantly higher in the PCO and PCOS groups. This could be partially explained by the fact that there was a greater choice in the embryos selected for transfer in these two groups. However, women with PCO and PCOS were significantly younger and had more embryos transferred than women with normal ovaries. Furthermore, Child et al180 also reported a casecontrol study comparing 170 IVM and 107 IVF cycles for women with PCOS. IVM yields significantly less mature oocytes than IVF cycles (7.8 vs 12, p <0.01)

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and less embryos per retrieval (6.1 vs 9.3, p <0.01). The pregnancy rate per retrieval were similar between the groups. However, the implantation rate in the IVM group was significantly lower than in the IVF group (9.5% vs 17.1%, p <0.01) despite patients in the IVM cycles receiving more embryos than in the IVF cycles (3.2 ± 0.7 vs 2.7 ± 0.8, p <0.01). Similar observations were reported by a French team.182 The lower implantation rates may be due to a reduced oocyte potential, a higher frequency of abnormal meiotic spindle and chromosomal alignment, or a reduced endometrial receptivity.183 Continuous improvements in the culture medium and synchrony between endometrial and embryonic development will hopefully result in better IVM success rates in the future. It is also important that the infants born after IVM treatment should have a long-term follow-up to ensure the safety of this new technology. A recent prospective observational study on 41 pregnancies resulting from IVM treatment showed that there were no increases in preterm birth, birth weight, or major structural malformation compared with pregnancies resulting from conventional IVF.184 However, a much larger cohort study is required to confirm the safety of this new technology.

Summary Women with PCOS undergoing IVF cycles respond differently from women with normal ovaries. For the obese group of patients, weight loss before commencement of ART should remain the pivotal part of the management in order to maximize the outcomes and to reduce the risks associated with IVF treatment. Early findings indicate that co-treatment with metformin improves the pregnancy outcome and reduces the incidence of OHSS. A low-dose step-up stimulation regimen in a long protocol, combined with regular US monitoring, still forms a key part of ART.

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136. Acevedo B, Gomez-Palomares JL, Ricciarelli E, Hernandez ER. Triggering ovulation with gonadotropin-releasing hormone agonists does not compromise embryo implantation rates. Fertil Steril 2006; 86(6): 1682–7. 137. Babayof R, Margalioth EJ, Huleihel M, et al. Serum inhibin A, VEGF and TNFalpha levels after triggering oocyte maturation with GnRH agonist compared with HCG in women with polycystic ovaries undergoing IVF treatment: a prospective randomized trial. Hum Reprod 2006; 21(5): 1260–5. 138. Griesinger G, Diedrich K, Devroey P, Kolibianakis EM. GnRH agonist for triggering final oocyte maturation in the GnRH antagonist ovarian hyperstimulation protocol: a systematic review and meta-analysis. Hum Reprod Update 2006; 12(2): 159–68. 139. Homburg R. Pregnancy complications in PCOS. Best Pract Res Clin Endocrinol Metab 2006; 20(2): 281–92. 140. Dokras A, Bochner M, Hollinrake E, et al. Screening women with polycystic ovary syndrome for metabolic syndrome. Obstet Gynecol 2005; 106(1): 131–7. 141. Tian L, Shen H, Lu Q, Norman RJ, Wang J. Insulin resistance increases the risk of spontaneous abortion after assisted reproduction technology treatment. J Clin Endocrinol Metab 2007; 92(4): 1430–3. 142. Boomsma CM, Eijkemans MJ, Hughes EG, et al. A meta-analysis of pregnancy outcomes in women with polycystic ovary syndrome. Hum Reprod Update 2006; 12(6): 673–83. 143. Fanchin R, de Ziegler D, Taieb J, et al. Human chorionic gonadotropin administration does not increase plasma androgen levels in patients undergoing controlled ovarian hyperstimulation. Fertil Steril 2000; 73(2): 275–9. 144. Check JH, Nazari A, Dietterich C. Comparison of androgen levels in conception vs. non-conception cycles following controlled ovarian stimulation using the luteal phase gonadotropin-releasing hormone agonist protocol. Gynecol Endocrinol 1995; 9(3): 209–14. 145. Takeuchi T, Nishii O, Okamura T, et al. Free testosterone and abortion in early pregnancy. Int J Gynaecol Obstet 1993; 43(2): 151–6. 146. Kodaman P, Taylor H. Hormonal regulation of implantation. Obstet Gynecol Clin North Am 2004; 31(4): 745–55. 147. Okon MA, Laid SM, Tuckerman EM, Li TC. Serum androgen levels in women who have recurrent miscarriages and their correlation with markers of endometrial function. Fertil Steril 1998; 69(4): 682–90. 148. Westergaard L, Wiberg N, Ansersen C, et al. Circulating concentrations of placenta protein 14 during the natural menstrual cycle in women significantly reflect endometrial receptivity to implantation and pregnancy during successive assisted reproduction cycles. Hum Reprod 1998; 13(9): 2612–19. 149. Lessey BA. Endometrial receptivity and the window of implantation. Baillières Best Pract Res Clin Obstet Gynaecol 2000; 14(5): 775–88.

150. Westergaard L, Wiberg N, Ansersen C, et al. Placenta protein 14 concentrations in circulation related to hormonal parameters and reproductive outcome in women undergoing IVF/ICSI. Reprod Biomed Online 2004; 8(1): 91–8. 151. Suzuki Y, Sugiyama R, Fukumine N, et al. Clinical applications of serum placental protein 14 measurement in IVF-ET cycle. J Obstet Gynaecol Res 2000; 26(4): 295–302. 152. Tuckerman EM, Okon MA, Li T, Laird SM. Do androgens have a direct effect on endometrial function? An in vitro study. Fertil Steril 2000; 74(4): 771–9. 153. Maliqueo M, Bacallao K, Quezada S, et al. Sex hormone-binding globulin expression in the endometria of women with polycystic ovary syndrome. Fertil Steril 2007; 87(2): 321–8. 154. Apparao KB, Lovely LP, Gui Y, et al. Elevated endometrial androgen receptor expression in women with polycystic ovarian syndrome. Biol Reprod 2002; 66(2): 297–304. 155. Giudice LC. Endometrium in PCOS: implantation and predisposition to endocrine CA. Best Pract Res Clin Endocrinol Metab 2006; 20(2): 235–44. 156. Rose G, Dowsett M, Mudge J, et al. The inhibitory effects of danazol, danazol metabolites, gestrinone and testosterone on the growth of human endometrial cells in vitro. Fertil Steril 1988; 49(2): 224–8. 157. Fleming R, Hopkinson ZE, Wallace AM, et al. Ovarian function and metabolic factors in women with oligomenorrhea treated with metformin in a randomized double blind placebocontrolled trial. J Clin Endocrinol Metab 2002; 87(2): 569–74. 158. Costello MF, Eden JA. A systematic review of the reproductive system effects of metformin in patients with polycystic ovary syndrome. Fertil Steril 2003; 79(1): 1–13. 159. Nestler JE, Jakubowicz DJ, Evans WS, Pasquali R. Effects of metformin on spontaneous and clomiphene-induced ovulation in the polycystic ovary syndrome. N Engl J Med 1998; 338(26): 1876–80. 160. Lord JM, Flight IM, Norman RJ. Insulin-sensitising drugs (metformin, troglitazone, rosiglitazone, pioglitazone, D-chiro-inositol) for polycystic ovary syndrome. Cochrane Database Syst Rev 2003(3): CD003053. 161. Kocak M, Caliskan E, Simsir C, Haberal A. Metformin therapy improves ovulatory rates, cervical scores, and pregnancy rates in clomiphene citrate-resistant women with polycystic ovary syndrome. Fertil Steril 2002; 77(1): 101–6. 162. Vandermolen DT, Ratts VS, Evans WS, et al. Metformin increases the ovulatory rate and pregnancy rate from clomiphene citrate in patients with polycystic ovary syndrome who are resistant to clomiphene citrate alone. Fertil Steril 2001; 75(2): 310–15. 163. Tang T, Glanville J, Hayden CJ, et al. Combined lifestyle modification and metformin in obese patients with polycystic ovary syndrome. A randomized, placebo-controlled, double-blind multicentre study. Hum Reprod 2006; 21(1): 80–9. 164. Moll E, Bossuyt PM, Korevaar JC, et al. Effect of clomifene citrate plus metformin and clomifene

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165.

166.

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169.

170.

171.

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173.

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citrate plus placebo on induction of ovulation in women with newly diagnosed polycystic ovary syndrome: randomised double blind clinical trial. BMJ 2006; 332(7556): 1485. Legro RS, Barnhart HX, Schlaff WD, et al. Clomiphene, metformin, or both for infertility in the polycystic ovary syndrome. N Engl J Med 2007; 356(6): 551–66. Stadtmauer LA, Toma SK, Riehl RM, Talbert LM. Metformin treatment of patients with polycystic ovary syndrome undergoing in vitro fertilization improves outcomes and is associated with modulation of the insulin-like growth factors. Fertil Steril 2001; 75(3): 505–9. Kjotrod SB, von During V, Carlsen SM. Metformin treatment before IVF/ICSI in women with polycystic ovary syndrome; a prospective, randomized, double blind study. Hum Reprod 2004; 19(6): 1315–22. Tang T, Glanville J, Orsi N, et al. The use of metformin for women with PCOS undergoing IVF treatment. Hum Reprod 2006; 21(6): 1416–25. Nestler JE, Jakubowicz DJ. Lean women with polycystic ovary syndrome respond to insulin reduction with decreases in ovarian P450c17 alpha activity and serum androgens. J Clin Endocrinol Metab 1997; 82(12): 4075–9. Mansfield R, Glanville J, Orsi N, et al. Metformin has direct effects on human ovarian steroidogenesis. Fertil Steril 2003; 79(4): 956–62. Attia GR, Rainey WE, Carr BR. Metformin directly inhibits androgen production in human thecal cells. Fertil Steril 2001; 76(3): 517–24. Costello MF, Chapman M, Conway U. A systematic review and meta-analysis of randomized controlled trials on metformin co-administration during gonadotrophin ovulation induction or IVF in women with polycystic ovary syndrome. Hum Reprod 2006; 21(6): 1387–99. Tarin JJ, Pellicer A. Consequences of high ovarian response to gonadotropins: a cytogenetic analysis of unfertilized human oocytes. Fertil Steril 1990; 54(4): 665–70. Ekpebegh CO, Coetzee EJ, van der Merwe K, Levitt NS. A 10-year retrospective analysis of pregnancy outcome in pregestational Type 2 diabetes: comparison of

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insulin and oral glucose-lowering agents. Diabet Med 2007; 24(3): 253–8. Gilbert C, Valois M, Koren G. Pregnancy outcome after first-trimester exposure to metformin: a metaanalysis. Fertil Steril 2006; 86(3): 658–63. Glueck CJ, Bornovali S, Pranikoff, et al. Metformin, pre-eclampsia, and pregnancy outcomes in women with polycystic ovary syndrome. Diabet Med 2004; 21(8): 829–36. Trounson A, Wood C, Kausche A. In vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients. Fertil Steril 1994; 62(2): 353–62. Child TJ, Abdul-Jalil AK, Gulekli B, Tan SL. In vitro maturation and fertilization of oocytes from unstimulated normal ovaries, polycystic ovaries, and women with polycystic ovary syndrome. Fertil Steril 2001; 76(5): 936–42. Chian RC. In-vitro maturation of immature oocytes for infertile women with PCOS. Reprod Biomed Online 2004; 8(5): 547–52. Child TJ, Phillips SJ, Abdul-Jalil T, Tan SL. A comparison of in vitro maturation and in vitro fertilization for women with polycystic ovaries. Obstet Gynecol 2002; 100(4): 665–70. Chian RC, Buckett WM, Tulandi T, et al. Prospective randomized study of human chorionic gonadotrophin priming before immature oocyte retrieval from unstimulated women with polycystic ovarian syndrome. Hum Reprod 2000; 15(1): 165–70. Le Du A, Kadoch IJ, Bourcigaux N, et al. In vitro oocyte maturation for the treatment of infertility associated with polycystic ovarian syndrome: the French experience. Hum Reprod 2005; 20(2): 420–4. Li Y, Feng HL, Cao YJ, et al. Confocal microscopic analysis of the spindle and chromosome configurations of human oocytes matured in vitro. Fertil Steril 2006; 85(4): 827–32. Cha KY, Chung HM, Lee DR, et al. Obstetric outcome of patients with polycystic ovary syndrome treated by in vitro maturation and in vitro fertilization–embryo transfer. Fertil Steril 2005; 83(5): 1461–5.

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54 Prognostic testing for ovarian reserve Frank J Broekmans, Bart CJM Fauser, Nick S Macklon

believed to decrease after the age of 31 years old, a decrease which may accelerate after age 37 years old, leading to sterility at a mean age of 41 years old.5 As with menopause, the rate of decline in fertility may vary considerably between women of the same age. This implies that a woman of 35 years old either may be close to natural sterility or have a normal fertility comparable to a 25-year-old woman. The decrease of female fertility is believed to exhibit the same range of variation as for the occurrence of menopause.2,6,7 This implies that the age at menopause, which is determined by the remaining follicle numbers, is considered a proxy variable for age at loss of natural fertility, with a fixed time period of 10 years in between. The correct prediction of menopause in an individual woman would therefore provide valuable information regarding a woman’s fertile life span and hence aid in preventing future subfertility. At present, however, reliable means of prediction remain elusive (Fig 54.2).

Female reproductive aging Age-related subfertility and ovarian reserve With the postponement of childbearing in Western societies, rates of subfertility related to female age have increased considerably.1 An increasing proportion of couples, therefore, will depend on assisted reproductive technologies (ART) in order to achieve a pregnancy. The increase of subfertility with increasing female age is mainly based on changes in ovarian function, referred to as decreasing ovarian reserve. Ovarian reserve can be defined as the number and quality of the remaining follicles and oocytes in both ovaries at a given age. Decline in follicle numbers dictates the occurrence of irregular cycles and menopause, whereas quality decay results in decreasing fertility, which is defined as the capacity to conceive and give birth to a child2 (Fig 54.1).

Variability of reproductive aging There is substantial individual variation in the onset of menopause, varying roughly between 40 and 60 years old, with a mean age of 51 years old. This variation has been shown to be rather constant over time and in populations worldwide.3,4 Female fecundity is

Natural and assisted fertility decline The human species can be considered as relatively subfertile compared with animals.8,9 The average monthly fecundity rate of about 20% implies that

Optimal fertility

Number of follicles

106

Declining fertility

End of fertility

Menopause

Irregular oocytes

105

100

104 75

50

103

25

102

0

10

20

30 Age (years)

40

50

60

Proportion of poor quality oocytes %

107

Fig 54.1 Quantitative (solid line) and qualitative (dotted line) decline of the ovarian follicle pool, which is assumed to dictate the onset of the important reproductive events. (Reproduced and adapted from de Bruin JP, te Velde ER. Female reproductive aging: concepts and consequences. In: Tulandi T, Gosden RG, eds. Preservation of Fertility. London: Taylor & Francis, 2004: 3.)

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2

3

for the chance of an IVF embryo to implant after IVF.12 A poor response to ovarian hyperstimulation for IVF, especially in those with abnormal ovarian reserve test results, is a strong predictor of poor prospects of becoming pregnant, and also of clearly reduced spontaneous fecundity and early menopause.13–15

4

50

use opa Men

Cyc le

Ster il

Sub fe

Cumulative %

75

ity

r tilit

y

irreg ular it

y

100

Ovarian reserve prediction 25

The knowledge and insights into the process of ovarian aging imply that for ovarian reserve testing prior to ART, female age remains the predictor of first choice. The availability of a test capable of providing reliable information regarding a woman’s individual ovarian reserve within a certain age category would enable the clinician to provide an individually tailored treatment plan. For instance, in older women, the finding of a high ovarian reserve may justify the decision to allow ART treatment, whereas in young women with exhausted reserve either early application or even refusal of ART could be the consequence. Ultimately, the response to maximal ovarian stimulation may provide further information on the reserve capacity of the ovaries. In the following two sections the biological rationale behind ovarian reserve testing and the accuracy and clinical value of several of these tests are discussed.

0 21

31

41 Age (years)

51

61

Fig 54.2 Variations in age at the occurrence of specific stages of ovarian aging. For explanation of the background data, see te Velde and Pearson.2 (Reprinted with permission from te Velde and Pearson.2)

among human couples trying to conceive, many exposure months may be needed to achieve their goal, especially if monthly fecundity has dropped with increasing female age.10 The proportion of subfertile couples (failing to achieve a vital pregnancy within 1 year) will amount to 10–20% in the age group of women over 35 years old compared with only 4% for women in their 20s. These subfertility rates may rise to 30–50% for only moderately fecund women of ≥35 years old who have tried to conceive for several years.10,11 The maintenance of regular menstrual cycles until an age when natural fecundity has already been reduced to zero means that women are largely unaware that this process is taking place. The age-related decline in female fertility has also been shown in numerous reports concerning ART (i.e. in vitro fertilization [IVF]) programs. After a mean female age of ∼34 years old, the chance of producing a live birth in ART programs decreases steadily, and will become under 10% per cycle in women >40 years old (Fig 54.3). The effect of female age has also been shown

The physiological background to ovarian reserve testing Follicle quantity Ovarian reserve can be considered normal in conditions where stimulation with the use of exogenous gonadotropins will result in the development of some 5–13 follicles and the retrieval of a corresponding number of healthy oocytes at follicle puncture.16,17 With such a yield, the chances of producing a live birth through IVF are considered optimal.18 In addition to the number of recruitable follicles, which deter-

40 35

Percentage

30 25 20 15 10 5 0 22

24

26

28

30

32

34

36

Female Age (Years)

38

40

42

44

46

Fig 54.3 Effect upon average singleton live birth rates of female age, showing a steady decrease after the age of 34 years old. The dotted line represents the average singleton live birth rate after oocyte donation as a function of the recipient age. It underlines the potential of oocyte donation in the treatment of women who remained unsuccessful in previous IVF treatment. Data were drawn from the 2003 CDC ART report: http: //www.cdc.gov/ART/ ART2003/section2a. htm#f12.

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mines the ovarian reserve status, follicle sensitivity to follicle-stimulating hormone (FSH) and the pharmacodynamics of FSH also determine a woman’s extent of ovarian response to stimulation. The dose of FSH used may be another factor, although the therapeutic range is generally considered as quite narrow. Higher doses of FSH may lead to higher numbers of oocytes retrieved in younger patients,19 but not in all cohorts.20 Such an approach will certainly fail in older women21 or in women expected to have a poor response to stimulation based on an abnormal ovarian reserve test.22

Female age In general, as outlined above, the age of a woman is a simple way of obtaining information on the extent of her ovarian reserve, both regarding quantity and quality.23 However, in view of the substantial variation in the decay of reproductive capacity of a woman with age, there is a need to identify women with clearly accelerated ovarian aging at a relatively young age. In addition, it would also be useful to identify women around the mean age at which natural fertility on average is lost (41 years old) with still adequate ovarian reserve. In clinical terms, the aim should be to identify women with a high risk of producing a poor response to ovarian hyperstimulation and/or a very low probability of becoming pregnant through IVF, as well as those who still produce enough oocytes and are likely to become pregnant even if female age is advanced. If it is going to be possible to identify such women, fertility management could be effectively individualized. For instance, stimulation dose or treatment scheme could be adjusted,24 counseling against initiation of IVF treatment or pertinent refusal could be effected, or treatment initiated early before the reserve has diminished too far.

Tests and their valuation Most tests examined in the literature are evaluated by their capacity to predict some defined outcome related to ovarian reserve. The preferred, or gold standard, outcome of prediction studies would be live birth after a series of ART exposure cycles, but other outcomes (especially oocyte yield or follicle number and pregnancy after one IVF/ICSI [intracytoplasmic sperm injection cycle) are in fact the most common. As the occurrence of pregnancy in a single exposure to IVF and embryo transfer will be dependent on many other factors besides ovarian reserve, such as laboratory performance and transfer technique, focus has been mostly upon the capacity of these tests to predict the occurrence of poor ovarian response. Indeed, most if not all ovarian reserve tests relate to the size of the follicle cohort that is at any time responsive to FSH. The antral follicle count (AFC) assessed by transvaginal ultrasonography provides direct visual assessment of the cohort.25 The endocrine markers anti-Müllerian hormone (AMH) and inhibin B, which are released from

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the antral follicles, provide other direct markers of quantity.26,27 Basal FSH, extensively studied in the past decades, provides the most indirect marker. FSH levels increase with advancing age, by a reduction in the release of inhibin B, thereby reducing the negative feedback on FSH release from the pituitary.28 High FSH levels therefore represent a small cohort size. Endocrine challenge tests in which the growth of antral follicles is stimulated by endogenous or exogenous FSH and response is assessed in terms of output of estradiol or inhibin B are also principally related to cohort size.26 However, they are considered as too laborious for screening purposes and will not add much predictive value compared to static tests such as AMH or the AFC.29,30 The same may be true for the clomiphene citrate (CC) challenge test, in which a CC-induced rise in FSH levels is counteracted by release of estradiol and inhibin B from growing antral follicles. The size of the antral follicle cohort will determine the degree of subsequent FSH suppression. Like the other challenge tests, the CC challenge test does not provide much additional information compared to basal FSH31,32 (Fig 54.4).

Accuracy and clinical value Ovarian reserve (OR) test evaluation, using response and/or pregnancy as reference or outcome variables, should imply the assessment of predictive accuracy and clinical value of the test. Predictive accuracy refers to the degree by which the outcome condition is predicted correctly. Summary statistics of accuracy include sensitivity (rate of correct identification of cases with, for example, poor response), specificity (rate of correct identification of cases without poor response), and the likelihood ratio (LR; how many times more likely particular test results are in patients with poor response compared to those without poor response).33,34 Using the calculated sensitivity and specificity for each cut-off level, a receiver operating characteristic (ROC) curve can be drawn and area under this curve (AUC) calculated to represent the overall predictive accuracy of the test. A value of 1.0 implies perfect and value of 0.5 indicates completely absent discrimination. If a test is to identify all cases that will respond poorly to stimulation without falsely labeling normal responders, it must have high sensitivity and high specificity. Positive LRs >10 and negative <0.1 are considered as indicators of an adequate diagnostic test, while values between 5 and 10 and <0.2 are considered to indicate a moderate test. As such, the LR can be considered a clinically useful tool to help judge the performance of the test, as the value will change when the cut-off for an abnormal test is shifted.

Clinical value Clinical value incorporates the question of whether application of the test at a certain cut-off will really change management, or costs, or safety, or success

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Textbook of Assisted Reproductive Technologies Hypothalamus

Hypothalamus

Younger

Older

Pituitary

Pituitary

Inhibin A AMH

Ovary

LH

FSH Inhibin B

Inhibin A E

AMH

FSH LH

Inhibin B

Ovary

rates, on a population basis. Assessment of the clinical value is a complex process in which the applicability in daily practice should become clear. The overall accuracy represented by the ROC curve, the choice of a cut-off for abnormality, the rate of abnormal tests at that cut-off, the post-test probability of disease (i.e. poor response or nonpregnancy), the valuation of false-positive and false-negative test results, and the consequence for patient management of an abnormal test will all contribute to the process of deciding whether a test is useful or not. The cost of carrying out the test as a routine measure and the burden to the patient balanced against the reduction in costs by excluding cases with low pregnancy prospects need to be incorporated in the decision process. Finally, clinical value may also be influenced by valuation from the patients’ and health insurance preference regarding the consequences that should be drawn from abnormal tests.35 Studies on the predictive accuracy and clinical value of OR tests should preferably be prospective in design. Moreover, they should examine cohorts of patients in IVF settings without exclusion of cases with signs of diminished ovarian reserve, and patient management should not be influenced by the test under study (verification bias). Also, evaluation should be equally weighted for every case; thus, every case should contribute the same amount of cycles to the analysis. In most studies only one IVF cycle is studied. A case-control design for the purpose of OR testing bears the disadvantage of retrospection and the absence of a reliable estimate of disease prevalence. The tests under study should, in principle, be reproducible, both at the laboratory (hormone assays)

E

Fig 54.4 Illustration of the changes in follicle reserve with increasing female age and the effect of these quantitative changes upon several endocrine factors. AMH, anti-Müllerian hormone; LH, lutenizing hormone; FSH, follicle stimulating hormone; E, estradiol. (Adapted from Soules et al.53)

as well as at the operator level (ultrasound examination). Also, the outcome of treatment (response and pregnancy), serving as the reference for ovarian reserve, should be clearly defined.

Screening or diagnosis One aspect of clinical value deserves special attention. Ovarian reserve tests are mostly used as a diagnostic test, indicating that in case of an abnormal test result the diagnosis of diminished ovarian reserve is made.36,37 For the valuation of the test, only proxy variables of true ovarian reserve (poor ovarian response and nonpregnancy) are used. Also, false-positive test results may eliminate couples from the IVF trial that do have adequate prospects. Therefore, ovarian reserve tests may better be considered as screening tests, where an abnormal test necessitates confirmation by another test. This other test may, for instance, be a first IVF attempt where ovarian response is the additional test. Alternatively, combinations of independently predictive tests or repeating of the initial test could improve the diagnostic performance of the single test.17,38–43

The value of ovarian reserve testing Systematic review In a recent review the predictive performance of all the listed tests was analyzed by using the approach of the systematic review and meta-analysis.26 A strict approach was followed, where the accuracy of the prediction of two distinct outcomes in IVF treatment was presented and the clinical value was assessed, as summarized in Table 54.1. Also, the role of the test

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Table 54.1

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Simplified stepwise approach to the meta-analysis of diagnostic tests

Step

Description

1. Data extraction 2. Homogeneity test: Homogeneity not rejected Homogeneity rejected

Contingency table: quality/methodology characteristics Chi square test on Sens and Spec of included studies Calculate summary point estimates for Sens and Spec and 95% CI Logistic regression analysis on relation quality/methodology characteristics and test accuracy: If present, subgroup analysis; if absent, assume cut-off point effect Spearman correlation between Sens and Spec (present if r<−0.5) Summary ROC curve estimation using random-effects regression model No pooling possible: subgroup analysis? Positive predictive value of abnormal test at various prevalence values using various cut-offs based on Summary ROC curve, in correspondence with abnormal test rate

3. Data pooling: Sens and Spec related Sens and Spec not related 4. Assess clinical value

Sens, sensitivity; Spec, specificity; CI, confidence interval; ROC, receiver operating characteristic.

Accuracy of poor response prediction

(a) 1 0.9 0.8

Sensitivity

0.7 0.6 0.5 0.4 sROC curve AFC sROC curve AMH sROC curve FSH AFC AMH FSH

0.3 0.2 0.1 0 0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1−Specificity

(a)

Accuracy of nonpregnancy prediction 1

0.9 0.8

Sensitivity

0.7 0.6 0.5 0.4 0.3 sROC curve AFC sROC curve AMH sROC curve FSH AFC AMH FSH

0.2 0.1 0 0

0.1

0.2

0.3

0.4

0.5

0.6

1−Specificity

0.7

0.8

0.9

1

Fig 54.5 Example of ovarian reserve test performance – antral follicle count (AFC), anti Müllerian hormone (AMH), and follicle-stimulating hormone (FSH) – showing receiver operating characteristic (ROC) curves for the prediction of (a) poor response and (b) non pregnancy in IVF. Data were based on a recent meta-analysis of ovarian reserve tests.26

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result in the choice of management options for the couple was included in the final judgment of the test. For example, a test identifying couples with a very poor prognosis, leading to denial of treatment, is certainly more valuable than the same test used only for counseling of the couple without strict consequences.

Poor response prediction From the extensive analysis in the review, it appeared that most of the tests demonstrate a reasonable capacity to predict poor responders to ovarian hyperstimulation in IVF. The areas under the receiver operating characteristic curve (AUC-ROC) for baseline FSH, the AFC, AMH, and ovarian volume indicate that the overall accuracy is sufficient (AUC-ROC: >0.70, Fig 54.5). From the clinical value analysis it was suggested that the AFC, AMH, and basal FSH had the best sensitivity and specificity combination for predicting ovarian response.26 For instance, if the prevalence of poor response was set at 20% and the cut-off chosen implied a positive LR >6, an abnormal AFC would indicate a post-test probability of poor ovarian response of around 67%. This would make the AFC test a clinically valuable test, especially as an abnormal test result would be found in 12% of patients. For FSH, a positive LR of ≥6 in a clinical setting where the prevalence of poor response was 20% would imply a post-test likelihood of about 67%, but this positive LR implies such a high basal FSH level that it would occur in only 3% of patients (Table 54.2). If poor response were to be the endpoint of interest, the clinical value of these tests would be satisfactory, provided that a change in management, such as using a higher gonadotropin stimulation dose, would benefit

Table 54.2

those with a predicted poor response and not harm those with a false-positive test by producing extreme responses (Fig 54.5). However, even a normalized response to ovarian stimulation may not alter the prognosis regarding the chances of pregnancy.44 Indeed, several studies have shown that in observed poor responders in a first IVF cycle no clear benefit can be expected from various changes in management, such as increasing the dosage, applying co-medication, or changing the approach of the gonadotropin-releasing hormone (GnRH) agonist administration. This implies that a prior prediction of poor response is to be considered useless, unless this prediction would identify cases with a poor response due to FSH under dosing related to obesity or FSH receptor polymorphisms. In IVF/ICSI cases without signs of ovarian aging, the use of a prediction model for ovarian response to FSH, containing the AFC, ovarian volume, power Doppler score, female age, and smoking habit, was developed for individualization of the FSH dose from the first cycle onwards.17 To test whether this FSH dosage score performed well in predicting ovarian response, a randomized trial compared ovarian response in women assigned either to an individual dose of FSH based on their score or a ‘standard’ dose of 150 IU/day.43,46 Women in the individual dose group, had more oocytes retrieved and a higher proportion of appropriate ovarian response than women in the standard dose group. Even ongoing pregnancy rates were higher in the individualized compared to the standard dose group, and dose adjustments were less frequently necessary than in the standard dose group. All this means that in poor responders, owing to other factors than ovarian aging, some benefit can be expected from adapted treatment schedules.

The clinical value of several ovarian reserve tests for outcome prediction in IVF Prediction of poor response in IVF (pre-test probability = 20%)

Test

FSH AMH AFC Inhibin B Estradiol OVVOL

Positive test rate (for the pLR value ≥ 6) 5% 12% 12% 1% 1% 0%

Post-test probability (of poor response) >60% >60% >60% >60% >60% >60%

Prediction of nonpregnancy after IVF (pre-test probability = 80%) Positive test rate (for the pLR value ≥ 6) 3% 1% 1% 1% 0% 0%

Post-test probability (of nonpregnancy) >96% >96% >96% >96% >96% >96%

Shown is the occurrence of abnormal ovarian test results, given a positive likelihood ratio (pLR) value of ³ 6, and the concomitant post-test probabilities of poor response and nonpregnancy, given a prevalence of poor response of 20% and nonpregnancy of 80%. Data were based on a recent meta-analysis of ovarian reserve tests.26 In poor response prediction only for the AFC, a reasonable proportion of positive tests is observed at cut-off levels with a moderate to good levels of the pLR, leading to a substantial change in the chance of producing a poor response in IVF. For nonpregnancy prediction, the abnormal test rate is clearly low at the cut-off levels that lead to an appropriate overall test performance and probability of nonpregnancy shifts moderately in case of such a test result. FSH, follicle stimulating hormone; AMH, anti-Müllerian hormone; AFC, antral follicle count; OVVOL, ovarian volume.

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Pregnancy prediction From the meta-analysis of the various reserve tests, the predictive ability towards the occurrence of pregnancy after one IVF cycle was shown to be only marginal, as only a small proportion of the nonpregnant cases were predicted correctly and false-positives remained even with extreme cut-offs for an abnormal test (see Fig 54.5, Table 54.2). This finding should not be regarded as a surprise, as most tests relate to the quantitative aspects of ovarian reserve that are constantly present (i.e. antral follicle cohort size), while the quality perspective is only tested against a single exposure, which certainly will not be a good expression of a couple’s fertility potential (only tested properly in a series of ART cycles). In general, therefore, ovarian reserve testing prior to starting ART treatment should be regarded as useful only if the occurrence of poor response to ovarian stimulation is to be predicted and with the assumption that this foreseen poor response can be effectively prevented with improvement of pregnancy chances.

First cycle poor response Testing for ovarian reserve may also be possible by using the quantity of the ovarian response to maximal ovarian stimulation in the first ART cycle. A poor response to stimulation, defined as a low number of mature follicles developed or oocytes obtained after a conventional long GnRH agonist suppression protocol, will generally be interpreted as a proof of diminished ovarian reserve and reduced prognosis for pregnancy. Also, poor responders in IVF/ICSI treatment experience an earlier transition into menopause compared with normal responders, confirming the relationship between response and fertility potential. Still, a poor response may also be caused by conditions such as submaximal stimulation in obese women, or carriers of an FSH receptor polymorphism, or simply by chance. In such poor responders, prospects in the actual and subsequent cycles are not so unfavorable that refusal of treatment is justified. Only if a poor response occurs in cases with an unfavorable additional profile (female age >38 years old, abnormal ovarian reserve test, repeated poor response) does prognosis for subsequent cycles becomes cumbersome enough for further denial of treatment.14,15,47 If the policy would be to allow any couple with female age <40 years old to proceed to ART, then a poor response combined with an appropriate ovarian reserve test may be the best policy to direct further management.

Test combinations Improvement of ovarian reserve test performance in the identification of women with a reduced quantitative ovarian reserve for their age category may come from combining endocrine and imaging tests. Combination of several endocrine and imaging tests into predictive

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models has shown to improve the accuracy of poor response prediction in single studies.26 In a meta-analysis on several models combining various single tests, no clear improvement compared to the AFC and basal FSH could be found. With the possible finding of genetic markers for ovarian reserve status in the near future the performance of these models may improve.48 Currently, studies concerning AMH and ovarian aging are rapidly accumulating.49–51 In view of the high correlation between the AFC and AMH, widespread availability of the assay to measure AMH levels may lead to replacement of the AFC as the most direct test for quantitative reserve screening.52 This may be especially true in the opinion of a recent review where AMH was shown to be as predictive of poor responders as the AFC. The true challenge for ovarian reserve tests lies in the possibility of identifying women with a reduced reproductive life span at such a stage in their lives that adequate action can be taken. In such a test, the preferable outcome variable to judge the test upon is the age at which a woman will become menopausal. The relation between menopausal age and the end of natural fertility has been hypothesized as being fixed.2 If a test was able to predict age at menopause, family planning clinics where any young woman can be tested for her reproductive expectations or limits could become a reality.

Summary Age-related fertility decline varies considerably among women; therefore, chronological female age, although informative on pregnancy prospects in assisted reproduction, will not always correctly express a woman’s reproductive potential. The value of quantitative ovarian reserve tests added to the information of female age has not been established to date. Currently, several tests are considered adequate in poor response prediction. In combination with the actual response to hyperstimulation, these tests may identify true poor prognosis cases.

References 1. Stephen EH, Chandra A. Declining estimates of infertility in the United States: 1982–2002. Fertil Steril 2006; 86: 516–23. 2. te Velde ER, Pearson PL. The variability of female reproductive ageing. Hum Reprod Update 2002; 8: 141–54. 3. Morabia A, Costanza MC. International variability in ages at menarche, first livebirth, and menopause. World Health Organization Collaborative Study of Neoplasia and Steroid Contraceptives. Am J Epidemiol 1998; 148: 1195–205. 4. Thomas F, Renaud F, Benefice E, de Meeus T, Guegan JF. International variability of ages at menarche and menopause: patterns and main determinants. Hum Biol 2001; 73: 271–90. 5. Noord-Zaadstra BM, Looman CW, Alsbech H, et al. Delaying childbearing: effect of age on fecundity and outcome of pregnancy. BMJ 1991; 302: 1361–5.

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6. Broekmans FJ, Faddy MJ, Scheffer G, te Velde ER. Antral follicle counts are related to age at natural fertility loss and age at menopause. Menopause 2004; 11: 607–14. 7. Eijkemans MJ, Polinder S, Mulders AG, et al. Individualized cost-effective conventional ovulation induction treatment in normogonadotrophic anovulatory infertility (WHO group 2). Hum Reprod 2005; 20: 2830–7. 8. Viudes-de-Castro MP, Vicente JS. Effect of sperm count on the fertility and prolificity rates of meat rabbits. Anim Reprod Sci 1997; 46: 313–19. 9. Moce E, Lavara R, Vicente JS. Influence of the donor male on the fertility of frozen–thawed rabbit sperm after artificial insemination of females of different genotypes. Reprod Domest Anim 2005; 40: 516–21. 10. Evers JL. Female subfertility. Lancet 2002; 360: 151–9. 11. Menken J, Trussell J, Larsen U. Age and infertility. Science 1986; 233: 1389–94. 12. van Kooij RJ, Looman CW, Habbema JD, Dorland M, te Velde ER. Age-dependent decrease in embryo implantation rate after in vitro fertilization. Fertil Steril 1996; 66: 769–75. 13. de Boer EJ, den Tonkelaar I, te Velde ER, Burger CW, van Leeuwen FE; Omega-project group. Increased risk of early menopausal transition and natural menopause after poor response at first IVF treatment. Hum Reprod 2003; 18: 1544–52. 14. Lawson R, El Toukhy T, Kassab A, et al. Poor response to ovulation induction is a stronger predictor of early menopause than elevated basal FSH: a life table analysis. Hum Reprod 2003; 18: 527–33. 15. Klinkert ER, Broekmans FJ, Looman CW, te Velde ER. A poor response in the first in vitro fertilization cycle is not necessarily related to a poor prognosis in subsequent cycles. Fertil Steril 2004; 81: 1247–53. 16. Fasouliotis SJ, Simon A, Laufer N. Evaluation and treatment of low responders in assisted reproductive technology: a challenge to meet. J Assist Reprod Genet 2000; 17: 357–73. 17. Popovic-Todorovic B, Loft A, Lindhard A, et al. A prospective study of predictive factors of ovarian response in ‘standard’ IVF/ICSI patients treated with recombinant FSH. A suggestion for a recombinant FSH dosage normogram. Hum Reprod 2003; 18: 781–7. 18. van der Gaast MH, Eijkemans MJ, van der Net JB, et al. Optimum number of oocytes for a successful first IVF treatment cycle. Reprod Biomed Online 2006; 13: 476–80. 19. Out HJ, David I, Ron-El R, et al. A randomized, double-blind clinical trial using fixed daily doses of 100 or 200 IU of recombinant FSH in ICSI cycles. Hum Reprod 2001; 16: 1104–9. 20. Harrison RF, Jacob S, Spillane H, Mallon E, Hennelly B. A prospective randomized clinical trial of differing starter doses of recombinant follicle-stimulating hormone (follitropin-beta) for first time in vitro fertilization and intracytoplasmic sperm injection treatment cycles. Fertil Steril 2001; 75: 23–31. 21. Yong PY, Brett S, Baird DT, Thong KJ. A prospective randomized clinical trial comparing 150 IU and 225 IU of recombinant follicle-stimulating hormone

22.

23.

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25.

26.

27.

28.

29.

30.

31.

32.

33.

34. 35.

36.

(Gonal-F*) in a fixed-dose regimen for controlled ovarian stimulation in in vitro fertilization treatment. Fertil Steril 2003; 79: 308–15. Klinkert ER. Clinical significance and management of poor response in IVF. Academic thesis, Utrecht, 2005. Templeton A, Morris JK, Parslow W. Factors that affect outcome of in-vitro fertilisation treatment [see comments]. Lancet 1996; 348: 1402–6. Tarlatzis BC, Zepiridis L, Grimbizis G, Bontis J. Clinical management of low ovarian response to stimulation for IVF: a systematic review. Hum Reprod Update 2003; 9: 61–76. Hendriks DJ, Mol BW, Bancsi LF, te Velde ER, Broekmans FJ. Antral follicle count in the prediction of poor ovarian response and pregnancy after in vitro fertilization: a meta-analysis and comparison with basal follicle-stimulating hormone level. Fertil Steril 2005; 83: 291–301. Broekmans FJ, Kwee J, Hendriks DJ, Mol BW, Lambalk CB. A systematic review of tests predicting ovarian reserve and IVF outcome. Hum Reprod Update 2006; 12: 685–718. Seifer DB, MacLaughlin DT, Christian BP, Feng B, Shelden RM. Early follicular serum Müllerianinhibiting substance levels are associated with ovarian response during assisted reproductive technology cycles. Fertil Steril 2002; 77: 468–71. Klein NA, Houmard BS, Hansen KR, et al. Agerelated analysis of inhibin A, inhibin B, and activin a relative to the intercycle monotropic follicle-stimulating hormone rise in normal ovulatory women. J Clin Endocrinol Metab 2004; 89: 2977–81. Hendriks DJ, Broekmans FJ, Bancsi LF, et al. Single and repeated GnRH agonist stimulation tests compared with basal markers of ovarian reserve in the prediction of outcome in IVF. J Assist Reprod Genet 2005; 22: 65–73. Kwee J, Elting MW, Schats R, et al. Comparison of endocrine tests with respect to their predictive value on the outcome of ovarian hyperstimulation in IVF treatment: results of a prospective randomized study. Hum Reprod 2003; 18: 1422–7. Hendriks DJ, Mol BW, Bancsi LF, te Velde ER, Broekmans FJ. The clomiphene citrate challenge test for the prediction of poor ovarian response and nonpregnancy in patients undergoing in vitro fertilization: a systematic review. Fertil Steril 2006; 86: 807–18. Jain T, Soules MR, Collins JA. Comparison of basal follicle-stimulating hormone versus the clomiphene citrate challenge test for ovarian reserve screening. Fertil Steril 2004; 82: 180–5. Deeks JJ. Systematic reviews in health care: systematic reviews of evaluations of diagnostic and screening tests. BMJ 2001; 323: 157–62. Grimes DA, Schulz KF. Refining clinical diagnosis with likelihood ratios. Lancet 2005; 365: 1500–5. Mol BW, Verhagen TE, Hendriks DJ, et al. Value of ovarian reserve testing before IVF: a clinical decision analysis. Hum Reprod 2006; 21: 1816–23. Levi AJ, Raynault MF, Bergh PA, et al. Reproductive outcome in patients with diminished ovarian reserve. Fertil Steril 2001; 76: 666–9.

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Prognostic testing for ovarian reserve 37. Scott RT, Jr, Hofmann GE. Prognostic assessment of ovarian reserve [see comments]. Fertil Steril 1995; 63: 1–11. 38. Bancsi LFJ, Broekmans FJ, Eijkemans MJ, de Jong FH, Habbema JD, te Velde ER. Predictors of poor ovarian response in in vitro fertilization: a prospective study comparing basal markers of ovarian reserve. Fertil Steril 2002; 77: 328–36. 39. van Rooij IA, Broekmans FJ, te Velde ER, et al. Serum anti-Müllerian hormone levels: a novel measure of ovarian reserve. Hum Reprod 2002; 17: 101–7. 40. Bancsi LF, Broekmans FJ, Looman CW, Habbema JD, te Velde ER. Impact of repeated antral follicle counts on the prediction of poor ovarian response in women undergoing in vitro fertilization. Fertil Steril 2004; 81: 35–41. 41. Bancsi LF, Broekmans FJ, Looman CW, Habbema JD, te Velde ER. Predicting poor ovarian response in IVF: use of repeat basal FSH measurement. J Reprod Med 2004; 49: 187–94. 42. Ng EH, Tang OS, Ho PC. The significance of the number of antral follicles prior to stimulation in predicting ovarian responses in an IVF programme. Hum Reprod 2000; 15: 1937–42. 43. Popovic-Todorovic B, Loft A, Bredkjaeer HE, et al. A prospective randomized clinical trial comparing an individual dose of recombinant FSH based on predictive factors versus a ‘standard’ dose of 150 IU/day in ‘standard’ patients undergoing IVF/ICSI treatment. Hum Reprod 2003; 18: 2275–82. 44. Land JA, Yarmolinskaya MI, Dumoulin JC, Evers JL. High-dose human menopausal gonadotropin stimulation in poor responders does not improve in vitro fertilization outcome. Fertil Steril 1996; 65: 961–5. 45. Shanbhag S, Aucott L, Bhattacharya S, Hamilton MA, McTavish AR. Interventions for ‘poor responders’ to

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controlled ovarian hyperstimulation (COH) in in-vitro fertilisation (IVF). Cochrane Database Syst Rev 2007; CD004379. Popovic-Todorovic B, Loft A, Ziebe S, Andersen AN. Impact of recombinant FSH dose adjustments on ovarian response in the second treatment cycle with IVF or ICSI in ‘standard’ patients treated with 150 IU/day during the first cycle. Acta Obset Gynecol Scand 2004; 83: 842–9. de Boer EJ, den Tonkelaar, I, te Velde ER, et al. A low number of retrieved oocytes at in vitro fertilization treatment is predictive of early menopause. Fertil Steril 2002; 77: 978–85. Hefler LA, Grimm C, Bentz EK, et al. A model for predicting age at menopause in white women. Fertil Steril 2006; 85: 451–4. Fanchin R, Taieb J, Lozano DH, et al. High reproducibility of serum anti-Müllerian hormone measurements suggests a multi-staged follicular secretion and strengthens its role in the assessment of ovarian follicular status. Hum Reprod 2005; 20: 923–7. Hazout A, Bouchard P, Seifer DB, et al. Serum antiMüllerian hormone/Müllerian-inhibiting substance appears to be a more discriminatory marker of assisted reproductive technology outcome than follicle-stimulating hormone, inhibin B, or estradiol. Fertil Steril 2004; 82: 1323–9. La Marca A, Malmusi S, Giulini S, et al. AntiMüllerian hormone plasma levels in spontaneous menstrual cycle and during treatment with FSH to induce ovulation. Hum Reprod 2004; 19: 2738–41. Visser JA, Themmen AP. Anti-Müllerian hormone and folliculogenesis. Mol Cell Endocrinol 2005; 234: 81–6. Soules MR, Battaglia DE, Klein NA. Inhibin and reproductive aging in women. Am J Human Biol 1998; 30: 193–204.

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55 Management of hydrosalpinx Annika Strandell

Introduction In the beginning of the in vitro fertilization (IVF) era, tubal factor infertility was the sole indication for the treatment. Today, other indications constitute the majority of treatments and tubal disease may account for as little as 20% in some centers. It is notable that tubal factor infertility is often reported to yield worse results than other causes of infertility. We reported tubal factor infertility to be an independently negative predictive factor of pregnancy and birth, as compared with all other indications,1 in the debate on high multiple pregnancy rates in IVF. Hydrosalpinx is the severe condition that has attained special interest in research and clinical practice. Tubal diseases such as salpingitis isthmica nodosa and other types of proximal tubal occlusions have not been studied exclusively in connection with assisted reproductive technologies (ART) and will not be further explored here. This chapter will focus on the problems associated with hydrosalpinx and ART, including diagnosis, prognosis, possible mechanisms, and interventions.

Definitions and methods of diagnosis Hydrosalpinx is a commonly used term to describe a heterogeneous spectrum of pathology of distal tubal occlusion. A strict definition is a collection of watery fluid in the uterine tube, occurring as the end stage of pyosalpinx. However, hydrosalpinx is used for any distal tubal occlusion regardless of the cause, implying that a nontubal infection such as an adjacent appendicitis can also cause hydrosalpinx. Furthermore, the end stage of a tubal infection has different appearances: the hydrosalpinx simplex is characterized by excessive distention and thinning of the wall of the uterine tube, the plicae being few and widely separated, while the hydrosalpinx follicularis describes a tube without any central cystic cavity, the lumen being broken up into compartments as the result of fusion of the tubal plicae. Thus, the terminology is not consistent with the original translation since hydrosalpinx is also used in cases without any

obvious fluid in the tubes. Sactosalpinx is also used as a synonym, although the definition is slightly different: dilation of the inflamed uterine tube by retained secretions (saktos = stuffed). The diagnosis of hydrosalpinx can be suspected and, in many cases also confirmed by transvaginal ultrasound, if the tube is fluid-filled. Ultrasound has the obvious advantage to hysterosalpingography in detecting the condition without the instillation of fluid, which carries a high risk of subsequent infection (Figs 55.1 and 55.2). Both methods, including instillation of contrast, can be used to diagnose a distally occluded tube without any fluid prior to instillation. Antibiotic prophylaxis is mandatory! Laparoscopy is obviously the ultimate method for diagnosis of hydrosalpinx and associated pathology of pelvic adhesions. However, the method is highly invasive, and advantage should be taken to perform all diagnostic and therapeutic procedures at the same time. It has been proposed to establish cut-off values for the size of a hydrosalpinx, to decide when there is a need for intervention prior to IVF. However, the size of a hydrosalpinx, as measured by ultrasound, may vary during a cycle and it has not been possible to correlate IVF outcome to the precise size. Only two indices of size have been established – detection at ultrasound examination and bilateral affection – and these are discussed in the next section.

Hydrosalpinx – a sign of poor prognosis Since 1994 there have been a large number of retrospective studies dedicated to the influence of hydrosalpinges on pregnancy results in IVF, most of them showing an impaired outcome.2 Patients with hydrosalpinges have been identified as having significantly lower implantation and pregnancy rates than patients suffering from other types of tubal damage. The retrospective data have been compiled and summarized in meta-analyses, of which one is shown in Fig 55.3.3 There is a consistency in the results showing a reduction by half in clinical pregnancy and delivery rates and a doubled rate of spontaneous abortion in women

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Fig 55.1 Typical appearance of a hydrosalpinx beside the ovary at transvaginal ultrasound investigation.

Fig 55.2 The folds of the tubal wall in a distended hydrosalpinx depicted as typical spikes into the lumen.

with hydrosalpinx. In addition, thaw cycles demonstrated a significantly reduced pregnancy rate, which none of the separate studies has been able to show. The rate of ectopic pregnancy was non-significantly increased in hydrosalpinx patients (odds ratio [OR] = 1.3, 95% confidence interval [CI] 0.7–2.6). Patients with tubal infertility have an increased risk of ectopic pregnancy after IVF compared with patients with other indications, but it has not been possible to establish that patients with hydrosalpinges have an increased risk of ectopic pregnancy compared with patients suffering from other types of tubal infertility. Although retrospective cohort studies are not the best quality of evidence, it is obvious from the

overwhelming consistency in results that patients with hydrosalpinges have an impaired pregnancy outcome after IVF. Some of the retrospective studies have attempted to characterize further and subdivide the different features of hydrosalpinx. The first publication showed that the size was important by demonstrating that only largely distended hydrosalpinges were associated with significantly reduced pregnancy and delivery rates.2 DeWit et al. also demonstrated the importance of size by using ultrasound and allocated hydrosalpinges according to size, depending on whether they were visible or not.4 Pregnancy rates were significantly lower (15%) in patients with

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Clinical pregnancy/ cycle:

Odds ratio (95% CI)

Odds ratio (95% CI)

HSX

No HSX

187/1144

1544/5569

0.51 (0.41, 0.62)

3/44

39/180

0.39 (0.16, 0.94)

Clinical pregnancy/ thaw cycle: Implantation rate:

0.47 (0.32, 0.67)

Spontaneous abortion rate:

2.3 (1.56, 3.48)

Ectopic pregnancy rate:

1.3 (0.65, 2.57)

0.1

0.2

Higher rate in controls

1

749

5

10

Higher rate in hydrosalpinx patients

Fig 55.3 Meta-analysis of retrospective studies on IVF outcome in hydrosalpinx (HSX) patients compared with patients with other tubal infertility. (From Zeyneloglu et al.3)

visible hydrosalpinges compared with patients in whom the hydrosalpinges were not visible (31%). Wainer et al. demonstrated that the presence of bilateral as opposed to unilateral hydrosalpinx was associated with significantly lower pregnancy (12% vs 24%) and implantation rates (5% vs 11%).5 These findings suggest that the total amount of fluid in the hydrosalpinges is negatively correlated with the chance of achieving a pregnancy, and these aspects should be considered in the design of a prospective trial.

What is the mechanism of hydrosalpinx impairing implantation? The hydrosalpinx fluid may act on two different target systems: directly on the transferred embryos or on the endometrium and its receptivity for implantation, or both.

Embryotoxic properties of hydrosalpinx fluid Potential embryotoxic effects have been evaluated using either mouse or human embryos in human hydrosalpinx fluid. There is a discrepancy in the results of culture systems using human and murine models, but the results from different mouse studies are also diverging. In a review of hydrosalpinx studies, five out of eight studies using a murine model described embryotoxicity at low concentrations of human hydrosalpinx fluid, and three studies demonstrated impaired development, but in undiluted

hydrosalpinx fluid only.6 There are only two studies on human embryos, neither of which have been able to demonstrate any obvious toxic effect on embryo development.7,8 The experimental models using mouse and human embryos do not seem to be comparable, and conclusions from studies based on mouse models are not obviously applicable to humans. From studies on embryo development, it may be concluded that hydrosalpinx fluid does not appear to host a common potent factor deleterious to embryo development, and the lack of essential substrates is more likely to be responsible for the impaired development of embryos in undiluted hydrosalpinx fluid.

Is hydrosalpinx fluid toxic in individual cases? Even though there may not be a common toxic factor in all fluids, the presence of factors inhibitory to embryo development in fluids from certain individuals cannot be excluded. Most experiments are based on small numbers of hydrosalpinx fluids, and individual variations in content may reflect the differences in embryo development. In a study on the effect of hydrosalpinx fluid on gametes and fertilization, one out of four fluids was directly cytotoxic to murine spermatozoa when incubated in 50% hydrosalpinx fluid during capacitation.9 No pathogenic microorganisms have been detected in any of the published studies, but slightly elevated concentrations of endotoxin have been demonstrated in individual fluids as a sign of previous infection.7 If

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a toxic substance was responsible for the negative influence, assay of the aspirated hydrosalpingeal fluid before stimulation would be useful in selecting patients for salpingectomy. An assay of mouse embryo culture in 50% hydrosalpinx fluid has been suggested to predict IVF outcome.10 In a population of 39 hydrosalpinx patients, the test had a sensitivity of 64%, a specificity of 86%, and a positive likelihood ratio of 4.5, suggesting the test to be fairly good in detecting toxicity. The diagnostic performance was not improved by including important factors like age and number of good-quality embryos transferred. The use of this technique requires transvaginal puncture, preferably before the start of any stimulation, when the result may be helpful in the decision concerning prophylactic salpingectomy. The technique still awaits clinical evaluation. The hydrosalpingeal fluid may also exhibit growthpromoting properties, as seen by a study in which the production of tropho-uteronectin by human cytotrophoblasts was significantly increased by the presence of hydrosalpinx fluid, suggesting promotion of early embryo–integrin interactions.11 Also, a significant increase in trophoblast cell viability, as well as in the production of β-human chorionic gonadotropin (βhCG) in the presence of hydrosalpinx fluid, suggested growth-promoting properties of hydrosalpinx fluid.

Oxidative stress The presence of oxidative and antioxidant systems in various reproductive tissues has evoked interest in the role of oxidative stress in reproductive diseases. Oxidative stress has been defined as an elevation in the steady-state concentration of various reactive oxygen species on a cellular level and has been suggested to be of importance in hydrosalpinx cases. A first report on this issue described a positive effect of low levels of reactive oxygen species in relation to blastocyst development, as compared with absence of reactive oxygen species in hydrosalpinx fluid.12 The low levels were suggested to be within a physiological range, and no high levels were detected to demonstrate a negative effect. This hypothesis will need further evaluation.

Endometrial receptivity The cross-talk between the embryo and the endometrium, essential for allowing the embryo to implant, and mediated by the secretion and expression of certain cytokines and other substances during the implantation window, may be disturbed under the presence of hydrosalpinx fluid. Cytokines such as interleukin-1 (IL-1), leukemia inhibitory factor (LIF), colony-stimulating factor-1 (CSF-1), and the integrin αvβ3 are all factors which have been shown to be of importance to implantation; they and some of their receptors are secreted or expressed by either the

embryo or the endometrium in an increased manner during the implantation window.13,14 Chlamydia trachomatis is the most common pathogen, and antibodies to chlamydial heat-shock proteins were found to be more prevalent in patients with hydrosalpinx compared with women in couples of male infertility.15 Heat-shock proteins elicit intense immune and inflammatory reactions, and are thought to be responsible for a local immune response, leading to inflammatory reactions, impaired implantation, and immune rejection after embryo transfer.16 A recent study demonstrated impaired endometrial and subendometrial blood flow among hydrosalpinx patients, supporting the theory of simultaneous damage to the endometrium as the original infection.17

Mechanical explanations Leakage of hydrosalpingeal fluid through the uterine cavity, resulting in embryo disposal, has been suggested as a mechanism by several authors.18–20 The clinical feature of hydrorrhea was shown to be a sign of poor prognosis among patients with hydrosalpinx undergoing IVF.19 The existence of a hydrosalpingeal fluid interface on the endometrial surface, sometimes seen during ART, has been suggested to be a hindrance to implantation.19,21 One study has demonstrated an association between endometrial cavity fluid and increased cancellation rates and lower clinical pregnancy rates in ART cycles, but without any association with hydrosalpinges visible on ultrasound.22 These findings suggest that leakage of hydrosalpingeal fluid through the uterine cavity is only one of several possible explanations of endometrial cavity fluid. It has also been suggested that hydrosalpinx fluid may cause an increase in endometrial peristalsis. In one report, uterine dynamics of five patients with hydrosalpinx were analyzed by image-processing techniques and compared with healthy volunteers.23 The authors describe, from a mathematical simulation model, a reflux phenomenon (opposing the cervix-to-fundus intrauterine peristalsis) generated by a pressure gradient from tubal fluid accumulation. It was suggested that this reflux phenomenon could explain the reduced implantation rate associated with hydrosalpinx.

Interventions against hydrosalpinx in conjunction with IVF According to the theory that the hydrosalpingeal fluid plays a causative role in impairing implantation and/or embryo development, any surgical intervention interrupting the communication to the uterus would remove the leakage of the hydrosalpingeal fluid and restore pregnancy rates. Treatment with salpingectomy prior to IVF is the only surgical method that has been evaluated in a sufficiently large randomized controlled trial (RCT), supplying us with

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a high level of evidence to formulate our recommendation. Other suggested treatments for hydrosalpinges prior to IVF, such as tubal ligation and transvaginal aspiration, may also be considered, but they need further evaluation in large prospective trials.

Salpingectomy Hitherto, salpingectomy is the only method of prophylactic surgery in patients with hydrosalpinx that has been properly evaluated in a large randomized trial.24 A multicenter study in Scandinavia compared laparoscopic salpingectomy to no intervention prior to the first IVF cycle; the study demonstrated a significant improvement in pregnancy and birth rates after salpingectomy in patients with hydrosalpinges that were large enough to be visible on ultrasound. Clinical pregnancy rates were 46% vs 22% (p = 0.049), and birth rates were 40% vs 17% (p = 0.040) in salpingectomized patients vs patients without any surgical intervention (Fig 55.4b). The difference in outcome was not statisti-

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cally significant in the total study population of 204 patients, which included patients with hydrosalpinges that were not visible on ultrasound (Fig 55.4a), demonstrating that the benefit of salpingectomy is only evident if the hydrosalpinx is fluid-filled. Within the group of hydrosalpinges visible on ultrasound, there can still be tubes that are suitable for reconstructive surgery, and the main rule must be that tubes with healthy-looking mucosa should not be removed (Figs 55.5 and 55.6). The psychological aspect of removing the tubes in an infertile patient is very important and has to be considered. Even if it is obvious that the patient would benefit from salpingectomy, it is crucial that she is psychologically prepared to undergo the procedure. In some cases, it takes one or several failed cycles before the patient is ready to give her consent. There are three additional RCTs on salpingectomy prior to IVF,25–27 all of smaller sample sizes as compared to the Scandinavian study. A systematic review in the Cochrane Library,28 included three of the hitherto four

(a) a 40 p = 0.083

30 Percentage

30 18

20 10 0 Salpingectomy

No intervention

Fig 55.5 A hydrosalpinx without adjacent adhesions is easy to assess at laparoscopy.

(b) b p = 0.019 55

60

Percentage

50 40

p = 0.057 33

p = 0.040 40

30 20

17

15

16

10 0 Bilateral

US visible

Salpingectomy

Bilateral US visible

No intervention

Fig 55.4 Live birth rate in 204 patients’ first transfer cycle in the Scandinavian multicenter trial on salpingectomy prior to IVF in hydrosalpinx patients. (a) The total study population. (b) The a priori decided subgroups of bilateral and/or ultrasound (US) visible hydrosalpinges.

Fig 55.6 Assessment of mucosal status through a distal opening of the hydrosalpinx is recommended before the final decision of salpingectomy or distal tuboplasty is taken.

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A cost-effectiveness analysis, based on the Scandinavian RCT, showed that the strategy to perform salpingectomy prior to the first IVF cycle was more cost-effective than the strategy to suggest surgery after one or two cycles had failed.30

published RCTs, and the summary estimates from the meta-analyses demonstrated a significant improvement in pregnancy (OR = 1.8, 95% CI 1.1–2.9) and live birth (OR = 2.1, 95% CI 1.2–3.7) after IVF if salpingectomy was performed compared with no surgical intervention. If the most recent study is also included in a metaanalysis, the common OR for ongoing pregnancy is 2.7 (95% CI 1.6–4.6), as shown in Fig 55.7. In the Scandinavian study, the cumulative result, including all subsequent cycles, was evaluated.29 Patients were offered up to three stimulated cycles, and those who were randomized to undergo salpingectomy achieved a cumulative birth rate of 55%. When all subsequent cycles were considered, including all patients regardless of the size of the hydrosalpinx, salpingectomy implied a doubled birth rate as compared to patients with persistent hydrosalpinges (hazard ratio = 2.1, 95% CI 1.6–3.6, p = 0.014). This result, as well as the compiled data from the Cochrane Review, suggests that all patients with hydrosalpinx, regardless of size or fluid accumulation, should undergo salpingectomy. However, the cumulative data from the Scandinavian study revealed that the benefit of salpingectomy mainly affected patients with hydrosalpinges visible on ultrasound, and consequently, those are the only patients to be recommended prophylactic salpingectomy prior to IVF.

Author

Déchaud 199825

Surgery: Birth or ongoing pregnancy/ patients 13/30

Effect on ovarian function after salpingectomy The effect of salpingectomy on ovarian function has been debated, and the results of hitherto published studies are not entirely in consensus.24,31–36 A summary of the published studies is presented in Table 55.1. The close anatomical association of the vascular and nervous supply to the tube and ovary constitute the theoretical rationale for the risk of impaired ovarian function after surgery. The majority of studies have analyzed the ovarian performance in IVF cycles subsequent to salpingectomy due to ectopic pregnancy. None of them demonstrate an effect on the overall performance, although one study has shown a decreased response in the ovary, ipsilateral to the salpingectomy.32 In the Scandinavian RCT on salpingectomy prior to IVF, a subset of patients, who underwent a stimulated cycle both before and after the salpingectomy, were included in an analysis of the effect of salpingectomy on the ovarian performance by measuring the need for follicle-stimulating hormone (FSH) and number of retrieved

No surgery: Birth or ongoing pregnancy/ patients

Odds ratio (95% CI)

Weight

6/30

20%

Odds ratio (95% CI)

3.06 (0.97, 9.66)

Strandell 199924

31/116

15/88

49% (0.89, 3.54)

Goldstein 199826

4/15

1/16

5%

5.45 (0.53, 55.80)

Kontoravdis 2006 27

Total

23/47

1/14

26%

12.45 (1.51, 103.05)

71/208

23/148

100%

2.66 (1.55, 4.56)

0.1

0.2

Favors control

1

5

10

Favors surgery

Fig 55.7 Meta-analysis of four randomized trials of laparoscopic salpingectomy vs no surgery in hydrosalpinx patients due to undergo IVF, with primary outcome live birth or ongoing pregnancy.

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753

Table 55.1 Summary of nine studies examining the effect of salpingectomy on ovarian function by measuring the number of reterived oocytes after controlled ovarian hyperstimulation. Controls are either the same patient before surgery, the contralateral ovary, or patients without previous tubal surgery No. of oocytes

Verhulst 199431

Lass 1998

32

Strandell 199924

Dar 200033 Stadtmauer 200039

Strandell 200134

Ipsilateral vs contralateral

overall (two ovaries)

No. of patients

Reason for surgery

26 vs 134

Ectopic pregnancy hydrosalpinx sterilization

n.s.

29 vs 73

Ectopic pregnancy

3.8 vs 6.0 p<0.01

9.9 vs 9.1 n.s.

108 vs 79

Hydrosalpinx

n.s.

10.6 vs 10.6 n.s.

6.1 vs 5.3 n.s.

11.1 vs 9.7 n.s.

Hydrosalpinx

n.s.

14.0 vs 12.9 n.s.

Hydrosalpinx

n.s.

9.4 vs 8.7 n.s.

n.s.

16.2 vs 17.5 n.s.

6.3 vs 6.2 n.s.

8.6 vs 8.4 n.s.

n.s.

10.2 vs 12.9 n.s.

26 15 vs 34

26

Ectopic pregnancy after IVF

Surrey and Schoolcraft 200140

32 vs 35

Hydrosalpinx

Tal 200235

26 vs 52

Ectopic pregnancy

Gelbaya 200636

40 vs 103

Hydrosalpinx

11.2 vs 11.2 n.s.

Study design Retrospective comparison with controls Prospective comparison with controls Randomized controlled trial Analysis before and after surgery Retrospective cohort Analysis before and after surgery Retrospective cohort Comparison with matched controls Retrospective cohort

n.s., not studied.

oocytes.34 There were no significant differences in either the amount of FSH used or the number of retrieved oocytes. In the cycle after salpingectomy, in mean 0.7 fewer oocytes were retrieved compared with the cycle before surgery. In the most recent study,36 comparing different surgical methods for hydrosalpinx, the mean number of retrieved oocytes were significantly lower after salpingectomy, in comparison with tubal ligation, but not compared with no surgery. From the results, we cannot conclude that patients with a low ovarian reserve are at greater risk of suffering from poor response after salpingectomy. However, theoretically, it seems important to be very careful not to damage the vascular and nervous supply when performing a salpingectomy. A laparoscopic salpingectomy should be performed with cautious use of electrocautery, with no unnecessary excision of the mesosalpinx, but resection very close to the actual tube to avoid damage to the medial tubal artery; it is preferable to leave a portion of an adherent tube on the ovary rather than to perform an excessively radical salpingectomy. The risk of dehiscence in the uterine wall and subsequent protrusion of the fetus has

been described, suggesting that resection not too close to the uterus is to be recommended.37

Tubal ligation Surgical treatment requiring laparoscopy also includes proximal ligation and salpingostomy. There is one recent randomized trial, in which 115 patients with hydrosalpinx were allocated to proximal tubal occlusion, salpingectomy, or no surgery prior to IVF.27 Both surgical methods demonstrated significantly higher ongoing pregnancy rates (34% and 46%) compared with women having no surgery (6.6%), analyzed on an intention-to-treat basis (p = 0.049). Although this study was underpowered, the result supports the findings of previous retrospective studies, suggesting that proximal occlusion is effective. Two out of four small retrospective studies, summarized in Table 55.2, indicate that tubal ligation may be better than no surgical intervention.36,38–40 According to the theory of the hydrosalpingeal fluid affecting the endometrium negatively, the procedure of tubal ligation is likely to be effective in improving

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Table 55.2 Clinical pregnancy rates per embryo transfer in four retrospective studies comparing tubal ligation to salpingectomy and to no surgery in hydrosalpinx patients, prior to IVF Salpingectomy

Tubal ligation

No surgery

Author publication year

n/n

(%)

n/n

(%)

n/n

Murray 199838

9/23

(39)

9/15

(60)

4/47

(%) (8.5) p<0.05

Stadtmauer 200039

7/15

(47)

22/30

(73)

2/15

(13) p<0.05

Surrey and Schoolcraft 200140 36

Gelbaya 2006

16/28

(57)

7/15

(47)

6/40

(15)

4/25

(16)

14/103

(14)

concern the disposal of the fluid. The simplest way, vaginal aspiration of fluid, has not been evaluated in a prospectively controlled manner. There are several case reports on the subject, describing both positive and negative outcomes concerning pregnancy. The two largest retrospective studies have reached different conclusions regarding the benefit of drainage at the time of oocyte retrieval in terms of improved pregnancy and implantation rates.42,43 The main results from the two studies, including first cycle, are shown in Table 55.3. In the first and largest study, aspiration has no effect on pregnancy rates, while the latter study shows a significant improvement. However, the results are based on very few pregnancies, which increases the risk of the result being highly affected by chance. There is a rapid reoccurrence of fluid already noticeable at the time of transfer in many cases, which most likely compromises any beneficial effect of drainage.44 Aboulghar et al evaluated transvaginal aspiration before ovarian stimulation was initiated and demonstrated that there was no improvement in pregnancy rates.45 There is a need for proper evaluation of the method, including the risk of infection associated with the puncture of a hydrosalpinx. The occurrence of infections in association with puncture of hydrosalpinx seems to be rare when antibiotics have been given, according to the published reports. The method has the obvious advantage of being less invasive than the aparoscopic methods.

pregnancy results, but it still remains to be proven in an adequately powered randomized study. The procedure is currently recommended when pelvic adhesions are too extensive to perform a salpingectomy. Tubal ligation by hysteroscopy has been described as an alternative when patients are unsuitable for laparoscopy.40a

Salpingostomy There are no separate studies on salpingostomy prior to IVF, although it has been performed in a few cases and reported to be part of a control group to hydrosalpinx patients in retrospective studies. Salpingostomy is naturally the method of choice if the tube is suitable for reconstructive surgery. The selection of patients suitable for surgical repair has to be based on the evaluation of the tubal mucosa through an endoscopic technique, and tubes with more than half of the mucosa in a good condition may have a fair chance of spontaneous conception.41 These patients should be given sufficient time to await spontaneous conception, although the woman’s age may hasten the need for IVF.

Transvaginal aspiration Whatever the exact mechanism of the negative influence of hydrosalpinx fluid, the treatment options

Table 55.3 first cycle

–

Summary of two retrospective studies on transvaginal aspiration of hydrosalpingeal fluid prior to IVF, including only

Treatment group

Author

Outcome

n/n

Aspiration (%)

(n/n)

No aspiration (%)

Significance p-value

Sowter et al. 199742

Pregnancy Birth

6/30 5/30

(20.0) (16.7)

3/18 3/18

(16.7) (16.7)

1.0 1.0

Van Voorhis et al. 199843

Pregnancy Birth

5/16 5/16

(31.3) (31.3)

1/18 0

(5.6)

0.078 0.015

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Antibiotic treatment The use of antibiotics has also been discussed, not only as prophylactics when a hydrosalpinx has been punctured but also when given as a routine before oocyte retrieval to all patients. However, antibiotic treatment specifically in hydrosalpinx patients has never been prospectively evaluated. One retrospective study suggested that extended doxycycline treatment during an IVF cycle would minimize the detrimental effect of hydrosalpinx.46 When patients with hydrosalpinx who received extended doxycycline treatment during an IVF cycle were compared with patients with other indications (tubal occlusion/adhesions or endometriosis/unexplained infertility) who did not receive any antibiotics, implantation and pregnancy rates were similar in all groups. This design does not allow for any recommendation of antibiotic treatment as an effective treatment. The method is, however, advantageously cheap and simple, but its benefit still needs to be evaluated in a prospective trial.

Interventions against hydrosalpinx without IVF As IVF developed and the results improved, the importance of using surgical methods for treating tubal infertility declined. It is well known that the success rate was closely related to the status of the tubal mucosa; the less damage to the tubes, the better chance of a subsequent intrauterine pregnancy. Today, IVF is often offered as a first-line treatment also to patients with mild tubal damage. Whether surgery is discussed or not is mainly a question of surgical competence, availability of IVF, and the patient’s financial situation, and is not primarily a medical issue. The work-up of the subfertile couple has also changed over time, so that laparoscopy is no longer a compulsory investigation, due to limited resources and the fact that laparoscopy is a very invasive procedure. This new mode implies that fewer patients will be evaluated laparoscopically during the work-up, unless a hydrosalpinx is detected. The result from the Scandinavian multicenter study to recommend salpingectomy prior to IVF has raised a number of concerns by Puttemans et al,47 who fear that tubes that are suitable for functional surgery could be sacrificed. In the scenario where laparoscopy is not routinely used, this fear might be justified, if the tubes are not properly evaluated before a salpingectomy is performed. Even if a patient is scheduled for a laparoscopic salpingectomy, it is necessary to open the distally occluded tube for evaluation of the mucosa before a final decision of salpingectomy is taken. If it is appropriate to perform a salpingostomy, time for spontaneous conception should be given instead of immediate IVF. Salpingectomy of a unilateral hydrosalpinx may imply an increased chance of spontaneous conception.

755

Two women in the Scandinavian study conceived spontaneously after long-lasting infertility followed by a unilateral salpingectomy and achieved a full-term pregnancy; at least three additional case reports on the same theme have been published.48–50

Summary and conclusions In patients with severe tubal disease presented as a hydrosalpinx on ultrasound and with a destroyed mucosa upon endoscopic inspection, IVF is the method of choice, but should be preceded by a discussion of laparoscopic salpingectomy, which will double the patient’s chances of a subsequent birth after IVF. In cases of extensive adhesions, rendering the salpingectomy difficult and bearing a risk of complications, proximal ligation and distal fenestration is the preferred method. Psychological aspects of removing or interrupting the tubes are very important and always have to be considered. If no surgical intervention is performed prior to IVF, transvaginal aspiration of the fluid can be performed in conjunction with oocyte retrieval under antibiotic cover. Patients with a preserved mucosa in the hydrosalpinx may have a good chance of spontaneous conception if salpingostomy is performed. In the presence of a unilateral hydrosalpinx and a contralateral healthy tube, a unilateral salpingectomy can be recommended, followed by sufficient time to await spontaneous conception, before proceeding to IVF.

Implications for research The underlying mechanisms of impaired implantation and/or development of embryos in the presence of hydrosalpinx need further exploration. Basic research on endometrial receptivity and implantation are very intense research fields, and as more general knowledge is gained, more specific hypotheses may be directed to the negative role of hydrosalpinx. In addition, the formation of hydrosalpinx following pelvic infection needs to be elucidated. A better understanding of the mechanisms would provide prerequisites for a more rational therapy. As of today, we recommend very robust surgical methods, but it is possible that the treatment should be more individualized. Only salpingectomy has been evaluated according to evidence-based concepts, and all other methods lack proper evaluation. In particular, the use of transvaginal aspiration needs to be tested in a randomized trial.

References 1. Strandell A, Bergh B, Lundin K. Selection of patients suitable for one-embryo transfer may reduce the rate of multiple births by half without impairment of overall birth rates. Hum Reprod 2000; 15: 2520–5.

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2. Strandell A, Waldenström U, Nilsson L, Hamberger L. Hydrosalpinx reduces in-vitro fertilization/ embryo transfer rates. Hum Reprod 1994; 9: 861–3. 3. Zeyneloglu HB, Arici A, Olive DL. Adverse effects of hydrosalpinx on pregnancy rates after in vitro fertilization–embryo transfer: Fertil Steril 1998; 70: 492–9. 4. deWit W, Gowrising CJ, Kuik DJ, et al. Only hydrosalpinges visible on ultrasound are associated with reduced implantation and pregnancy rates after invitro fertilization. Hum Reprod 1998; 13: 1696–701. 5. Wainer R, Camus E, Camier B, et al. Does hydrosalpinx reduce the pregnancy rate following in vitro fertilization. Fertil Steril 1997; 68: 1022–6. 6. Strandell A. The influence of hydrosalpinx on invitro fertilisation and embryo transfer – a review. Hum Reprod Update 2000; 6: 387–95. 7. Strandell A, Sjögren A, Bentin-Ley U, et al. Hydrosalpinx fluid does not adversely affect the normal development of human embryos and implantation in vitro. Hum Reprod 1998; 13: 2921–5. 8. Granot I, Dekel N, Segal I, et al. Is hydrosalpinx fluid cytotoxic? Hum Reprod 1998; 13: 1620–4. 9. de Vantéry Arrighi C, Lucas H, El-Mowafi D, et al. Effects of human hydrosalpinx fluid on in-vitro murine fertilization. Hum Reprod 2001; 16: 676–82. 10. Chen CD, Yang JH, Lin KC, et al. The significance of cytokines, chemical composition, and murine embryo development in hydrosalpinx fluid for predicting the in-vitro fertilization outcome in women with hydrosalpinx. Hum Reprod 2002; 17: 128–33. 11. Sawin SW, Loret de Mola JR, Monzon-Bordonaba F, et al. Hydrosalpinx fluid enhances human trophoblast viability and function in vitro: implications for embryonic implantation in assisted reproduction. Fertil Steril 1997; 68: 65–71. 12. Bedaiwy MA, Goldberg JM, Singh M, et al. Relationship between oxidative stress and embryotoxicity of hydrosalpingeal fluid. Hum Reprod 2002; 17: 601–4. 13. Lessey BA, Castelbaum AJ, Buck CA,, et al. Further characterization of endometrial integrins during the menstrual cycle and in pregnancy. Fertil Steril 1994; 62: 497–506. 14. Sjöblom C, Wikland M, Robertson SA. Granulocytemacrophage colony-stimulating factor promotes human blastocyst development in vitro. Hum Reprod 1999; 14: 3069–76. 15. Spandorfer SD, Neuer A, LaVerda D, et al. Previously undetected Chlamydia trachomatis infection, immunity to heat shock proteins and tubal occlusion in women undergoing in-vitro fertilization. Hum Reprod 1999; 14: 60–4. 16. Ajonuma LC, Ng EH, Chan HC. New insights into the mechanisms underlying hydrosalpinx fluid formation and its adverse effect on IVF outcome. Hum Reprod Update 2002; 8: 255–64. 17. Ng EH, Chan CC, Tang OS, Chung PC. Comparison of endometrial and subendometrial blood flows among patients with and without hydrosalpinx shown on scanning during in vitro fertilization treatment. Fertil Steril 2006; 85: 333–8. 18. Mansour RT, Aboulghar MA, Serrour GI, Riad R. Fluid accumulation of the uterine cavity before

embryo transfer: a possible hindrance for implantation. J In Vitro Fert Embryo Transfer 1991; 8: 157–9. 19. Andersen AN, Lindhard A, Loft A, et al. The infertile patient with hydrosalpinges – IVF with or without salpingectomy? Hum Reprod 1996; 11: 2081–4. 20. Bloeche M, Schreiner T, Lisse K. Recurrence of hydrosalpinges after transvaginal aspiration of tubal fluid in an IVF cycle with development of serometra. Hum Reprod 1997; 12: 703–5. 21. Sharara FI. The role of hydrosalpinx in IVF: simply mechanical? Hum Reprod 1999; 14: 577–8. 22. Levi AJ, Segars JH, Miller BT, Leondires MP. Endometrial cavity fluid is associated with poor ovarian response and increased cancellation rates in ART cycles. Hum Reprod 2001; 16: 2610–5. 23. Eytan O, Azem F, Gull I, et al. The mechanism of hydrosalpinx in embryo implantation. Hum Reprod 2001; 16: 2662–7. 24. Strandell A, Lindhard A, Waldenström U, et al. Hydrosalpinx and IVF outcome: a prospective, randomized multicentre trial in Scandinavia on salpingectomy prior to IVF. Hum Reprod 1999; 14: 2762–9. 25. Déchaud H, Daures JP, Arnal F, et al. Does previous salpingectomy improve implantation and pregnancy rates in patients with severe tubal factor infertility who are undergoing in vitro fertilization? A pilot prospective randomized study. Fertil Steril 1998; 69: 1020–5. 26. Goldstein DB, Sasaran LH, Stadtmauer L, Popa R. Selective salpingostomy–salpingectomy (SSS) and medical treatment prior to IVF in patients with hydrosalpinx. (Abstracts) Fertil Steril 1998; 70(3 Suppl 1): S320. 27. Kontoravdis A, Makrakis E, Pantos K, et al. Proximal tubal occlusion and salpingectomy results in similar improvement in in vitro fertilization outcome in patients with hydrosalpinx. Fertil Steril 2006; 86: 1642–9. 28. Johnson NP, Mak W, Sowter MC. Surgical treatment for tubal disease in women due to undergo in vitro fertilisation (Cochrane Review). In: The Cochrane Library, 2003; Issue 2. Oxford: Update Software. 29. Strandell A, Lindhard A, Waldenstrom U, Thorburn J. Hydrosalpinx and IVF outcome: cumulative results after salpingectomy in a randomized controlled trial. Hum Reprod 2001; 16: 2403–10. 30. Strandell A, Lindhard A, Eckerlund I. Cost-effectiveness analysis of salpingectomy prior to IVF, based on a randomized controlled trial. Hum Reprod 2005; 20: 3284–92. 31. Verhulst G, Vandersteen N, van Steirteghem AC, Devroey P. Bilateral salpingectomy does not compromise ovarian stimulation in an in-vitro fertilization/embryo transfer programme. Hum Reprod 1994; 9: 624–8. 32. Lass A, Ellenbogen A, Croucher C, et al. Effect of salpingectomy on ovarian response to superovulation in an in vitro fertilization–embryo transfer program. Fertil Steril 1998; 70: 1035–8. 33. Dar P, Sachs GS, Strassburger D, Bukovsky I, Arieli S. Ovarian function before and after salpingectomy in artificial reproductive technology patients. Hum Reprod 2000; 15; 142–4.

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Management of hydrosalpinx 34. Strandell A, Lindhard A, Waldenstrom U, Thorburn J Salpingectomy prior to IVF does not impair the ovarian response. Hum Reprod 2001; 16: 1135–9. 35. Tal J, Paltieli Y, Korobotchka R, et al. Ovarian response to gonadotropin stimulation in repeated IVF cycles after unilateral salpingectomy. J Assist Reprod Genet 2002; 19: 451–5. 36. Gelbaya TA, Nardo LG, Fitzgerald CT, et al. Ovarian response to gonadotrophins after laparoscopic salpingectomy or the division of fallopian tubes for hydrosalpinges. Fertil Steril 2006; 85: 1464–8. 37. Inovay J, Marton T, Urbancsek J, et al. Spontaneous bilateral cornual uterine dehiscence early in the second trimester after bilateral laparoscopic salpingectomy and in-vitro fertilization. Hum Reprod 1999; 14: 2471–3. 38. Murray DL, Sagoskin AW, Widra EA, et al. The adverse effect of hydrosalpinges on in vitro fertilization pregnancy rates and the benefit of surgical correction. Fertil Steril 1998; 69: 41–5. 39. Stadtmauer LA, Riehl RM, Toma SK, Talbert LM. Cauterization of hydrosalpinges before in vitro fertilization is an effective surgical treatment associated with improved pregnancy rates. Am J Obstet Gynecol 2000; 183: 367–71. 40. Surrey ES, Schoolcraft WB. Laparoscopic management of hydrosalpinges before in vitro fertilization– embryo transfer; salpingectomy versus proximal tubal occlusion. Fertil Steril 2001; 75: 612–17. 40a. Rosenfield RB, Stones RE, Coates A, et al. Proximal occlusion of hydrosalpinx by hysteroscopic placement of microinsert before in vitro fertilization-embyro transfer. Fertil Steril 2005; 83: 1547. e11–14. 41. Vasquez G, Boeckx W, Brosens I. Prospective study of tubal mucosal lesions and fertility in hydrosalpinges. Hum Reprod 1995; 10: 1075–8.

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42. Sowter MC, Akande VA, Williams JA, et al. Is the outcome of in-vitro fertilization and embryo transfer treatment improved by spontaneous or surgical drainage of a hydrosalpinx? Hum Reprod 1997; 12: 2147–50. 43. Van Voorhis BJ, Sparks AE, Syrop CH, et al. Ultrasound-guided aspiration of hydrosalpinges is associated with improved pregnancy and implantation rates after in-vitro fertilization cycles. Hum Reprod 1998; 13: 736–9. 44. Sharara FI, McClamrock HD. Endometrial fluid collection in women with hydrosalpinx after human chorionic gonadotrophin administration: a report of two cases and implications for management. Hum Reprod 1997; 12: 2816–19. 45. Aboulghar MA, Mansour RT, Serour GI, et al. Transvaginal ultrasonic needle guided aspiration of pelvic inflammatory cystic masses before ovulation induction for in vitro fertilization. Fertil Steril 1990; 53; 311–14. 46. Hurst BS, Tucker KE, Awoniyi CA, Schlaff WD. Hydrosalpinx treated with extended doxycycline does not compromise the success of in vitro fertilization. Fertil Steril 2001; 75: 1017–19. 47. Puttemans P, Campo R, Gordts S, Brosens I. Hydrosalpinx and ART: hydrosalpinx – functional surgery or salpingectomy? Hum Reprod 2000; 15: 1427–30. 48. Choe J, Check JH. Salpingectomy for unilateral hydrosalpinx may improve in vivo fecundity. Gynecol Obstet Invest 1999; 48: 285–7. 49. Kiefer DG, Check JH. Salpingectomy improves outcome in the presence of a unilateral hydrosalpinx in a donor oocyte recipient: a case report. Clin Exp Obstet Gynecol 2001; 28: 71–2. 50. Aboulghar MA, Mansour RT, Serour GI. Spontaneous intrauterine pregnancy following salpingectomy for a unilateral hydrosalpinx. Hum Reprod 2000; 17: 1099–100.

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56 Severe ovarian hyperstimulation syndrome Zalman Levine, Daniel Navot

Introduction Ovarian hyperstimulation syndrome (OHSS) is the gravest complication of so-called controlled (far too often uncontrolled) ovarian hyperstimulation (COH).1 From a perspective of priorities in reproductive medicine in general and assisted reproductive technologies in particular, OHSS is second only to high-order multiple birth on the list of adverse outcomes that need to be minimized or completely eliminated. Ovarian hyperstimulation syndrome consists of ovarian enlargement accompanied by an overproduction of ovarian hormones and a host of other ovarian vasoactive substances, which alone or in concert may produce the hyperpermeability state responsible for the signs, symptoms, and complications of OHSS.

Classification Over the past 25 years, several classification systems have been suggested to better categorize the condition and disseminate uniform guidelines for prevention and treatment. The original classification, suggested by Rabau et al and later expanded by other authors,2,3 categorizes the syndrome by severity (mild, moderate, and severe) and further subdivides the categories into six grades. While this classification seemed at the time to be comprehensive, it incorporated unnecessarily cumbersome subdivisions; simple classification as mild, moderate, and severe OHSS is adequately descriptive and clinically useful. The mild form of the syndrome involves supraphysiologic levels of estradiol (E2) and progesterone (P4) accompanied by slight ovarian enlargement of <5 cm and abdominal discomfort. Since these findings are routinely observed in a large proportion of women undergoing so-called COH, mild OHSS is often nothing more than an acknowledgment that COH indeed has been achieved. The moderate form of OHSS (Fig 56.1) includes significant ovarian enlargement (5–12 cm), and accompanying symptoms such as abdominal pain, significant bloating, nausea, and diarrhea. Most of these symptoms

may be directly ascribed to marked ovarian enlargement and the dramatically elevated levels of E2. Moderate OHSS is of concern only in the context of the risk of progression to severe OHSS. Golan et al4 incorporated into the definition of moderate OHSS any amount of ascites detectable ultrasonographically but not clinically. OHSS is classified as severe in the presence of hepatic dysfunction and anasarca, and severe OHSS is accompanied by a variety of symptoms and signs that include marked abdominal distention, dyspnea, tachypnea, lower abdominal pain, hypotension, oliguria, and hydrothorax in addition to a host of laboratory abnormalities such as hyponatremia and hyperkalemia. To the original classification of severe OHSS, Navot et al5 added a critical stage denoting a life-threatening phase of the syndrome. The critical stage is defined by a severely contracted blood volume with hemoconcentration, multiorgan failure, and/or thromboembolic phenomena (Table 56.1).

Etiology While the exact etiological factor responsible for the pathogenesis of OHSS is unknown, the syndrome is known to be dependent on human chorionic gonadotropin (hCG). OHSS does not occur if hCG is withheld, and ongoing hCG stimulation by early pregnancy is a significant risk factor for persistent and severe OHSS. This hCG dependence underlies some of the major preventive strategies for the syndrome. More recently, numerous vasoactive substances have been implicated in the pathophysiology of the disease, including prorenin, renin, prostaglandins, angiotensin II, vascular endothelial growth factor (VEGF), tumor necrosis factor α (TNF-α), insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), basic fibroblast growth factor (BFGF), plateletderived growth factor (PDGF), transforming growth factors (TGF) α and β, and interleukins 1β, 2, and 6.6–17 Many of these substances are proangiogenic, and are probably responsible for the physiological neovascularization that occurs during folliculogenesis and luteinization within the ovary. VEGF seems to play a

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(a)

(b)

Fig 56.1. Transvaginal ultrasound depicting the ovaries and uterus of a 26-year-old woman who had undergone IVF for unexplained infertility. Peak E2 was 2336 pg/ml on the ninth day of stimulation using a GnRH-antagonist protocol; hCG (10 000 IU) was used for oocyte maturation; 20 oocytes were retrieved, 2 blastocysts were transferred on the fifth day following oocyte retrieval, and 6 blastocysts were cryopreserved. Two days after embryo transfer, the woman presented with abdominal pain and nausea, and was found to have ovarian enlargement, moderate ascites, hemoconcentration, and leukocytosis. She was diagnosed with moderate OHSS and treated as an outpatient with isotonic fluids. Serum hCG was positive 10 days after embryo transfer. A clinical twin intrauterine pregnancy was observed ultrasonographically on the 17th day after embryo transfer, as seen in (c). An enlarged left ovary can be seen in (a), and an enlarged right ovary with ascites in (b). The patient continued to be monitored as an outpatient until resolution of her OHSS, and was discharged to obstetric care with an ongoing twin pregnancy at 11 weeks’ gestational age.

(c)

Table 56.1

Classification of OHSS

Mild

Moderate

Severe

Critical

Bloating Nausea Abdominal distention Ovaries ≤ 5 cm

Vomiting Abdominal pain U/S evidence of ascites Hct >41% WBC >10 000/mm3 Ovaries >5 cm

Massive ascites Hydrothorax Hct >45% WBC >15 000/mm3 Oliguria Creatinine 1–1.5 mg/dl Creatinine clearance ≥50 ml/min Hepatic dysfunction Anasarca Ovaries variably enlarged

Tense ascites Hypoxemia Pericardial effusion Hct >55% WBC >25 000/mm3 Oliguria or anuria Creatinine >1.5 mg/dl Creatinine clearance <50 ml/min Renal failure Thromboembolic phenomena ARDS Ovaries variably enlarged

Hct, hematocrit; WBC, white blood count; U/S, ultrasound; ARDS, acute respiratory distress syndrome.

particularly critical role in the pathophysiology of OHSS. Evidence for this critical role are that VEGF is secreted by granulosa cells, VEGF levels correlate with OHSS severity, recombinant VEGF has been shown to induce OHSS, and specific VEGF antiserum

has been shown to reverse the effects of VEGFinduced OHSS. Furthermore, hCG has recently been shown to increase VEGF secretion by granulosa cells, and to increase serum levels of VEGF.18–20 Indeed, many of the angiogenic factors implicated in the

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pathophysiology of OHSS probably act directly or indirectly through VEGF. Since angiogenic factors are so strongly associated with OHSS, ongoing research will likely identify antiangiogenic strategies for prevention and treatment.

Prevention of severe OHSS The role of the stimulatory agent and protocol Ovarian hyperstimulation syndrome has intrigued clinicians for many years because of its devastating consequences in otherwise healthy young women. As an iatrogenic condition resulting from elective ovarian stimulation in the quest for pregnancy, the need to completely prevent the syndrome is evident. In order to promulgate safe or controlled ovarian stimulation, it is essential to first define the ‘at-risk population.’ Table 56.2 delineates the risk factors for severe OHSS that should alert the clinician contemplating COH. Because oocyte retrieval for in vitro fertilization (IVF), presumably due to the follicular trauma inherent in the procedure, decreases the risk of OHSS, the criteria defining high vs low risk may differ depending on whether the COH is for the purpose of IVF or for conventional ovulation induction or superovulation. The single most important risk factor for OHSS is a polycystic appearance of the ovaries on transvaginal ultrasound, with a high antral follicle count and a ‘necklace sign’ or ‘string of pearls’ appearance (Fig 56.2). In contrast, relatively quiescent ovaries with few antral follicles usually predict a slow COH response with little risk of OHSS. Early reports suggested a relationship between the type of gonadotropin preparation utilized and the risk of OHSS. More recent comparisons between recombinant follicle-stimulating hormone (rFSH) and human menopausal gonadotropins (hMG) did not show significant differences among variable drug regimens. A large study by Bergh et al21 compared 119 cycles of rFSH (Gonal-F) to 114 cycles of highly purified urinary FSH (uFSH-HP; Metrodin HP). Both groups were downregulated by a long gonadotropin-releasing hormone agonist (GnRH-a) protocol. All parameters studied, including E2 serum concentrations, ampules utilized, days of stimulation, number of oocytes retrieved, and number of embryos obtained, were significantly in favor of rFSH, and although clinical pregnancy rates and implantation rates were similar, significantly more embryos were frozen subsequent to rFSH stimulation. The respective rates of OHSS for rFSH and uFSH-HP were 5.2% and 1.7%. Another large study compared 585 patients receiving rFSH with 396 patients receiving uFSH.22 This report demonstrated similar advantages of rFSH regarding length of treatment and ampules utilized, but also showed a significantly higher ongoing pregnancy rate for rFSH when frozen–thawed embryos were added to the equation. The rate of OHSS in this study was 3.2% vs 2.0% for rFSH and uFSH, respectively, and the difference was not statistically significant.

Table 56.2

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Risk factors associated with OHSS

High risk

Low risk

Young (<35 years old) Polycystic-appearing ovaries Asthenic habitus High serum estradiol (ART >4000 pg/ml, OI >1700 pg/ml) Multiple stimulated follicles (ART >20, OI >6) Necklace sign Pregnancy hCG luteal supplementation GnRH-agonist downregulatory protocol

Older (>35 years old) Hypogonadotropic Heavy build Low serum estradiol Poor response to gonadotropins Few antral follicles Elevated baseline FSH Progesterone or no luteal supplementation Clomiphene citrate and/or hMG protocol

The capacity of rFSH to enhance both follicular recruitment and serum E2 concentrations may indeed carry a slightly increased risk of OHSS. However, the seemingly increased risk in these studies may also be due to early inexperience with rFSH. With increased awareness and understanding of the unique features of rFSH, the actual rate of OHSS with rFSH use has since decreased, as has been borne out in more recent published studies.23,24 Indeed, numerous studies have shown that the method of stimulation (chronic low dose, step up, or step down) carries far more weight as a risk factor than the type of injectable gonadotropin used.25,26 Specifically, the so-called chronic low-dose regimen is more likely to result in a mono- or bifollicular response and therefore a significantly lower rate of OHSS. Similarly, unlike a step-up protocol, which continuously rescues follicles from atresia, a stepdown protocol will allow more follicles to undergo atresia, thus reducing the overall number of follicles capable of secretory activity by the time hCG is administered. A reduction in the rate of OHSS will naturally follow. An extension of the step-down concept is ‘coasting’ or ‘controlled drift’ championed by Sher27,28 and later practiced widely by several other researchers with variable results. Benadiva et al and Tortoriello et al have reported significant reduction in OHSS,29–32 while Shapiro et al and others have found no benefit in coasting.33,34 This discrepancy in the result of coasting is most probably due to differences in the coasting protocol. A recent review of 10 studies showed that <2% of women developed OHSS while maintaining acceptable pregnancy rates (36.5–63%) when coasting was continued until serum E2 levels fell below 3000 pg/ml.35 Others have shown diminished oocyte collection rates36 and implantation and pregnancy rates37 when coasting is prolonged, particularly >3 days.38 Coasting may work to prevent or reduce the severity of OHSS by altering the capacity of the granulosa cells to produce VEGF.39 Additional research is required to

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Figure 56.2. Transvaginal ultrasound depicting the right ovary of a 31-year-old woman with amenorrhea and polycystic ovarian syndrome. The picture features the major high-risk factors for the development of severe OHSS:

• • • •

evaluate the efficacy of coasting and to determine the optimal protocol for withholding hCG in high-risk patients.40 The use of GnRH-a in conjunction with COH, either as a ‘long’ or ‘short’ protocol, profoundly affects the risk of OHSS. Gonadotropin-releasing hormone agonists play a paradoxical role in OHSS by virtue of the control they afford, despite their overall suppressive effect on ovarian stimulation. Both the long and the short GnRH-a protocols uniformly abolish the midcycle luteinizing hormone (LH) surge. This suppression of the LH surge allows continued stimulation by gonadotropins, which in turn will drive more follicles to either full or quasi maturation, with a consequent rise in serum E2 values and a markedly increased risk of OHSS.5 In contrast, during cycles without GnRH-a suppression, either a significant LH surge or at least marked luteinization will limit continued gonadotropin stimulation and thus lead to a concomitantly lesser risk of OHSS. On the other hand, because of the suppressive effect of GnRH-a on ovarian function, some have advocated continuing GnRH-a administration for 1 week following hCG administration in GnRH-a down-regulation cycles where all embryos are electively cryopreserved because of a high risk of OHSS.41 This strategy can be extended easily to oocyte donation cycles.

The role of hCG and its substitutes Once the prerequisite for severe hyperstimulation is established, namely a multifollicular, high-estrogenic milieu, the occurrence of OHSS is utterly hCG dependent; either exogenously-administered or pregnancyderived hCG is absolutely essential for the development

‘string of pearls’ appearance of antral follicles on the left panel dense stroma occupying the center of the ovary enlarged ovary measuring 49 × 44.6 mm total of 60 antral follicles in one ovary.

of OHSS. In contrast, avoidance of hCG or substitution by a low-affinity, shorter-acting compound is the mainstay for the prevention of OHSS. Indeed, the acknowledgment of the role of hCG in OHSS has led to all but complete discontinuation of the foul habit of hCG administration for luteal supplementation. Cessation of this practice eliminated a major risk factor for OHSS in assisted reproduction. However, hCG as a surrogate for the midcycle LH surge is still universally used in COH for both ovulation induction and IVF. The standard dosage of hCG used to trigger ovulation is 5000–10 000 IU, or 250 µg of recombinant hCG (rhCG). In these dosages hCG takes 6–9 days to clear from the circulation, thus exerting continuous ovarian stimulation up to the stage at which endogenous pregnancy-derived hCG is perceived. Since hCG has a very long half-life and high affinity for the ovarian LH receptor, it sustains the function of multiple corpora lutea to the point of rescue by endogenous hCG. This ovarian action of hCG exerts a stimulatory effect on the putative ovarian substances which directly promote, or may even be the causal factors in, ovarian hyperstimulation. Indeed, angiotensin II, VEGF, TNF-α, and interleukins 1β, 2, or 6 are all either directly or indirectly enhanced by hCG.42,43

Recombinant LH and OHSS The critical role of hCG in OHSS has prompted many researchers to look for an alternate substance to trigger ovulation while reducing the prolonged and often excessive stimulation of hCG. Although exogenous native LH would constitute a physiological replacement, it has several theoretical disadvantages, including its very short half-life of about 20 minutes. Because of this short half-life, either huge doses or repeated

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administration would be needed to create a sustained surge of at least 24 hours. Recently, recombinant LH (rLH) has become commercially available. A dosefinding study, in which rLH was utilized to trigger ovulation, showed the engineered product to be highly sialated, greatly extending its half-life in vivo. Consequently, the use of rLH as a midcycle substitute to natural LH surge is now feasible in all kinds of COH, whether using gonadotropins alone, clomiphene citrate in conjunction with gonadotropins, down-regulation with GnRH-a, or LH suppression with gonadotropinreleasing hormone antagonist (GnRH-ant). While this prolonged half-life, on the one hand, allows rLH to be clinically useful, it might also theoretically render the rLH similar to hCG, and therefore may not substantially reduce the incidence of OHSS. One preliminary comparison of rLH and rhCG in IVF demonstrated a lower incidence of moderate to severe OHSS in women receiving a single dose of rLH.44 Loumaye et al used doses of rLH between 5000 IU and 30 000 IU to compare with 5000 IU of hCG.45 All doses of rLH in the Loumaye et al study successfully induced final follicular maturation and yielded similar numbers of oocytes and equal fertilization rates, but the incidence of ovarian enlargement and some ascites seemed to be directly dose dependent. Whereas no ovarian enlargement or ascites were seen up to 10 000 IU of rLH, 1 of 26 women (3.8%) who took 30 000 IU of rLH, and 13 of 121 women (10.7%) who took hCG, had ovarian enlargement, ascites, or accompanying symptomatology. One patient in the hCG group had severe OHSS. The group concluded that rLH may be safer than hCG as far as OHSS is concerned. This dose-finding study suggests that the rLH used probably has a relatively long half-life compared with native LH. Alternatively, it is possible that a single peak of rLH is sufficient to induce final oocyte aturation, as opposed to the 24-hour-long naturally occurring LH surge.

GnRH agonists and OHSS Alternatively, final follicular maturation and ovulation may be triggered using a GnRH agonist to stimulate an endogenous LH surge in patients at risk for OHSS. Early attempts to elicit an LH surge with synthetic GnRH in an hMG-stimulated cycle yielded variable results.46,47 Recently, however, attempts to trigger ovulation with GnRH analogs have been more consistent in their results. Lanzone et al48 and Imoedemhe et al49 were the first to report the successful use of GnRH-a for induction of an endogenous LH/FSH surge for final follicular maturation following exogenous gonadotropin stimulation of the ovaries. Since then, there have been numerous reports of the successful use of GnRH-a to successfully induce follicular maturation in IVF cycles,50,51 as well as ovulation in non-IVF cycles.52,53 Several authors have addressed the efficacy of GnRHa in preventing OHSS. Most reports support the hypothesis that GnRH-a induces adequate ovulation while

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avoiding OHSS. Emperaire and Ruffie studied 37 of 126 cycles in 48 patients undergoing ovulation induction with a regimen of either hMG or hMG with clomiphene citrate.54 All cycles were considered to be at high risk for OHSS and/or multiple pregnancy (E2 level >1000–1200 pg/ml and >3 follicles of >17 mm mean diameter). In these at-risk cycles, an endogenous LH surge was provoked by intranasal buserelin 200 mg three times at 8-hourly intervals. Ovulation was documented in all cycles except one (97%). Eight pregnancies resulted (21.6%), and there were no cases of OHSS. Imoedemhe et al used two doses of GnRH-a (Suprefact 100 mg) by nasal spray 8 hours apart to induce follicular maturation 34–36 hours prior to oocyte recovery in 38 women considered at risk of OHSS (E2 >4000 pg/ml) in an IVF program.49 Of the 707 oocytes recovered at egg retrieval, 93% were scored as mature, and 46% were successfully fertilized. Twenty-six women had embryos replaced; 11 pregnancies occurred (28.9%), and there were no cases of OHSS. Itskovitz et al used buserelin acetate in dosages of either 250 mg or 500 mg injected subcutaneously in either a single or two divided doses 12 hours apart.51 Approximately 78% of all eggs recovered were considered mature. Three of 13 patients conceived (21.4%), and none developed any signs or symptoms of OHSS. Shalev et al53 and Balasch et al55 used a single subcutaneous injection of triptorelin or leuprolide (0.5 mg), respectively, to trigger ovulation in gonadotropin-stimulated cycles that would otherwise have been cancelled due to a high risk for OHSS. Conception rates of 50% and 17.4% were achieved, respectively, while no patient developed OHSS. Kulikowski and colleagues used a single dose of 0.3 mg GnRH-a subcutaneously in 32 patients undergoing ovulation induction for IVF and in 16 patients undergoing ovulation induction for ovulatory disturbances, all of whom they felt were at risk of OHSS (E2 >2500 pg/ml).56 All patients had ovulation induced with a clomiphene/hMG protocol. There were no cases of OHSS in the GnRH-a group, and four pregnancies occurred (12.5%). In the control IVF group, there were four cases of OHSS, and three pregnancies occurred (8.8%). In the 16 patients who had undergone ovulation induction with clomiphene/hMG/GnRH-a, no OHSS was detected, while four patients became pregnant (25%). Recently, Engmann et al randomized 66 IVF patients at risk for OHSS to a GnRH-ant protocol with GnRH-a trigger, or a GnRH-a down-regulatory protocol with hCG trigger.57 All patients received luteal support using intramuscular progesterone, and the GnRH-a trigger group received luteal estrogen supplementation as well; 31% of patients in the hCG trigger group developed some form of OHSS, compared with none in the GnRH-a trigger group. Moreover, there were no significant differences in implantation, clinical pregnancy, and ongoing pregnancy rates. The study concluded that the use of a GnRH-a trigger reduces, if not eliminates, the risk of OHSS in high-risk patients, without compromising pregnancy outcomes. In summary, a number of investigators have used midcycle GnRH-a, in

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varying dosages and time intervals, for cycles considered to be at high risk for development of OHSS. Pregnancy rates of 12.5–50% were achieved, with a 0% incidence of OHSS. Because some studies have suggested a degradation in pregnancy outcomes with the use of GnRHa triggers, Griesinger et al recently proposed the use of a GnRH-a trigger to prevent OHSS, together with pronuclear cryopreservation and frozen–thawed embryo transfer at a later date. In their prospective observational study, cumulative ongoing pregnancy rates were high, and no cases of moderate or severe OHSS were observed.58 In contrast to the above reports showing an absence of development of OHSS in high-risk patients given GnRH-a for follicular maturation and ovulation, van der Meer et al published a study in which three patients who used buserelin to induce a preovulatory endogenous LH surge, in lieu of hCG, nevertheless, developed moderate OHSS.59 Severe ascites, hypovolemia, or electrolyte imbalance did not occur, and no patients were hospitalized. These authors concluded that OHSS is due to a massive luteinization of the follicles after exaggerated follicular stimulation, and can occur independent of the ovulation triggering agent. Gerris et al also reported the occurrence of moderate OHSS in one patient following GnRH-a administration,52 but in this case native GnRH was used, resulting in successful ovulation triggering but without the critical ovarian suppression that is thought to be at least equally important in the prevention of OHSS.60 Casper surveyed a total of 163 cycles in which GnRHa was used to trigger ovulation in the context of preventing OHSS.61 He stipulated that 900 cycles should have been randomized in order to detect a significant difference between GnRH-a and hCG. However, his analysis relies on an assumed 2% risk for severe OHSS and, while a 2% risk may be applicable to the average woman undergoing COH, most women in his survey likely had far greater risk, possibly in the 10– 20% range. The preponderance of evidence to date supports a decreased incidence of OHSS with GnRh-a compared with hCG as the triggering agent for ovulation, although a small possibility of moderate OHSS remains, particularly in conception cycles. Most importantly, there have been no reports of severe or critical OHSS after triggering ovulation with midcycle GnRH-a. Clinicians using GnRH-a to trigger ovulation must realize that the ensuing luteal phase is dramatically deficient, and full luteal progesterone support must be employed. Clinicians must also be aware that because of pituitary desensitization, GnRH-a cannot be used as an ovulation trigger for cycles in which GnRH-a was previously used for down-regulation. If a patient at high risk for OHSS is identified and GnRH-a triggering is contemplated, a GnRH-ant protocol, rather than a long-GnRH-a protocol, should be used for suppression of the endogenous midcycle LH surge.

GnRH antagonists and OHSS GnRH antagonists seem to be associated with a decreased risk of OHSS compared with the GnRH-a long protocol in patients undergoing IVF. Although one meta-analysis found no differences in the incidence of OHSS with the use of GnRH-ant compared with long GnRH-a down-regulatory protocols,62 another more recent meta-analysis of 27 randomized controlled trials revealed a significant reduction in the incidence of OHSS with the use of GnRH-ant compared with GnRHa down-regulation protocols, with a relative risk of 0.61.63 Furthermore, fewer interventions to prevent OHSS were administered in GnRH-ant protocols vs GnRH-a protocols (odds ratio [OR] = 0.44). Another meta-analysis confirmed a decreased risk of OHSS with the use of GnRH-ant, but particularly for cetrorelix and less so for ganirelix.64 A recent prospective multicenter study demonstrated a reduction in the incidence of OHSS, and a decreased cycle cancellation rate for risk of OHSS, in high-risk IVF patients treated with a GnRHant protocol as compared with historical controls down-regulated with GnRH-a.65 Interestingly, since the degree of ovarian suppression with GnRH-ant may be more profound at high doses, the dose of GnRH-ant may thereby be adjusted to minimize the development of OHSS in high-risk patients. de Jong et al employed this strategy when they used the GnRH-ant ganirelix to prevent OHSS by increasing the dose of the antagonist when target E2 values were inadvertently exceeded.66 Indeed, E2 values rapidly decreased with a concomitant decrease in ovarian size. Although this group’s suggestion is novel, far more intriguing is the potential use of GnRH-ant in conjunction with either rLH or GnRH-a to trigger ovulation. Because of the competitive nature of GnRH-ant suppression and lack of desensitization, it is possible to trigger ovulation with GnRH-a during co-treatment with gonadotropins and GnRH-ant. The respective dosages of each agent still awaits further studies, although 0.25 mg of ganirelix or cetrorelix daily seems to be sufficient to eliminate the LH surge, and seems to result in favorable clinical outcomes. Theoretically, a larger dose of GnRH-a would be needed to induce an LH surge in cycles suppressed by a GnRHant than in cycles using gonadotropins alone without a GnRH-ant. It cannot be stressed enough that full progesterone support is mandatory throughout the luteal phase when GnRH-a is used to trigger ovulation.

Embryology strategies Liberal application of embryo cryopreservation for patients showing early signs of hyperstimulation can be an important safety net in guarding against severe OHSS,67 although the efficacy of cryopreservation as a preventive measure for OHSS has recently been questioned.68 With routine culture of embryos to the blastocyst stage, it is possible to accurately assess the degree of OHSS prior to embryo transfer; because blastocyst

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transfer takes place on the seventh day after hCG, absence of even a moderate degree of OHSS is reassuring, and one may safely proceed with embryo transfer.5 The higher implantation rates associated with blastocyst transfer have led some clinicians to employ single blastocyst transfer in patients at risk of developing severe OHSS.69,70 Such a strategy results in a negligible multiple gestation rate, which purportedly is associated with more severe OHSS, presumably secondary to higher hCG levels. Although theoretically plausible, the utility of such an approach remains to be confirmed. Improvements in in vitro maturation of immature oocytes might also enable women, particularly those with polycystic ovary syndrome (PCOS) who are at greatest risk of OHSS, to undergo assisted reproduction using minimal if any gonadotropin stimulation. This may dramatically reduce or eliminate the risk of OHSS.71,72

Prophylactic use of colloid agents to prevent OHSS Third spacing and intravascular volume depletion due to increased capillary leakage are hallmarks of OHSS. Several investigators have administered intravenous colloidal agents such as albumin and hydroxyethyl starch (HES) at the time of oocyte retrieval, as prophylactic intravascular volume and oncotic pressure enhancers, to minimize the risk of developing OHSS.73–75 In contrast to the significant value of albumin for treatment of the fully developed syndrome, colloids are of questionable benefit as preventive measures. Recently, a meta-analysis of five randomized clinical trials has validated the use of intravenous albumin administration at the time of oocyte retrieval in high-risk patients.76 In this meta-analysis, albumin infusion to 18 at-risk women was necessary to prevent one case of severe OHSS. Because albumin treatment inheres risks such as allergic reactions and transmission of viruses and prions, the relative merits of albumin infusion compared with other preventive strategies remain unclear.

Miscellaneous techniques to prevent OHSS Other modalities that have been suggested for the prevention of OHSS include unilateral or bilateral follicular aspiration as a rescue for cycles not otherwise intended to undergo oocyte retrieval.77 Egbase advocated ovarian diathermy prior to initiation of COH.78 Ovarian diathermy should, however, be reserved for young patients with severe PCOS who tend to hyperstimulate even on a prolonged low-dose FSH regimen. Recently, metformin, the second-generation biguanide insulin sensitizer, has been advocated for the treatment of women with severe PCOS and insulin resistance. Although a more favorable response to ovulation enhancement would be expected, it is unclear yet whether a reduction in the incidence of OHSS will

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follow. One small study, comparing combined treatment using clomiphene and metformin with clomiphene alone, showed a lower incidence of OHSS with the addition of metformin, although this difference did not achieve statistical significance.79 A randomized prospective trial showed that adding metformin to gonadotropin regimens for ovulation induction yields fewer large follicles, lower E2 levels on the day of hCG administration, and a reduced rate of cycle cancellation for over-response.80 These effects would probably result in a reduced risk of OHSS as well. Indeed, a recent study of 287 women undergoing in vitro fertilization demonstrated a significant reduction in the incidence of OHSS in those taking metformin.81 Clearly, though, more data are needed to fully elucidate the effects of metformin on OHSS in women with PCOS. Suppression of ovarian steroidal secretion, either through continued administration of GnRh-a following oocyte retrieval coupled with cryopreservation, or through administration of intramuscular hydroxyprogesterone caproate and estradiol valerate following embryo transfer, has been suggested to minimize the risk of developing OHSS. Both approaches currently remain experimental.41,82 The anti-inflammatory action of corticosteroids has also been hypothesized to be beneficial in preventing OHSS. However, because of conflicting reports in the literature, there are currently insufficient data to recommend such an approach.83,84 Most recently, novel research has focused on preventive strategies aimed particularly at VEGF as the critical ovarian mediator of the syndrome. Encouragingly, treatment with a VEGF receptor antagonist prevented the increase in capillary permeability seen in an OHSS rat model.85 Likewise, treatment with fms-like tyrosine kinase, a soluble agent which binds VEGF with high affinity and thus decreases its availability for its endothelial effects, demonstrated the same effect in a similar OHSS rat model.86 Cabergoline, a dopamine agonist, inactivates VEGF receptor-2 in animal models,87 and a recent prospective, randomized, double-blind study investigated the effects of daily cabergoline treatment (0.5 mg/day) in oocyte donors at risk for OHSS. The study showed more than a 50% reduction in the incidence of moderate OHSS with the use of cabergoline from the day of hCG administration through 6 days post-oocyte retrieval.88 Such novel options make pathophysiologic sense and have the potential to play a future role in OHSS prevention. Obviously, there are many strategic options for the prevention of OHSS in high-risk patients. The increasing use of GnRH-ant in clinical practice holds great promise for preventing severe OHSS. As we master the complexity of GnRH-ant for LH surge suppression, together with GnRH-a for triggering ovulation, ovarian stimulation will likely become better controlled, and severe OHSS will become a rare if not forgotten entity.

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Treatment of severe OHSS Medical approach There are two possible approaches to the treatment of OHSS: one pathogenesis-oriented and one empiric. The former approach utilizes agents which specifically negate the putative causative factor(s) of OHSS. Indomethacin was hypothesized to be such an agent when prostaglandins were believed to play a role in OHSS. Angiotensin-converting enzyme (ACE) inhibitors are another group of specific pharmacological agents which were thought to have potential use in the treatment of OHSS because they inhibit the production of angiotensin II, a probable pathogenic factor for the syndrome. Unfortunately, indomethacin did not benefit the syndrome, and ACE inhibitors are teratogenic and thus contraindicated whenever a pregnancy is contemplated. Just as VEGF antagonists may become useful for the prevention of OHSS, similar cytokine inhibitors are being studied for treatment of the syndrome. To date, such therapies remain investigational, largely preclinical, and not yet compelling. One recent study found pentoxifylline, an inhibitor of the synthesis of TNF-α, to be ineffective in limiting ascites formation in an OHSS rabbit model, although it did decrease ovarian weight compared with controls.89 However, until such interventions are validated in human trials, the treatment of OHSS remains largely empiric in nature. The clinical manifestations of OHSS are a cascade of pathophysiological events resulting from a global increase in vascular permeability. This increased vascular permeability causes a change in extracellular fluid equilibrium, with fluid shifting into the extravascular or third space, often causing abdominal ascites, pleural and pericardial effusions, and hemoconcentration. Cardiac preload falls due to a combination of hypovolemia caused by fluid shifts, and compression of the inferior vena cava from the increasing intraperitoneal pressure. Falling cardiac preload reduces cardiac output, which in turn leads to a decrease in renal perfusion. Decreasing renal perfusion increases proximal tubule reabsorption of salt and water, leading to decreased urinary sodium excretion and oliguria. The proximal sodium reabsorption, and consequently diminished exposure of the distal tubule to sodium, impairs the sodium–hydrogen/potassium exchange in the distal tubule, causing hyperkalemic acidosis. A full-blown prerenal azotemia can develop. OHSS also produces a hypercoagulable state, possibly due to a combination of hemoconcentration and high levels of ovarian steroids. Individual treatment will depend on the severity of the syndrome. Mild forms of OHSS require little more than reassurance, since it is well established that mild symptoms usually resolve, in the absence of pregnancy, within 2 weeks after receiving hCG. If a pregnancy ensues, mild symptoms may progress but rarely more than one degree in severity. In patients with moderate

ascites and mild hemoconcentration (hematocrit <45%), bed rest and abundant liquid intake should be prescribed. The tendency for intravascular volume depletion and hyponatremia may be treated with oral isotonic electrolyte solutions; sports drinks, popular among athletes, are particularly suitable because they are engineered for optimal rehydration. The patient should be vigilant in noting any decreases in urine output, significant weight gain, or abdominal bloating as self-assessed by daily abdominal girth measurement. These findings, if present, may be the first warning signals of accumulation of ascitic fluid and worsening hemoconcentration. A hematocrit >45%, or 30% increased over baseline, indicates that the condition has entered the category of severe OHSS and that hospitalization is required. Dramatic clinical deterioration is most likely to manifest 8–9 days after hCG administration, when endogenous, pregnancy-derived hCG becomes perceptible. The single most important variable that indicates the severity of the OHSS is hemoconcentration, as reflected in the hematocrit. Because the hematocrit is actually the ratio between red cell volume and total blood volume, where total blood volume = red cell volume + plasma volume, the change in plasma volume must always be larger than the change reflected by the hematocrit.42 Thus, a change of 2 percentage points in the hematocrit from 42% to 44% is four times smaller than the actual 8% drop in plasma volume. This is extremely important to remember when one is treating patients with OHSS. Any increase in the hematocrit as it approaches 45% underestimates the magnitude of plasma volume depletion and thus the seriousness of the patient’s condition. One should therefore not be lulled into a false sense of security when only a small incremental rise in hematocrit between 40% and 45% is observed. Similarly, in the face of hemoconcentration, small reductions in hematocrit may represent a significant improvement in plasma volume.5 An additional measure of hemoconcentration is the magnitude of leukocytosis; white blood cell (WBC) counts higher than 25 000/mm3, largely reflecting a granulocytosis, may be seen. This massive neutrophilia may be attributed to hemoconcentration and generalized stress reaction. When oral isotonic fluid intake is insufficient to maintain plasma volume, intravenous fluid therapy becomes mandatory. Table 56.3 details the advantages and disadvantages of various therapies in the treatment of severe OHSS, and Fig 56.3 provides a clinical algorithm for the management of OHSS. Crystalloids alone, although seldom sufficient in restoring homeostasis because of massive protein loss through hyperpermeable capillaries, still remain the mainstay of treatment. Because of the tendency for hyponatremia, sodium chloride with or without glucose is the crystalloid of choice. The daily volume infused may vary from 1.5 L to >3.0 L. Although some authors advocate fluid restriction to

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Table 56.3

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Pros and cons of various therapies of OHSS

Therapy

Pros

Cons

IV crystalloids

Alleviates hemoconcentration Improves renal perfusion

Lost immediately from vascular tree Aggravates ascites

Fluid restriction

Controls ascites

Reduces renal perfusion Promotes hemoconcentration

Albumin

Improves colloid-oncotic pressure Improves renal perfusion

Risks of human blood product

Furosemide

Reduces total body water

Further reduces intravascular volume

Indomethacin

May block prostanglandin-induced hyperpermeability

Implicated in renal failure

ACE inhibitors

May block angiotensin II-induced hyperpermeability

Teratogenic

Paracentesis

Alleviates tense ascites Improves renal perfusion

Risks of hemorrhage, infection, and leakage

Heparin

Decreases risk of thromboembolic phenomena

Increases risk of hemorrhage

Peritoneo-venous shunt

Replaces lost electrolytes and proteins

Risk of self-toxicity Elaborate setup and risk of infection

Dopamine drip

Improves renal perfusion

Need for Intensive Care Unit management

minimize the accumulation of ascites,90 one should rather deal with the discomfort of ascites than face the consequences of hemoconcentration with the attendant risks of thromboembolism and renal shutdown. In order to maintain fluid balance, the patient’s urine output, oral and intravenous fluid intake, body weight, abdominal girth, hematocrit, and serum electrolytes must be monitored. In addition, coagulation parameters and liver enzymes should be periodically assessed. Intravenous volume replacement should aim to improve renal perfusion before fluid escapes into the peritoneal and/or pleural cavities; this transient hemodilution is achieved at the expense of increased third spacing and increased total body water. Whenever adequate fluid balance cannot be restored by crystalloid alone, plasma expanders should be utilized. Since albumin is the main protein lost in OHSS, human albumin is physiological and thus the colloid of choice (Table 56.3). Albumin at doses of 50–100 g at 25% concentration should be administered intravenously and repeated every 2–12 hours until the hematocrit falls below 45% and urine output increases. At a relatively advanced stage of OHSS, during treatment with crystalloids and colloids, gradual hemodilution is obtained at the expense of a tightening abdominal wall with the rapid accumulation of ascitic fluid. At this stage of restored intravascular volume and improved renal perfusion, there may occur a sudden, paradoxical

onset of oliguria, increasing serum creatinine, and rapidly falling creatinine clearance.91 This sudden deterioration in fluid balance is probably the result of a significant rise in intra-abdominal pressure produced by tense ascites. Increased intra-abdominal pressure may in turn impede renal venous outflow, causing congestion, renal edema, and decrease in renal function. Such tense ascites are best treated surgically via therapeutic paracentesis, although diuretics may also be effective. When oliguria persists despite evidence of adequate hemodilution, intravenous furosemide at a 10–20 mg dose is often beneficial. In practice, an albumin–furosemide chase protocol seems to yield the best results. Two units of albumin, 50 g each, followed immediately by intravenous furosemide, will often result in diuresis. In states of volume contraction, hemoconcentration, and hypotension, furosemide should be strictly avoided. In this precarious stage of OHSS, with impending renal failure, a renal-dose dopamine drip should be used for renal rescue.

Paracentesis The single most important treatment modality in lifethreatening OHSS that cannot be controlled by medical therapy is paracentesis. Rabau et al first proposed the use of paracentesis in the treatment of severe OHSS.2 Paracentesis was temporarily discredited, but later regained popularity (Table 56.3 and Figure

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(a) THROMBOEMBOLISM

PULMONARY

Heparin prophylaxis

Physical examination, chest X-ray, monitor for tachypnea, hypoxemia

If suspicion for DVT, or PE

Maintain to hospital Discharge

Initiate therapeutic anticoagulation, and full evaluation for DVT or PE

IV = Intravenous CVP = central venous pressure ARDS = acute respiratory distress syndrome

DVT = deep vein thrombosis PE = pulmonary embolus

Hydrothorax

ARDS

Thoracentesis

Consider glucocorticoid therapy

Repeat as needed

Mechanical ventilation as needed

(b) HEMODYNAMIC / RENAL

Assess blood pressure, body weight, hematocrit, electrolytes, creatinine

ASCITES

Persistent hemoconcentration

Persistent oliguria Severe discomfort

Foley catheter to assess urine output

IV isotonic fluids to maintain CVP and urine output

IV isotonic fluids to maintain CVP and urine output

Utrasound-guided paracentesis; repeat as needed

Large-bore IV or central line, monitor CVP

Plasma-expanding colloid therapy (albumin, hetastarch)

Consider renal-dose dopamine to maintain renal blood flow

Dialysis if uncontrollable hyperkalemia and metabolic acidosis

Consider peritoneovenous shunt

Consider percutaneous pigtail catheter for continuous drainage

Maintain adequate IV fluid intake

Figure 56.3 Algorithm for the intensive care of the patient with critical OHSS: (a) thromboembolism and pulmonary; (b) hemodynamic/renal and ascites.

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56.3).3 Thaler,92 Borenstein,91 and Forman93 have all promoted paracentesis as safe and exceptionally beneficial. Dramatic improvements in the clinical symptoms of severe OHSS, with almost instantaneous diuresis, were reported.91 In a series of seven patients in whom paracentesis was performed, urine output rose from 780 ± 407 ml to 1670 ± 208 ml (p <0.05), creatinine clearance rose from 75.4 ± 16 ml/min to 101 ± 15 ml/min (p <0.05), hematocrit decreased from 46.3 ± 2.2% to 37.1 ± 2.5% (p <0.05), and a mean weight loss of 5.3 kg was observed.94 In the study by Forman et al93 37 L of ascitic fluid with a protein content of 46–53 g/L (reflecting a total protein loss of 1.85 kg) was removed from a single patient, underscoring both the high protein content of ascitic fluid and the safety of the procedure. The indications for paracentesis include the need for symptomatic relief, tense ascites, oliguria, rising serum creatinine concentration or falling creatinine clearance, and hemoconcentration unresponsive to medical treatment. Paracentesis should be performed aseptically under ultrasound guidance. Careful monitoring of hemodynamic stability is also mandatory. Rizk and Aboulghar advocated transvaginal ultrasonically guided aspiration of ascitic fluid as an effective and equally safe method,94 but a transabdominal approach can be used as well. Up to 4 L may be removed either by slow drainage to gravity,92 or with negative pressure using large evacuated containers. Paracentesis is contraindicated in patients who are hemodynamicaly unstable, or in the presence of suspected hemoperitoneum. A new and innovative treatment for severe OHSS was suggested by Koike et al.90 These authors describe continuous peritoneovenous shunting in 18 patients with severe OHSS. This study group was compared with 36 control patients who had received intravenous albumin at a dose of 37.5 g/day. Recirculation of ascites fluid rich in proteins is not a novel idea;95 however, the reliance on a continuous shunt from the peritoneal cavity into the antecubital vein is a novel and logical way to replenish the vascular tree with the fluid, proteins, and electrolytes that were lost from the vasculature. The study reports faster hemodilution, shorter hospital stays, and prompt improvement in symptoms in the shunted patients due to diuresis and reduction in the amount of ascites. There are, however, some problems with the study besides the complexity of the setup. First, the reinfused ascites may contain the very substances which might be responsible for the profound hyperpermeability of OHSS, and thus may exacerbate the syndrome. Secondly, the group advocates fluid restriction, which may aggravate hemoconcentration and thus contribute to renal failure and thromboembolic phenomena.90 In addressing the hypercoagulable state of OHSS, most authors reserve anticoagulation for special circumstances in which thromboembolic events have already occurred, or in the setting of a hereditary coagulopathy. One study suggests that prophylactic screening for hereditary thrombophilias should perhaps be undertaken in all

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patients undergoing assisted reproduction, because of a higher prevalence of thrombophilia in women with severe OHSS.96 However, another conflicting study found no increase in such prevalence, and recommends against screening the general IVF population for thrombophilias.97 In any case, although prophylactic treatment of women with OHSS with unfractionated or low-molecular-weight heparin is of some theoretical value, rapid alleviation of the patient’s hemoconcentration is far more important. Rarely, as a last resort, when the critical stage of OHSS is complicated by renal failure, thromboembolism, acute respiratory distress syndrome, and multiorgan failure, there is no choice but to perform a potentially life saving termination of pregnancy.

References 1. Abramov Y, Elchalal U, Schenker JG. Severe OHSS: An ‘epidemic’ of severe OHSS: a price we have to pay? Hum Reprod 1999; 14: 2181–3. 2. Rabau E, David A, Serr DM, Mashiach S, Lunenfeld B. Human menopausal gonadotropins for anovulation and sterility. Results of 7 years of treatment. Am J Obstet Gynecol 1967; 98: 92–8. 3. Schenker JG, Weinstein D. Ovarian hyperstimulation syndrome: a current survey. Fertil Steril 1978; 30: 255–68. 4. Golan A, Ron-el R, Herman A, et al. Ovarian hyperstimulation syndrome: an update review. Obstet Gynecol Surv 1989; 44: 430–40. 5. Navot D, Bergh P, Laufer N. Ovarian hyperstimulation syndrome in novel reproductive technologies: prevention and treatment. Fertil Steril 1992; 58: 249–61. 6. Navot D, Margalioth EJ, Laufer N, et al. Direct correlation between plasma renin activity and severity of the ovarian hyperstimulation syndrome. Fertil Steril 1987; 48: 57–61. 7. McClure N, Healy DI, Rogers PA, et al. Vascular endothelial growth factor as a capillary permeability agent in ovarian hyperstimulation syndrome. Lancet 1994; 344: 235–6. 8. Friedlander MA, Loret de Mola JR, Goldfarb JM. Elevated levels of interleukin-6 in ascites and serum from women with ovarian hyperstimulation syndrome. Fertil Steril 1993; 60: 826–33. 9. Abramov Y, Schenker JG, Lewin A, et al. Plasma inflammatory cytokines correlate to the ovarian hyperstimulation syndrome. Hum Reprod 1996; 11: 1381–6. 10. Revel A, Barak V, Lavy Y, et al. Characterization of intraperitoneal cytokines and nitrates in women with severe ovarian hyperstimulation syndrome. Fertil Steril 1996; 66: 66–71. 11. Orvieto R, Ben-Rafael Z. The immune system in severe ovarian hyperstimulation syndrome. Isr J Med Sci 1996; 32: 1180–2. 12. Sealey JE, Atlas SA, Glorioso N, Manapat H, Laragh JH. Cyclical secretion of prorenin during the menstrual cycle: synchronization with luteinizing hormone and progesterone. Proc Natl Acad Sci USA 1985; 82: 8705–9.

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13. Derkx FH, Alberda AT, Zeilmaker GH, Schalekamp MA. High concentrations of immunoreactive renin, prorenin and enzymatically-active renin in human ovarian follicular fluid. Br J Obstet Gynaecol 1987; 94: 4–9. 14. Geva E, Jaffe RB. Role of vascular endothelial growth factor in ovarian physiology and pathology. Fertil Steril 2000; 74: 429–38. 15. Whelan LG 3rd, Vlahos NF. The ovarian hyperstimulation syndrome. Fertil Steril 2000; 73: 883–96. 16. Warren RS, Yuan H, Matli MR, Ferrara N, Donner DB. Induction of vascular endothelial growth factor by insulin-like growth factor I in colorectal carcinoma. J Biol Chem 1996; 271: 483–8. 17. Ferrara N, Davis-Smyth T. The biology of vascular endothelial growth factor. Endocrinol Rev 1997; 18: 4–25. 18. Levin ER, Rosen GF, Cassidenti DL, et al. Role of vascular endothelial growth factor in ovarian hyperstimulation syndrome. J Clin Invest 1998; 102: 1978–85. 19. Neulen J, Yan Z, Raczek S, et al. Human chorionic gonadotropin-dependent expression of vascular endothelial growth factor/vascular permeability factor in human granulosa cells: importance in ovarian hyperstimulation syndrome. J Clin Endocrinol Metab 1995; 80: 1967–71. 20. Pellicer A, Albert C, Mercader A, et al. The pathogenesis of ovarian hyperstimulation syndrome: in vivo studies investigating the role of interleukin-1β, inerleukin-6, and vascular endothelial growth factor. Fertil Steril 1999; 71: 482–9. 21. Bergh C, Howles CM, Borg K, et al. Recombinant human follicle stimulating hormone (r-hFSH; GonalF) versus highly purified urinary FSH (Metrodin HP): results of a randomized comparative study in women undergoing assisted reproductive techniques. Hum Reprod 1997; 12: 2133–9. 22. Out HJ, Mannaerts BM, Driessen SG, Bennink HJ. A prospective, randomized, assessor-blind, multicentre study comparing recombinant and urinary follicle stimulating hormone (Puregon versus Metrodin) in in-vitro fertilization. Hum Reprod 1995; 10: 2534–40. 23. Frydman R, Howles C, Truong F. A double-blind, randomized study to compare recombinant follicle stimulating hormone (FSH; Gonal-F) with highly purified urinary FSH (Metrodin HP) in women undergoing assisted reproductive techniques including intracytoplasmic sperm injection: on behalf of The French Multicentre Trialists. Hum Reprod 2000; 15: 520–5. 24. Schats R, Sutter P, Bassil S, et al. Ovarian stimulation during assisted reproduction treatment: comparison of recombinant and highly purified urinary human FSH. On behalf of The Feronia and Apis study group. Hum Reprod 2000; 15: 1691–7. 25. Hedon B, Hugues J. A comparative prospective study of a chronic low dose versus a conventional ovulation stimulation regimen using r-FSH in anovulatory infertile women. Report no GF 8220. Geneva: Ares Serono International SA. 26. Homburg R, Levy T, Ben-Rafael Z. A comparative prospective study of conventional regimen with chronic low-dose administration of follicle-stimulating

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ovarian hyperstimulation syndrome and multiple pregnancy. Gynecol Endocrinol 1994; 8: 7–12. Kulikowski M, Wolczynski S, Kuczynski W, Grochowski D. Use of GnRH analog for induction of the ovulatory surge of gonadotropins in patients at risk of the ovarian hyperstimulation syndrome. Gynecol Endocrinol 1995; 9: 97–102. Engmann L, DiLuigi A, Schmidt D, et al. The use of gonadotropin-releasing hormone (GnRH) agonist to induce oocyte maturation after cotreatment with GnRH antagonist in high-risk patients undergoing in vitro fertilization prevents the risk of ovarian hyperstimulation syndrome: a prospective randomized controlled study. Fertil Steril 2008; 89: 84–91. Griesinger G, von Otte S, Schroer A, et al. Elective cryopreservation of all pronuclear oocytes after GnRH agonist triggering of final oocyte maturation in patients at risk of developing OHSS: a prospective, observational proof-of-concept study. Hum Reprod 2007; 22: 1348–52. Van der Meer S, Gerris J, Joostens M. Triggering of ovulation using a gonadotrophin-releasing hormone agonist does not prevent ovarian hyperstimulation syndrome. Hum Reprod 1993; 8: 1628–31. Kol S, Lewit N, Itskovitz-Eldor J. Ovarian hyperstimulation: effects of GnRH analogues. Ovarian hyperstimulation syndrome after using gonadotrophin-releasing hormone analogue as a trigger of ovulation: causes and implications. Hum Reprod 1996; 11: 1143–4. Casper RF. Ovarian hyperstimulation: effects of GnRH analogues. Does triggering ovulation with gonadotrophin-releasing hormone analogue prevent severe ovarian hyperstimulation syndrome? Hum Reprod 1998; 11: 1144–6. Griesinger G, Diedrich K, Tarlatzis BC, Kolibianakis EM. GnRH-antagonists in ovarian stimulation for IVF in patients with poor response to gonadotrophins, polycystic ovary syndrome, and risk of ovarian hyperstimulation: a meta-analysis. Reprod Biomed Online 2006; 13: 628–38. Al-Inany H, Abou-Setta AM, Aboulghar MA. Gonadotropin-releasing hormone antagonists for assisted reproduction: a Cochrane review. Reprod Biomed Online 2007; 14: 640–9. Ludwig M, Katalinic A, Diedrich K. Use of GnRH antagonists in ovarian stimulation for assisted reproductive technologies compared to the long protocol. Arch Gynecol Obstet 2001; 265: 175–82. Ragni G, Vegetti W, Riccaboni A, et al. Comparison of GnRH agonists and antagonists in assisted reproduction cycles of patients at high risk of ovarian hyperstimulation syndrome. Hum Reprod 2005; 20: 2421–5. de Jong D, Macklon NS, Mannaerts BM, Coelingh Bennink HJ, Fauser BC. High-dose gonadotrophinreleasing hormone antagonist (ganirelix) may prevent ovarian hyperstimulation syndrome caused by ovarian stimulation for in-vitro fertilization. Hum Reprod 1998; 13: 573–5. Garrisi G, Navot D. Cryopreservation of semen, oocytes, and embryos. Curr Opin Obstet Gynecol 1992; 4: 726–31. D’Angelo A, Amso NN. Embryo freezing for preventing ovarian hyperstimulation syndrome: a Cochrane review. Hum Reprod 2002; 17: 2787–94.

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69. Kinget K, Nijs M, Cox AM, et al. A novel approach for patients at risk for ovarian hyperstimulation syndrome: elective transfer of a single zona-free blastocyst on day 5. Reprod Biomed Online 2002; 4: 51–5. 70. Trout SW, Bohrer MK, Deifer DB. Single blastocyst transfer in women at risk of ovarian hyperstimulation syndrome. Fertil Steril 2001; 76: 1066–7. 71. Child TJ, Phillips SJ, Abdul-Jalil AK, Gulekli B, Tan SL. A comparison of in vitro maturation and in vitro fertilization for women with polycystic ovaries. Obstet Gynecol 2002; 100: 665–70. 72. Tan SL, Child TJ. In vitro maturation of oocytes from unstimulated polycystic ovaries. Reprod Biomed Online 2002; 4 (Suppl 1): 18–23. 73. Shalev E, Giladi Y, Matilsky M, Ben-Ami M. Decreased incidence of severe ovarian hyperstimulation syndrome in high risk in vitro fertilization patients receiving intravenous albumin: a prospective study. Hum Reprod 1995; 10: 1373–6. 74. Isik AZ, Gokmen O, Zeyneloglu HB, Kara S, Gulekli B. Intravenous albumin prevents moderate–severe ovarian hyperstimulation in in vitro fertilization patients: a prospective, randomized and controlled study. Eur J Obstet Gynecol Reprod Biol 1996; 70: 179–83. 75. Gokmen O, Ugur M, Ekin M, et al. Intravenous albumin versus hydroxyethyl starch for the prevention of ovarian hyperstimulation in an in-vitro fertilization programme: a prospective randomized placebo controlled study. Eur J Obstet Gynecol Reprod Biol 2001; 96: 187–92. 76. Aboulghar M, Evers JH, Al-Inany H. Intravenous albumin for preventing severe ovarian hyperstimulation syndrome: a Cochrane review. Hum Reprod 2002; 17: 3027–32. 77. Egbase PE, Al Sharhan M, Grudzinskas JG. Early unilateral follicular aspiration compared to coasting for the prevention of severe OHSS; a prospective randomized study. Hum Reprod 1999; 14: 1421–5. 78. Egbase PE. Severe OHSS; How many cases are preventable? Hum Reprod 2000; 15: 8–10. 79. Malkawi HY, Qublan HS. The effect of metformin plus clomiphene citrate on ovulation and pregnancy rates in clomiphene-resistant women with polycystic ovarian syndrome. Saudi Med J 2002; 23: 663–6. 80. DeLeo D. Effects of metformin on gonadotropininduced ovulation in women with polycystic ovary syndrome. Fertil Steril 1999; 72: 282–5. 81. Khattab S, Fotouh IA, Mohesn IA, Metwally M, Moaz M. Use of metformin for prevention of ovarian hyperstimulation syndrome: a novel approach. Reprod Biomed Online 2006; 13: 194–7. 82. Rjosk HK, Abendstein BJ, Kreuzer E, Schwartzler P. Preliminary experience with steroidal ovarian suppression for prevention of severe ovarian hyperstimulation syndrome in IVF patients. Hum Fertil 2001; 4: 246–8. 83. Lainas T, Petsas G, Stavropoulou G, et al. Administration of methylprednisolone to prevent severe ovarian hyperstimulation syndrome in patients

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undergoing in vitro fertilization. Fertil Steril 2002; 78: 529–33. Tan SL, Balen A, el Hussein E, Campbell S, Jacobs HS. The administration of glucocorticoids for the prevention of ovarian hyperstimulation syndrome in in vitro fertilization: a prospective randomized study. Fertil Steril 1992; 58: 378–83. Gomez R, Simon C, Remohi J, Pellicer A. Vascular endothelial growth factor receptor-2 activation induces vascular permeability in hyperstimulated rats, and this effect is prevented by receptor blockade. Endocrinology 2002; 143: 4339–48. NcElhinney B, Ardill J, Caldwell C, McClure N. Preventing ovarian hyperstimulation syndrome by inhibiting the effects of vascular endothelial growth factor. J Reprod Med 2003; 48: 243–6. Gomez R, Gonzalez-Izquierdo M, Zimmermann RC, et al. Low-dose dopamine agonist administration blocks vascular endothelial growth factor (VEGF)mediated vascular hyperpermeability without altering VEGF receptor 2-dependent luteal angiogenesis in a rat ovarian hyperstimulation model. Endocrinology 2006; 147: 5400–11. Alvarez C, Marti-Bonmati L, Novella-Maestre E, et al. Dopamine agonist cabergoline reduces hemoconcentration and ascites in hyperstimulated women undergoing assisted reproduction. J Clin Endocrinol Metab 2007; 92: 2931–7. Serin IS, Ozcelik B, Bekyurek T, et al. Effects of pentoxifylline in the prevention of ovarian hyperstimulation syndrome in a rabbit model. Gynecol Endocrinol 2002; 16: 355–9. Koike T, Araki S, Minakami H, et al. Clinical efficacy of peritoneovenous shunting for the treatment of severe OHSS. Hum Reprod 2000; 15: 113–17. Borenstein R, Elhalal U, Lunenfeld B, Shoham Z. Severe OHSS; a reevaluated therapeutic approach. Fertil Steril 1989; 51: 791–5. Thaler I, Yoffe M, Kaftory JK, Brandes JM. Treatment of OHSS; the physiologic basis for a modified approach. Fertil Steril 1981; 36: 110–13. Forman RG, Frydman R, Egan D, Ross C, Barlow DH. Severe OHSS using agonists of gonadotropin-releasing hormone for in vitro fertilization; a European series and a proposal for prevention. Fertil Steril 1990; 53: 502–9. Rizk B, Aboulghar MA. Modern management of OHSS. Hum Reprod 1991; 6: 1082–7. Fukaya T, Funamaya Y, Chiba S, et al. Treatment of severe OHSS by ultrafiltration and reinfusion of ascitic fluid. Fertil Steril 1994; 61: 561–4. Dulitzky M, Cohen SB, Inbal A, et al. Increased prevalence of thrombophilia among women with severe ovarian hyperstimulation syndrome. Fertil Steril 2002; 77: 463–7. Fabregues F, Tassies D, Reverter JC, et al. Prevalence of thrombophilia in women with severe ovarian hyperstimulation syndrome and cost-effectiveness of screening. Fertil Steril 2004; 81: 989–95.

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57 The environment and reproduction Kenneth Barron, Machelle M Seibel

I used to walk along the beach, a favorite thing to do. Until the plastic and the trash completely spoiled my view. The place I take my rod and reel to catch my favorite dish. Has elevated mercury, so I can’t eat the fish. From Protect Environment by Machelle M Seibel Al Gore may have been unsuccessful in running for US president, but he has been a lightning rod in highlighting the impact of human industry on the environment, garnering an academy award for his documentary An Inconvenient Truth. Global warming, however, is not the only consequence of the 80 000 synthetic compounds used in the USA during the last half-century.1 One of our most basic human goals – reproduction – is also being affected. It is estimated that over 1000 new chemicals are being introduced into the world every year, yet less than 5% have been investigated for their effect on reproduction. Fertility studies in Pennsylvania have shown decreased total fertility rates from 1901 to 1985,2 and the overall US pregnancy rate in 1996 was 9% lower than it was in 1990.3 There is evidence that the quality and quantity of semen in normal men is also declining.4,5 What role do the thousands of compounds in our everyday environment play in these declining rates? What role does environmental exposure play in the spectrum of infertility patients that present to our clinics? What evidence is available for interpreting exposures, and what clinical considerations can we yield from such research? To address these questions we will consider the environment not as the world at large, but rather as three microenvironments: the follicle, the seminal fluid, and the amniotic sac. From this vantage point, the inhabitants are the egg, the sperm, and the unborn child. In this way, the impact of the environment is not a story of what the world will be like decades from now but rather an increased awareness of the impact of the environment on our reproductive-aged patients and children today.

Mechanisms of action Direct damage to the cell membrane or intracellular components is only one way compounds can cause

tissue injury. Some compounds alter the communication between different cells by mimicking or blocking normal pathways. Endocrine-disrupting chemicals (EDCs) are thought to effect reproduction by directly or indirectly mimicking, stimulating, antagonizing, altering, or displacing natural hormones.6 Exposure to such agents at critical stages of development can have a significant impact upon cellular, and ultimately fetal, development. Incomplete development of DNA repair mechanisms, detoxification enzymes, and the blood–brain barrier can exacerbate a chemical’s effect on the developing fetus. Moreover, there is an increasing body of research suggesting that epigenetic modulation may be an underlying mechanism of action. These effects, however, may not be seen for years. The theory of EDCs can be traced back to the publication of Silent Spring (1962), by Rachel Carson.7 In a serialized printing in the New Yorker she proposed a connection between the population changes in wildlife ecology and the increasing rates of human cancer. She associated both changes with widespread use of agricultural and manufacturing chemicals. Her work eventually prompted a government investigation that culminated in the banning of the pesticide DDT in 1972. It was 20 years later that Theo Colborn and colleagues advanced the EDC theory that is now widely accepted:8–11 EDCs can exact more influence over the development of affected offspring than the genes they inherit. EDCs have been shown to have deleterious effects on animal and fish reproduction,12 but primary outcome data are still being gathered in establishing an association between human exposure and infertility. Nevertheless, compelling data exist on the role of EDCs in other hormone-driven diseases, such as the rising prevalence of endometriosis in industrialized countries.13

Biological plausibility Xenoestrogens, alkylphenolic chemicals (bisphenol A, polychlorinated biphenyls), phthalates, dioxins, lead, mercury, and pesticides are ubiquitous in the global environment. They are unavoidable for the majority of us and have been reported to have a myriad of effects (Table 57.1). Many of these toxicants came under increased scrutiny when animal experiments began to demonstrate biological plausibility for human harm. Sharpe et al

Wash produce well, buy organic, vacuum frequently in agricultural areas

Avoid eating fish or game from areas known to be contaminated

Response to ovulation induction Fecundability Lactation Sperm quality Endometriosis Altered menstrual cycle Avoid fatty meats Avoid areas known to be contaminated

Cancer Birth defects Change in sex ratio Endometriosis

Results from industrial activities and fires. Found in fatty meats, fish, and dairy products

Dioxins

Unavoidable

Reproductive development disruption

Flame retardants in mattresses, furniture, pillows, carpets, electronic devices, TVs, DVD players, and computers

PBDEs

Avoid hard plastic bottles and food containers, but likely unavoidable

Breast cancer Prostate changes

Polycarbonate plastics: rigid water bottles, soda bottles, and plastic food containers

Bisphenols

Remove old paint and pressuretreated lumber. Be cautious/ limit certain fish consumption

IQ in offspring Semen quality/quantity Spontaneous abortion TTP Preterm labor

Lead: pre-1978 paint, old pipes. Mercury: old thermometers, and large fish such as tuna, shark, king mackerel, swordfish, and tilefish. Pressure-treated wood can leach chromium and arsenic

Heavy metals

PCBs, Polychlorinated biphenyls; PBDEs, polybrominated diphenyl ethers; DDE, dichlorodiphenyldichoroethylene; DDT dichlorodiphenyltrichelloroethane; IVF, in vitro fertilization; TTP, time to pregnancy; AGD, Anogenital distance; SGA, small for gestational age.

Unavoidable

Fecundability Success with IVF Semen quality Spontaneous abortion Preterm birth SGA

TTP AGD

Banned in the USA and most countries. PCBs have dioxin-like properties. They were used for cutting oils, as lubricants, and as electrical insulators. Sources are contaminated fish and game consumption

PCBs

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DDE, DDT, organochlorides, and ‘nonpersistent pesticides.’ DDT is banned in most countries, save Mexico, China, India, and parts of Africa. It is found in fruits, vegetables, flowers, antimicrobial soap, and air exposure in agricultural areas

Plastic toys, shampoos, soaps, nail polish, other personal care products, medical devices, timedrelease drug coatings, flooring, lacquers, and varnishes.

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Phhalates

Summary of common environmental toxicants

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demonstrated that gestational and lactational exposure of rats to xenoestrogens resulted in reduced testicular size and sperm production.14 Dicofol, an estrogenic organochloride pesticide, was observed to induce a significant decrease in ovarian follicles and the number of estrous cycles in rabbits15 while follicle destruction has been reported in rhesus monkeys exposed to polychlorinated biphenyls (PCBs).16 The list of mammalian studies linking subfertility to environmental toxicants is extensive, including studies demonstrating embryotoxicity for DDT (dichlorodiphenyltrichloroethane), methocychlor, and hexachlorocyclohexane.17 Most human exposure is through food, air, or, in the case of trihalomethanes (THMs), absorption through skin. Exposure to the aforementioned compounds has been well documented, but is only recently being monitored more closely. There were few data about non-occupational exposure to potential toxicants until the Centers for Disease Control (CDC) began testing in 1999 (116 compounds) and 2003 (148 compounds).18 National Geographic published an article in 2006 about a journalist who had his blood tested for levels of environmental toxicants to see what the average American accumulates in a lifetime. The tests, which cost around US$15 000, revealed 165 of 320 chemicals tested, including levels of a fire retardant used on airline seats 10 times higher than the average American, because of the many hours he spends in airplanes.19 The article highlights the fact that these chemicals are not only ubiquitous in the environment but also in our bodies. The correlation between animal experiments and human experience was nicely demonstrated by Swan et al when they examined the relationship between neonatal anogenital distance (AGD), a sexually dimorphic feature considered to be a sensitive indicator of masculinization, and phthalate metabolites.20 Phthalates (diesters of 1,2-benzenedicarboxylic acid) are a ubiquitous group of chemicals found in hundreds of products ranging from soft plastic vinyl toys and flooring to shampoos, soaps, and nail polish. High-molecular-weight phthalates are used in the manufacturing of flexible vinyl for flooring, wall coverings, food contact applications, and medical devices. Low-molecular-weight phthalates are used in personal care products as solvents and plasticizers, for making lacquers, varnishes, and coatings used in pharmaceuticals for timed-release drugs. Humans rapidly metabolize phthalate diesters (their half-lives are generally less than 24 hours), and thus do not accumulate them. Urinary biomarkers (phthalate monoesters), therefore, represent exposure in the last 1–2 days only. Swan et al evaluated mother–son pairs who had been recruited for an unrelated pregnancy cohort study (n = 85) and found a significant inverse relationship between the level of phthalate metabolites in the mother’s third trimester urine and the son’s AGD at birth. Higher prenatal phthalate metabolite levels correlated with a shorter AGD, which, in turn, was associated with incomplete testicular descent and smaller penile volume. These findings

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demonstrate the effect an environmental chemical can have on morphological development. Although implied, further investigation is needed to comment specifically on fertility or fecundity. Making the jump from biological plausibility to biological truth can be a difficult task. The randomized controlled trial (RCT) is widely recognized as the gold standard in medical research, but the use of such trials poses distinct challenges when studying toxicity. RCTs would be unethical: deliberately exposing individuals to potentially toxic chemicals is neither realistic nor to be condoned. Given these limitations, Stephen Genuis argues that clinical trials are not the only objective and credible way of establishing causality of a disease.21 He poses a simple analogy: it would be absurd to require an RCT to confirm the efficacy of parachutes to ‘prevent death and major trauma related to gravitational challenge.’22 In other words, not all research topics can be evaluated in identical manners. There are other challenges for interpreting environmental toxicant studies. Time-lag bias is a limitation that is highlighted by our experience with in utero DES exposure and vaginal cancer: compounds can have devastating effects in the long term that are not immediately recognizable. Variations in genetic vulnerability and phenotypic response can also mask a compound’s impact. For example, studies on bisphenol A metabolism have shown induction of hepatic cytochrome p450s in humans.23,24 Individuals have p450 isoenzyme variation and thus will respond to bisphenols with different levels of metabolic activity. Furthermore, it can be difficult to interpret dose–response curves because hormonal toxicants do not always respond according to the classic dose–response curve. Estradiol, for instance, has a negative feedback mechanism upon gonadotropin-releasing hormone (GnRH) release from the hypothalamus until it reaches a critical concentration at which it begins to increase the release of GnRH. This culminates in the luteinizing hormone (LH) surge that initiates ovulation. If EDCs are hormone mimickers, they probably act in the same fashion: effects may be seen at extremely low concentrations, but not at the higher concentrations used to test for chemical toxicity.25 This has been described as a non-monotonic, hormetic, or ‘biphasic’ dose–response curve (Fig 57.1) and is described frequently in the endocrinology literature but not in the assessment of environmental agents. Lastly, environmental health research is complicated by the phenomenon of bioaccumulation. Humans are exposed to thousands of compounds over a lifetime and it is therefore difficult to sort out the relationship between a specific compound and a specific outcome.26 The CDC’s National Report on Human Exposure to Environmental Chemicals14 is in its third edition and has only evaluated 148 compounds through blood and urine analysis of the known 80 000 synthetic compounds in our environment. With lag-time bias, phenotypic variation, the inability to perform RCTs, unpredictable dose– response mechanisms, and the bioaccumulation of

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Inhibition

Dose

Fig 57.1 Hormetic/biphasic dose–response curve. The nonmonotonic, hormetic, or ‘biphasic’ dose–response curve describes the action of certain agents at different doses such that a very low dose of a chemical agent may trigger the opposite response to a very high dose.

multiple compounds at once, it is apparent how difficult ‘proof’ of causality can be in environmental toxicology.

The seminal plasma microenvironment At levels measured in parts per trillion (ppt) and parts per billion (ppb), hormones such as insulin and estradiol are bioactive in cells and tissue. EDCs appear to be bioactive at equally low levels found in our blood stream.25 Of great concern is the postulation that the seminal plasma acts as a chemical concentrator, increasing levels of various environmental toxicants in the fluid surrounding our next generation. Men living in agrarian areas where use of pesticides is high have higher pesticide levels in their blood and semen, and lower sperm counts and motility than men living further away.27 As mentioned previously, humans rapidly metabolize phthalate diesters, and do not accumulate them. Despite fast metabolization, the omnipresence of phthalates in our environment raises concern. A study in 2003 from Columbia University Center for Children’s Environmental Health found that among 60 pregnant women tested, 100% had measurable urinary phthalate levels despite being sampled from different areas of both New York City (n = 30) and Krakow, Poland (n = 30).28 Concurrent research from the Harvard School of Public Health found a dose–response relationship between urine levels of phthalate metabolites and a decrease in sperm motility and concentration in a cohort of 168 infertile men.29 Additional studies demonstrated similar results.26,30 A conflicting study from Sweden, however, found no change in semen quality in 234 young men recruited at the time of their medical exam for entry into the military based on urine phthalate levels.31 Russ Hauser, a Harvard researcher, brings to light multiple methodological and analytical differences between these studies in a review article that calls into question the

validity of the Swedish study and comparability of the study results.30 Most notable are the study population differences: the Swedish study recruited young men, and the American studies had older infertile patients. Whereas not conclusive, the data suggest phthalates play a role in decreased sperm quality and possibly fertility. Conflicting data also exist for many other potential reproductive toxicants. The trihalomethanes are a group of chemical by-products of the chlorination processes used to disinfect drinking water. They have been a source of investigation for infertility and birth defects. One THM, bromodichloromethane, has been associated with low sperm counts and increased abnormal semen morphology,32 but the majority of studies in both rats and humans have not found conclusive evidence that trihalomethanes decrease sperm quality or quantity.33 Additional investigations have questioned a relationship between THM exposure and spontaneous abortion.34,35 In 2000, an international workshop gathered to assess the impact of disinfectant by-products on reproduction and concluded that more research on methods of exposure assessment needed to be done in order to properly evaluate exposure risk.36 Since that time, a well-documented case-control study of over 2400 pregnancies in North Carolina did not find an association between THMs and spontaneous abortion.37 The study enrolled patients at 7 weeks’ gestation or less, sampled weekly drinking water from three distinct THM-profile regions, and specifically analyzed exposure during critical periods of fetal development. Another chemical that causes concern is the pesticide dichlorodiphenyldichloroethylene (DDE), a persistent remnant of DDT, which, although no longer being produced in the USA, is sporadically used in Mexico,27 India, and China.38 Despite initially using pilot data, two recent studies were unable to demonstrate an association between sperm quality and DDE in both infertile US patients (n = 12) and older Swedish fishermen (n = 195).39,40 At this time, the evidence suggesting a risk of DDE to male fertility at casual exposure levels is still being evaluated. Polychlorinated biphenyls, much like DDT, have been banned in the USA since the late 1970s. PCBs are synthetic, persistent, halogenated, lipophilic substances that are still ubiquitous in our environment today. They have varying hormonal functions: some act as weak estrogens and some are antiestrogenic. PCBs were used as early as 1881 in cutting or thinning oils, as lubricants, and as electrical insulators. In the 1930s they were reported to cause ‘chloracne’ and even death from liver failure in occupational exposures.41 The Hudson River is perhaps the most famous site of PCB contamination from over 30 years of General Electric dumping PCBs until successfully sued to stop in 1975. We encounter PCBs primarily through the diet, but they can enter our systems by dermal contact (household dust) and inhalation. The half-life in some cases is >10 years; hence, their existence today.42

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The connection between seminal plasma PCB levels and sperm quality was first shown in 1986.43 It became clear in 2002 that PCBs were not the guilty molecules, but it was their active metabolites that were responsible for gamete abnormalities.44 Several environmental exposure studies show a consistent decrease in sperm quality in relation to seminal plasma PCB metabolite levels across different age groups: 18–21 year olds,45 30-year-old infertile couples,39 and 50-year-old fishermen.40 Russ Hauser at the Harvard School of Public Health has spent over 6 years studying PCBs and their effect on male factor infertility. He makes a strong case that the epidemiological data support an inverse association of PCBs with reduced semen quality, specifically reduced sperm motility. The associations found are generally consistent across studies, despite a range of PCB levels, methods of measuring PCB levels, and methods of measuring semen quality.30 Non-persistent pesticides or ‘contemporary-use’ pesticides are those that are currently in use for killing insects, weeds, and other pests. While non-persistent in the environment, heavy use of pest control in the developed world means that most people receive at least some exposure to low levels of these chemicals. Several epidemiological studies on occupational exposure to contemporary-use pesticides have been reported. In one cross-sectional study, greenhouse workers (n = 122) exposed to over a dozen pesticides were stratified into low, medium, or high exposure. The highest exposure group showed a higher proportion of abnormal sperm and lower median sperm counts in workers with more than 10 years of experience compared to those with less than 5 years.46 The study was appropriately adjusted for sexual abstinence and other potential cofounders. Juhler et al investigated dietary exposure to pesticides and semen quality in a cross-sectional study of organic farmers compared to traditional farmers.47 Through food frequency questionnaires and pesticide monitoring programs they found that men with a lower intake of organic food had lower proportions of normally shaped sperm using strict criteria after controlling for various confounders (2.5 vs 3.7%; p = 0.003). However, there were no differences between groups in 14 other semen parameters. Oliva et al had similar results in Argentina,48 but Larsen et al did not find significant differences in sperm quality between Danish farmers who sprayed pesticides and those who did not.49 Unfortunately for the sake of clarity, none of these studies looked at individual pesticide exposure, only exposure in general. This lack of specificity indicated the everpresent need for more controlled investigations that can link measurable quantities of these newer compounds to sperm quality and ultimately fertility. Several studies have specifically investigated exposure to organophosphate pesticides50,51 and found similar results to the broad cross-sectional studies mentioned previously. Whorton et al52 studied workers who packaged carbaryl (a common insecticide marketed under the

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name Sevin since 1958) and found an increased incidence of oligozoospermia (<20 million sperm/ml) compared with a reference group of chemical workers. Fifteen percent of exposed workers had sperm concentrations below the reference value of 20 million sperm/ml compared with 5.5% of nonexposed controls (p = 0.07). Wyrobek et al53 reported an association between carbaryl exposure and sperm morphology soon thereafter. The distribution of abnormal sperm morphology was significantly higher for exposed workers (p <0.005), and the proportion of teratospermic men (>60% abnormal) was larger in the exposed group (29%; n = 50) compared with controls (12%, n = 34; p = 0.06). More recently, Meeker et al54 found an inverse relationship between sperm concentration and motility in 272 men recruited from infertile couples and urinary levels of 1-naphthol, a metabolite of both carbaryl and naphthalene. They suggested that ‘an interquartile range increase in carbaryl metabolite levels in urine is associated with a 4% decrease in sperm motility,’ and may result in a significant increase in the number of subfertile men across the US population. In summary, there are human data supporting the association between contemporary-use pesticides and decreased semen quality, but the public health implications are yet to be determined. The estrogenic monomer bisphenol A (BPA) is used in the manufacture of polycarbonate plastic products, in resins lining metal cans, in dental sealants, and in blends with other types of plastic products. Typical products include polyvinyl chloride (PVC), medical tubing, water pipes, soda bottles, and baby bottles (see Table 57.1). Over time, the ester bonds linking BPA molecules in polycarbonate and resins undergo hydrolysis, resulting in the release of free BPA into food, beverages, and the environment. This hydrolysis is accelerated by heat or contact with acidic and basic substances, such that repeated washing or contact with substances of different acidity leads to increased leaching of BPA from polycarbonate. BPA levels have been found in rivers and streams, drinking water, indoor air, and leaching out of landfills.55 Numerous monitoring studies now show almost ubiquitous human exposure to biologically active levels of this chemical.56 The Food and Drug Administration (FDA) and Environmental Protection Agency (EPA) currently consider daily exposure of BPAs of <50 µg/kg to be safe based on megadose studies in which the lowest tested dose was 1000-fold higher. Now, over 40 studies have been published, reporting significant effects in rats and mice at doses <50 µg/kg.57 Previous ‘safe levels’ of exposure are now under scrutiny as older studies are being recognized as limited because of assay sensitivity. Moreover, it is difficult, but not impossible, to conduct laboratory experiments and avoid contamination from polycarbonate lab plastics. Mechanistically, BPAs exert estrogenic effects through the classic nuclear estrogen receptor, by acting as selective estrogen receptor modulators, and by initiating rapid responses via estrogen receptors presumably

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18

8.3

BPA concentration (ng/ml)

16 14 12 10 8 6 4

2

2.2 1.5

1.4

2.4

2

1.1

0

Fo llic flu ular n = id 36 A flu mn i d : io we 15 tic – n eks 18 =3 2 flu Amn id: io t n at te ic = 38 rm

Ma (no tern n-p al s e n = regn rum 3 0 a n t) M (e ater ar ly nal pr se n = egn rum 37 anc y) Ma (la ter te na pr l se n = egna rum 37 ncy ) F (u eta mb l s ili er n = cal c um 32 ord )

−2

associated with the plasma membrane.55 BPAs bind very little to sex hormone-binding protein and thus have an unconjugated free fraction of 8% that can be delivered to cells more easily than estradiol, which has a free fraction of 3.5%.58 Additionally, pregnant women have a significantly higher affinity for BPAs than nonpregnant women.59 Indeed, BPAs have been detected in fetal cord serum, maternal serum during pregnancy, and amniotic fluid.60 For unclear reasons, the level of BPAs found in the amniotic fluid of 15–18-week gestations is five times higher than serum levels, but returns to a concentration similar to fetal and maternal serum in the third trimester (Fig 57.2). Although the metabolism of BPAs is not completely understood, these findings can be explained by the development of fetal capacity to metabolize BPAs in the late second trimester, possibly by the liver. Metabolism research, like epidemiological studies, is just beginning to be published to accompany a large body of animal research already accessible. Whereas BPAs are newer molecules, heavy metals have long been implicated in impairing fertility. The most frequently studied metals are lead, and mercury. Physicians have recognized lead as a reproductive toxicant for well over a century. Lead salts, in fact, were once used as abortificants. The effects on reproduction were well summarized in 1944: It is generally agreed that if pregnancy does occur it is frequently characterized by miscarriage, intrauterine death of the fetus, premature birth and, if living children are born, they are usually smaller, weaker, slower in development, and have a higher infant mortality.61 Beyond broad generalizations, however, there is little evidence supporting the claim that lead affects fertility per se. Animal studies have shown altered spermatogenesis at 35 µg/dl (the CDC-cited safe level is <10 µg/dl), and a few case reports have observed similar findings in

Fig 57.2 Bisphenol A concentrations in human biological fluids. Columns represent mean bisphenol A (BPA) values, and ‘whiskers’ give the 95% confidence intervals of the values. The amniotic fluid at 15–18 weeks was significantly elevated (p <0.0001) compared with other biological fluids. (Reproduced from Tsutsumi,60a with permission.)

humans with levels over 40 µg/dl.62 It has been demonstrated that lead crosses into the seminal fluid, but in general, studies focused on male fertility and lead exposure are lacking. In Denmark, a prospective cohort of workers in a battery manufacturing plant had average serum lead levels of 35.9 µg/dl, but no decrease in the birth rate (odds ratio [OR] = 0.98, 95% confidence interval [CI] 0.88–1.12).63 A study of welders in Canada demonstrated a decrease in sperm quality, but did not correlate those findings with decreased fertility.64 Likewise, there is scant evidence of increased spontaneous abortion rates or increased time to pregnancy (TTP). No doubt, more information on the impact of lead on reproduction will be forthcoming as newer techniques of investigation are developed.

The follicle microenvironment One by-product of in vitro fertilization (IVF) has been access to follicular fluid for studies demonstrating the presence of toxicants.65–68 The pesticides DDE, mirex, hexachloroethane, and 1,2,4-trichlorobenzene, along with PCBs, BPA, and phthalates, have been implicated in infertility, but have not consistently demonstrated adverse IVF or pregnancy outcomes. Variables examined include number of oocytes retrieved, recovered, and fertilized, cleavage rates, and pregnancy rates. In a Canadian study of 21 IVF couples, higher DDE levels correlated with failed fertilization, but higher follicular PCB levels correlated with pregnancy success.65 A study of IVF patients in 1984 showed that oocyte recovery and embryo cleavage rates were inversely related to chlorinated hydrocarbon concentrations,68 although a subsequent study showed a positive relationship.67 Most remarkable, however, is the fact that in all of these studies, pesticides are present in follicular fluid at the time of resumption of meiosis when chromosome susceptibility is at its highest.

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For the most part, follicular toxicant concentrations are lower than serum levels.59,65 Knowing the relationship between serum and follicle concentrations has allowed speculation on the fertility outcomes of nonIVF patients based on serum levels. Law et al pulled frozen third-trimester blood samples from 380 planned pregnancies recruited for the 1959–1965 Collaborate Perinatal Project and compared serum levels of PCBs and DDE with TTP and fecundability. Dose–response curves with proportional hazards suggested that as PCB and DDE levels increase, the probability of pregnancy decreases. Since DDE and PCBs are lipophilic, the serum levels obtained in this study were adjusted for maternal lipid volume (an appropriate adjustment not done in most published reports). Once adjusted, the increased TTP attributed to DDE disappeared and the PCB effect became considerably weaker, leaving no significant difference in TTP or fecundability based on either substance’s concentration.69 These results echo the findings of a cohort of Swedish fishermen from which multiple papers have been published showing no relationship between fish consumption (including persistent organochlorine and PCB exposure) and TTP, miscarriage rate, stillbirths, or subfertility.70 In the end, there is little evidence to support the association between DDE, PCBs, and subfertility. Once again, however, the presence of such toxic substances bathing the preovulatory oocyte is worrisome given the protective barrier the reproductive organs have to passage of most substances. More studies will be required to understand if there is any adverse impact of these substances on oocyte DNA. Just as paternal lead and mercury exposure is widely thought to impair fertility, so is maternal exposure. Lead has been shown to destroy oocytes and lead to follicular atresia in rodents and primates,71 but has only been measured in follicular fluid in two published human studies available on MEDLINE,72,73 while mercury measurement has not been reported at all. Various rodent and nonhuman primate studies have demonstrated suppression of menarche, decreased circulating progesterone levels, and less frequent menstrual cycles with lead exposure.62 Nevertheless, examination of human epidemiological data is less conclusive. First, the mechanism of lead’s toxicity is not well understood: it is unclear if there is a direct toxic effect on the ovary, the effect is mediated through a central neuroendocrine dysfunction, or both. Secondly, older studies demonstrate an association between high-dose occupational exposure and spontaneous fetal loss,74 but a more recent study was unable to find an association between the two when 304 women living near a lead smelter in Yugoslavia were compared with 335 women from a nearby town with low serum lead levels.75 As mentioned, mercury levels have not been reported in follicular fluid, but mercury’s effect on the developing fetus in utero has been subject to a great deal of scrutiny.

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The amniotic sac microenvironment Over a decade ago, the endocrinologist Howard Bern of UC Berkeley in California coined the phrase the ‘fragile fetus’ while explaining the vulnerability of the developing fetus in utero to insult and exposure. This phrase has proven true when it comes to lead and mercury exposure. Mercury is a common atmospheric element that is released from the earth’s crust. Inorganic mercury gets converted to soluble forms that are deposited into soil and water by precipitation. Sources such as coal mining ‘rain down’ mercury across the earth, depositing it on both land and sea. These soluble mercury forms are then methylated via microbes or non-enzymatic processes, and readily taken up by proteins. Methylmercury then rapidly accumulates in the food chain through predatory fish. Humans, at the top of the food chain, acquire mercury through food consumption as well. The greatest concern with mercury is not fertility – there is little evidence correlating mercury poisoning and infertility – but the developmental effects of in utero exposure. In theory, the fetus is particularly vulnerable to mercury.76 In adults, methylmercury can be converted to inorganic mercury by intestinal flora and 90% eventually removed from our system through the feces. The fetus, with neither gut flora, a fully functional liver, nor the ability to defecate, is virtually guaranteed to rapidly accumulate this heavy metal. We must also consider the process of urination: in utero urine is cycled from the amniotic fluid into the developing fetus’ nose and mouth, and back into the amniotic fluid, unlike in adults, where urination is an essential mechanism for clearing toxicants from the body. Couple higher toxicant levels with deficient excretion mechanisms and there is the potential for alarming toxicant accumulation. Moreover, because levels of circulating binding proteins are lower in the fetus, there is a higher concentration of circulating unbound toxicants. Lastly, the blood–brain barrier is more permeable during development and thus the developing brain proportionally receives greater exposure to toxicants than the adult brain. In theory, this can increase the vulnerability of the fetus to neurotoxins such as mercury. We have known for decades that lead and mercury pass through the placenta, into the amniotic fluid, and directly to the baby in concentrations near maternal blood serum. Umbilical cord blood levels of lead are usually only 10–20% lower than maternal serum levels,77 but mercury (methylmercury) levels are generally higher than maternal serum levels.62 Mercury has been the most publicized environmental toxicant in relation to reproductive health, and explains the FDA’s recommended limitations on fish consumption in pregnancy. Current FDA recommendations are for women of childbearing age to avoid fish containing >1 µg/g, including swordfish, shark, tilefish, and king mackerel. Fortunately, many US sportfish have levels lower than this (Fig 57.3). In Massachusetts, though, there are no bodies of water that have safe levels of mercury for fish consumption by women of childbearing age. A statement to this effect was

Fig 57.3

0.3

0.4

Average tissue mercury concentrations in noncommercial fish.

National Fish and Wildlife Contamination Program

0.2

0.5

0.7

0.8

Mercury Concentration (ppm)

0.6

0.9

1

11

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* Noncommercial Fish: Fish caught and consumed by family and friends. Data source: US EPA National Listing of Fish and Wildlife Advisory (NLFWA) fish tissue database, October 2003. The NLFWA database represents samples collected and analyzed by state and tribal advisory programs, over the period 1987–2003. Note: These results do not represent a statistical average, as state sampling is generally oriented towards areas of known or expected sport/subs istence fishing activity. Calculation Method: Average values presented here are calculated as the arithmetic mean of all sampling station means for species with at least 100 sampling stations of data. Means calculated using fillet samples only for adult fish (all lengths and weights) as adult fillets are the sample types most relevant for human health risk assessment. Caution: Mercury concentrations in fish vary considerably from region to region and waterbody to waterbody. Consumers should, first and foremost, consider any local advisories.

Species

780

No Sampling Average Stations Conc (ppm) 241 0.06 English sole 0.09 151 Gizzard shad 0.10 130 Black bullhead 0.11 119 Rainbow trout 0.11 714 White sucker 0.13 214 Brown bullhead 0.13 Pumpkinseed sunfish 107 0.14 737 Common carp 426 0.14 Carp 1,062 0.15 Bluegill sunfish 131 0.16 Brown trout 116 0.18 Rainbow smelt 1,213 0.18 Channel catfish 376 0.19 Rock bass 652 0.19 Black crappie 352 0.19 White crappie 212 0.21 White bass 226 0.22 Freshwater drum 133 0.22 White perch 604 0.22 Yellow perch 215 0.26 Redear sunfish 146 0.27 Striped bass 185 0.27 Yellow bullhead 738 0.27 Smallmouth bass 109 0.28 Sauger 160 0.30 Lake trout 1,322 0.35 Northern pike 163 0.36 Spotted bass 158 0.37 Flathead catfish 147 0.39 Warmouth sunfish 1,520 0.40 Walleye 2,425 0.43 Largemouth bass 250 0.61 Chain pickerel 358 0.96 Bowfin

Average Tissue Mercury Concentrations in Noncommercial Fish*

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issued by the Massachusetts Department of Public Health in 2004. This was an extension on an advisory from 1994 and 2001: Previously issued statewide fish consumption advisory which cautioned pregnant women to avoid eating fish from all freshwater bodies due to concerns about mercury contamination … now include women of childbearing age who may become pregnant, nursing mothers and children under 12 years of age.78 Epidemiological studies show that an increase of only 1 ppm of mercury lowers the average cognitive score of a child,79 while high-dose exposure can lead to neonatal central nervous system (CNS) damage and even death. Minamata disease, mercury poisoning caused by dumping in Japan’s Bay of Minamata, is an example of the potential effects of high-dose exposure.80 However, fish are an important source of omega-3 fatty acids, which are essential for fetal neurodevelopment, and there is some evidence that higher fish consumption is correlative with greater cognitive development. A cohort study of 8947 women in England found that women who consumed over 340 g (12 oz) of fish per week during pregnancy had children with significantly greater outcomes on 14 of 23 neurodevelopmental measures, including various fine-motor, communication, social development milestones, and verbal IQ at 8 years of age.81 Unfortunately, recommendations about limiting fish consumption in pregnancy may overestimate the risk of chronic low-dose mercury intake and underestimate the benefit of omega-3 fatty acids. The challenge is to find the correct balance. Two important prospective observational studies, the Seychelles Child Development Study 82 (n = 779) and the Faroe Islands Cohort Study83 (n = 878), followed fish consumption and cognitive function for 9 and 14 years, respectively, and report conflicting results. The Seychelles study has not shown an association between in utero methylmercury levels and neurocognitive function, while the Faroe Islands Cohort has consistently shown a negative correlation after correcting for postnatal exposure. In response to these two studies, the World Health Organization (WHO) published a new maternal serum cut-off level of 5.6 µg/dl, and a tolerable weekly mercury intake of 1.6 µg/kg to ‘protect the developing fetus and embryo, the most sensitive subgroup of the population.’80 A second way of evaluating fetal mercury exposure is by cord blood mercury levels. The US National Research Council has issued concern for cord blood levels >58 ppm, or (5.8 µg/l).84 Large fish-consuming populations have been shown to have mercury levels average high above this. In Taiwan, 65 pregnant women filled out a questionnaire in the third trimester and gave blood samples, placenta tissue, and cord blood after

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delivery. Eighty-nine percent of the maternal blood mercury concentrations exceeded the US National Research Council recommended value. Levels were highest in women who ate fish more than three times a week while pregnant.85 In Hawaii, a study of 308 newborns showed a mean cord blood concentration of 4.8 µg/l, with 28% above the recommended safety value.86 While it is clear that fish consumption is correlated with both maternal and fetal mercury levels, the developmental significance is still being evaluated. Other amniotic sac microenvironment toxicants have been implicated in affecting fertility. Guo et al studied one of the only prospective cohorts of highdose PCB exposure when contaminated cooking oil was used in Taiwan in 1979.87 In 1998 they contacted children exposed to PCBs in utero and performed sperm analysis. They found abnormal sperm motility, morphology, and decreased ability to penetrate hamster eggs. Fertility rates have yet to be reported. The translation of subfertility across generations is one of the most interesting and concerning concepts to arise out of reproductive environmental health research. Belgian investigators failed to show a difference in serum concentration of the pesticide by-product DDE in fertile men (n = 73) and infertile men (n = 82), but a subanalysis of the blood of mothers of the patients demonstrated higher serum pesticide levels in mothers of subfertile men (n = 19) than in those of fertile men (n = 23).88 While these results can only offer hypotheses on the role of exposure to pesticides in utero and fertility, they do provide reason for caution toward potential toxicants and increase the need to verify their potential risks.

The global environment The global environment is the most difficult to deconstruct. In truth, the collection of studies presented here probably reflects alteration in the functionality of semen or ovarian follicles. There is, however, the potential for environmental agents to affect the systems that support pregnancy. For example, environmental estrogens may change the hormonal balance that allows sufficient endometrial growth, affect angiogenesis necessary to support a developing placenta, or cause/worsen endometriosis and tubal patency. These details are sure to be elucidated in the near future. Nevertheless, regional differences in fertility rates highlight the potential effect of the global environment on fertility.89 A study that falls into this category of the ‘global environment’ was published in 1999 by Khattak et al. Their prospective case-controlled trial on the effect of occupational maternal exposure to organic solvents involved pregnant women exposed to solvents and matched by age, gravity, smoking, and alcohol usage to comparable pregnant women exposed to a recognized non-teratogenic agent. In addition to an increased incidence of miscarriage (54/117 [46.2%] vs 24/125

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[19.2%]; p <0.001), the women in the exposure group were 13 times more likely to have children with major cardiovascular or CNS malformations.90 The authors concluded that occupational exposure to organic solvents in pregnancy is associated with increased risk of major fetal malformations. Where this effect takes place is not clear: it could be either the gametes or the amniotic microenvironment or both. Animal research has demonstrated that fetal alterations may continue to impact future generations through persistent epigenetic changes. Genome methylation or histone acylation can alter gene expression without modifying the DNA sequence and can transmit from generation to generation with a higher penetrance than DNA mutations themselves.91 Anway et al illustrated this concept when they observed that rats exposed to vinclozolin (an antiandrogenic compound that is used as a fungicide in wine vineyards) or methoxychlor (an estrogenic compound that has replaced DDT as a pesticide) resulted in increased male infertility in F1 generation rats and persisted in over 90% of all male rats through the next three generations.92 These kind of results in humans have only been hinted at by the association of elevated maternal serum levels of DDE with grown children’s fertility rates,88 and the transgenerational effect of DES and vaginal cancer.93 Even more interesting (and complicating) are reports that environmental agents thought to be toxic may actually enhance reproduction. Higher follicular fluid levels of PCBs have been associated with better IVF outcomes,65 higher DDE levels associated with reduced TTP,94 and in vitro work has shown that DDE stimulates the aromatase enzyme system of granulosa cells in synergy with follicular-stimulating hormone (FSH)95 and thus may speed follicle maturation. Additionally, in Denmark, 192 IVF couples with paternal exposure to pesticides, fungicides, and herbicides had a 21% spontaneous abortion rate compared with 28% in the reference population of 2925.96 How these findings will ultimately be interpreted is not presently clear.

Clinical considerations It is relatively easy to accept research indicating the presence of environmental toxicants in the semen, the oocyte, and the amniotic fluid. Translation of the epidemiological and observational research to the bedside is challenging. Clinically, how do we counsel our patients? We agree that providers should be cautious, not alarming, given that the literature is suggestive, not conclusive, of a link between EDCs and decreased reproductive performance.97 One way to incorporate knowledge about environmental exposures is to include questions during history-taking about exposure to solvents, pesticides, or heavy metals. Ask patients where they live and where they have lived, where they work, what they eat, how much fish they consume, and what exposures their parents may have

had to plastics, heavy metals, pesticides, and industrial solvents. Several formal exposure evaluations have been published for this purpose.98,99 The knowledge gained from history-taking can be used to advise patients according to the precautionary principle:100 educate patients on potential toxic exposures, and let them make efforts to avoid them. The data gathered can also become the basis for observational research opportunities to understand better which substances are most deleterious, and at what levels. The role of toxin decontamination is neither well studied nor reported, but certainly is not new. Hippocrates wrote of solariums; religious groups recommend fasting; Aborigines wrote of sweat lodges and hot baths; Egyptians applied body wraps; and some Scandinavian cultures utilized saunas and steam baths.21 None of these decontamination methods have been studied and reported upon in the scientific literature, despite the many services advertised in every community in the world. In the end, the scientific communities at large remain unimpressed with the impact of EDCs. The National Academy of Science report on ‘Hormonally Active Agents in the Environment,’101 was unable to come to a consensus opinion on the topic; the WHO stateof-the-science assessment in 2002102 concluded that organochlorines (PCBs, DDE) do affect pregnancy in wildlife, but was uncertain about the effect on humans. Congress mandated the EPA, through the advisory of the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC), to expand its current mandate to test all food-use pesticides and drinking water contaminants for hormonal activity. This is to include evaluation of the 80 000+ registered chemicals under the toxic substances control act of 1998 – a daunting task that requires prioritization. Currently, the EPA is reporting on the first 200 of these chemicals. There has been much progress and expansion in investigating the role that different compounds play in our reproductive health. There is much more work needed, however, to draw any definitive conclusions. We must ask ourselves: Why are we allowing an ‘innocent until proven guilty’ approach for chemical agents dispersed into our environment, when we insist the opposite for all pharmaceuticals?21

References 1. Trubo R. Endocrine-disrupting chemicals probed as potential pathways to illness. JAMA 2005; 294: 291–3. 2. Nonaka K, Miura T, Peter K. Recent fertility decline in Dariusleut Hutterites: an extension of Eaton and Mayer’s Hutterite fertility study. Hum Biol 1994; 66: 411–20. 3. Ventura SJ, Mosher WD, Curtin SC, Abma JC, Henshaw S. Trends in pregnancies and pregnancy rates by outcome: estimates for the United States, 1976–96. Vital Health Stat 2000; Jan: 1–47.

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21. Genuis SJ. Health issues and the environment – an emerging paradigm for providers of obstetrical and gynaecological health care. Hum Reprod 2006; 21: 2201–8. 22. Smith GC, Pell JP. Parachute use to prevent death and major trauma related to gravitational challenge: systematic review of randomised controlled trials. BMJ 2003; 327: 1459–61. 23. Niwa T, Tsutsui M, Kishimoto K, et al. Inhibition of drug-metabolizing enzyme activity in human hepatic cytochrome P450s by bisphenol A. Biol Pharm Bull 2000; 23: 498–501. 24. Niwa T, Fujimoto M, Kishimoto K, et al. Metabolism and interaction of bisphenol A in human hepatic cytochrome P450 and steroidogenic CYP17. Biol Pharm Bull 2001; 24: 1064–7. 25. Welshons WV, Thayer KA, Judy BM, et al. Large effects from small exposures. I. Mechanisms for endocrine-disrupting chemicals with estrogenic activity. Environ Health Perspect 2003; 111: 994–1006. 26. Hauser R, Williams P, Altshul L, Calafat AM. Evidence of interaction between polychlorinated biphenyls and phthalates in relation to human sperm motility. Environ Health Perspect 2005; 113: 425–30. 27. Younglai EV, Holloway AC, Foster WG. Environmental and occupational factors affecting fertility and IVF success. Hum Reprod Update 2005; 11: 43–57. 28. Adibi JJ, Perera FP, Jedrychowski W, et al. Prenatal exposures to phthalates among women in New York City and Krakow, Poland. Environ Health Perspect 2003; 111: 1719–22. 29. Duty SM, Silva MJ, Barr DB, et al. Phthalate exposure and human semen parameters. Epidemiology 2003; 14: 269–77. 30. Hauser R. The environment and male fertility: recent research on emerging chemicals and semen quality. Semin Reprod Med 2006; 24: 156–67. 31. Jonsson BA, Richthoff J, Rylander L, Giwercman A, Hagmar L. Urinary phthalate metabolites and biomarkers of reproductive function in young men. Epidemiology 2005; 16: 487–93. 32. Fenster L, Waller K, Windham G, et al. Trihalomethane levels in home tap water and semen quality. Epidemiology 2003; 14: 650–8. 33. Shaw GM, Ranatunga D, Quach T, et al. Trihalomethane exposures from municipal water supplies and selected congenital malformations. Epidemiology 2003; 14: 191–9. 34. Waller K, Swan SH, DeLorenze G, Hopkins B. Trihalomethanes in drinking water and spontaneous abortion. Epidemiology 1998; 9: 134–40. 35. Waller K, Swan SH, Windham GC, Fenster L. Influence of exposure assessment methods on risk estimates in an epidemiologic study of total trihalomethane exposure and spontaneous abortion. J Expo Anal Environ Epidemiol 2001; 11: 522–31. 36. Arbuckle TE, Hrudey SE, Krasner SW, et al. Assessing exposure in epidemiologic studies to disinfection by-products in drinking water: report from an international workshop. Environ Health Perspect 2002; 110 (Suppl 1): 53–60.

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37. Savitz DA, Singer PC, Herring AH, et al. Exposure to drinking water disinfection by-products and pregnancy loss. Am J Epidemiol 2006; 164: 1043–51. 38. Garcia AM. Pesticide exposure and women’s health. Am J Ind Med 2003; 44: 584–94. 39. Hauser R, Chen Z, Pothier L, Ryan L, Altshul L. The relationship between human semen parameters and environmental exposure to polychlorinated biphenyls and p,p′-DDE. Environ Health Perspect 2003; 111: 1505–11. 40. Rignell-Hydbom A, Rylander L, Giwercman A, et al. Exposure to CB-153 and p,p′-DDE and male reproductive function. Hum Reprod 2004; 19: 2066–75. 41. Schroeder M. Did Westinghouse keep mum on PCBs? Business Week 1991. 42. Phillips DL, Smith AB, Burse VW, et al. Half-life of polychlorinated biphenyls in occupationally exposed workers. Arch Environ Health 1989; 44: 351–4. 43. Bush B, Bennett AH, Snow JT. Polychlorobiphenyl congeners, p,p’-DDE, and sperm function in humans. Arch Environ Contam and Toxicol 1986; 15: 333–41. 44. Dallinga JW, Moonen EJ, Dumoulin JC, et al. Decreased human semen quality and organochlorine compounds in blood. Hum Reprod 2002; 17: 1973–9. 45. Richthoff J, Rylander L, Jonsson BA, et al. Serum levels of 2,2′,4,4′,5,5′-hexachlorobiphenyl (CB-153) in relation to markers of reproductive function in young males from the general Swedish population. Environ Health Perspect 2003; 111: 409–13. 46. Abell A, Ernst E, Bonde JP. Semen quality and sexual hormones in greenhouse workers. Scand J Work Environ Health 2000; 26: 492–500. 47. Juhler RK, Larsen SB, Meyer O, et al. Human semen quality in relation to dietary pesticide exposure and organic diet. Arch Environ Contam Toxicol 1999; 37: 415–23. 48. Oliva A, Spira A, Multigner L. Contribution of environmental factors to the risk of male infertility. Hum Reprod 2001; 16: 1768–76. 49. Larsen SB, Joffe M, Bonde JP. Time to pregnancy and exposure to pesticides in Danish farmers. ASCLEPIOS Study Group. Occup Environ Med 1998; 55: 278–83. 50. Padungtod C, Savitz DA, Overstreet JW, et al. Occupational pesticide exposure and semen quality among Chinese workers. J Occup Environ Med 2000; 42: 982–92. 51. Kamijima M, Hibi H, Gotoh M, et al. A survey of semen indices in insecticide sprayers. J Occup Health 2004; 46: 109–18. 52. Whorton MD, Milby TH, Stubbs HA, Avashia BH, Hull EQ. Testicular function among carbarylexposed exployees. J Toxicol Environ Health 1979; 5: 929–41. 53. Wyrobek AJ, Watchmaker G, Gordon L, et al. Sperm shape abnormalities in carbaryl-exposed employees. Environ Health Perspect 1981; 40: 255–65. 54. Meeker JD, Ryan L, Barr DB, et al. The relationship of urinary metabolites of carbaryl/naphthalene and chlorpyrifos with human semen quality. Environ Health Perspect 2004; 112: 1665–70.

55. Welshons WV, Nagel SC, Vom Saal FS. Large effects from small exposures. III. Endocrine mechanisms mediating effects of bisphenol A at levels of human exposure. Endocrinology 2006; 147: S56–69. 56. Calafat AM, Kuklenyik Z, Reidy JA, et al. Urinary concentrations of bisphenol A and 4-nonylphenol in a human reference population. Environ Health Perspect 2005; 113: 391–5. 57. Vom Saal FS, Welshons WV. Large effects from small exposures. II. The importance of positive controls in low-dose research on bisphenol A. Environ Res 2006; 100: 50–76. 58. Nagel SC, Vom Saal FS, Welshons WV. The effective free fraction of estradiol and xenoestrogens in human serum measured by whole cell uptake assays: physiology of delivery modifies estrogenic activity. Proc Soc Exp Biol Med 1998; 217: 300–9. 59. Ikezuki Y, Tsutsumi O, Takai Y, Kamei Y, Taketani Y. Determination of bisphenol A concentrations in human biological fluids reveals significant early prenatal exposure. Hum Reprod 2002; 17: 2839–41. 60. Schonfelder G, Wittfoht W, Hopp H, et al. Parent bisphenol A accumulation in the human maternal– fetal–placental unit. Environ Health Perspect 2002; 110: A703–7. 60a. Tsutsumi O. Assessment of human contamination of estrogenic endocrine-disrupting chemicals and their risk for human reproduction. J Steroid Biochem Mol Biol 2005; 93: 325–30. 61. Cantarow A, Trumper M. Lead Poisoning. Baltimore: Williams & Wilkins, 1944. 62. Miller R, Bellinger D. Metals. In: Paul M, ed. Occupational and Environmental Reproductive Hazards: A Guide for Clinicians. Baltimore: Williams & Wilkins, 1993. 63. Bonde JP, Kolstad H. Fertility of Danish battery workers exposed to lead. Int J Epidemiol 1997; 26: 1281–8. 64. Bigelow PL, Jarrell J, Young MR, Keefe TJ, Love EJ. Association of semen quality and occupational factors: comparison of case-control analysis and analysis of continuous variables. Fertil Steril 1998; 69: 11–18. 65. Younglai EV, Foster WG, Hughes EG, Trim K, Jarrell JF. Levels of environmental contaminants in human follicular fluid, serum, and seminal plasma of couples undergoing in vitro fertilization. Arch Environ Contam Toxicol 2002; 43: 121–6. 66. Foster WG, Jarrell JF, Younglai EV, et al. An overview of some reproductive toxicology studies conducted at Health Canada. Toxicol Ind Health 1996; 12: 447–59. 67. Jarrell J, Villeneuve D, Franklin C, et al. Contamination of human ovarian follicular fluid and serum by chlorinated organic compounds in three Canadian cities. CMAJ 1993; 148: 1321–7. 68. Trapp M, Baukloh V, Bohnet HG, Heeschen W. Pollutants in human follicular fluid. Fertil Steril 1984; 42: 146–8. 69. Law DC, Klebanoff MA, Brock JW, Dunson DB, Longnecker MP. Maternal serum levels of polychlorinated biphenyls and 1,1-dichloro-2,2-bis (p-chlorophenyl)ethylene (DDE) and time to pregnancy. Am J Epidemiol 2005; 162: 523–32.

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86. Sato RL, Li GG, Shaha S. Antepartum seafood consumption and mercury levels in newborn cord blood. Am J Obstet Gynecol 2006; 194: 1683–8. 87. Guo YL, Hsu PC, Hsu CC, Lambert GH. Semen quality after prenatal exposure to polychlorinated biphenyls and dibenzofurans. Lancet 2000; 356: 1240–1. 88. Charlier CJ, Foidart JM. Comparative study of dichlorodiphenyldichloroethylene in blood and semen of two young male populations: lack of relationship to infertility, but evidence of high exposure of the mothers. Reprod Toxicol 2005; 20: 215–20. 89. Carpenter DO, Shen Y, Nguyen T, Le L, Lininger LL. Incidence of endocrine disease among residents of New York areas of concern. Environ Health Perspect 2001; 109 (Suppl 6): 845–51. 90. Khattak S, K-Moghtader G, McMartin K, et al. Pregnancy outcome following gestational exposure to organic solvents: a prospective controlled study. JAMA 1999; 281: 1106–9. 91. Nakao M. Epigenetics: interaction of DNA methylation and chromatin. Gene 2001; 278: 25–31. 92. Anway MD, Cupp AS, Uzumcu M, Skinner MK. Epigenetic transgenerational actions of endocrine disruptors and male fertility. Science 2005; 308: 1466–9. 93. Crews D, McLachlan JA. Epigenetics, evolution, endocrine disruption, health, and disease. Endocrinology 2006; 147: S4–10. 94. Cohn BA, Cirillo PM, Wolff MS, et al. DDT and DDE exposure in mothers and time to pregnancy in daughters. Lancet 2003; 361: 2205–6. 95. Younglai EV, Holloway AC, Lim GE, Foster WG. Synergistic effects between FSH and 1,1-dichloro2,2-bis(P-chlorophenyl)ethylene (P,P′-DDE) on human granulosa cell aromatase activity. Hum Reprod 2004; 19: 1089–93. 96. Hjollund NH, Bonde JP, Ernst E, et al. Pesticide exposure in male farmers and survival of in vitro fertilized pregnancies. Hum Reprod 2004; 19: 1331–7. 97. Giudice LC. Infertility and the environment: the medical context. Semin Reprod Med 2006; 24: 129–33. 98. Miller CS, Prihoda TJ. The Environmental Exposure and Sensitivity Inventory (EESI): a standardized approach for measuring chemical intolerances for research and clinical applications. Toxicol Ind Health 1999; 15: 370–85. 99. Rea WJ. Chemical Sensitivity: Tools of Diagnosis and Methods of Treatment, Vol 4. Boca Raton, FL: Lewis Publishers, 1992. 100. Cranor CF. Toward understanding aspects of the precautionary principle. J Med Philos 2004; 29: 259–79. 101. NRC. National Research Council: Hormonally Active Agents in the Environment. Washington, DC: National Academy Press, 1999. 102. IPCS. International Programme on Chemical Safety. In: Damstra T, Barlow S, Bergman A, Kavlock R, van der Kraak G, eds. Global Assessment of the State-of-theScience of Endocrine Disruptors. Geneva, Switzerland: World Health Organization, 2002.

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58 Bleeding, severe pelvic infection, and ectopic pregnancy Raoul Orvieto, Zion Ben-Rafael

Transvaginal ultrasound-guided aspiration of oocytes is a well-accepted and universally used method in assisted reproduction.1,2 Its major advantages include easy access to ovarian follicles, with excellent oocyte yield, and good visualization of the major pelvic vessels. It is perfomed as a day-care procedure, either under intravenous analgesia and sedation or under general anesthesia, and is usually atraumatic. Nevertheless, there are some inherent risks: namely, puncture of blood vessels and hemoperitoneum, bleeding from the vaginal-vault puncture site, rupture of adnexal cystic masses, bowel perforation, trauma to pelvic organs, and pelvic infection. In addition, embryo transfer (ET) itself may be associated with complications such as pelvic infection, multiple pregnancy (which is directly related to the number of transferred embryos), spontaneous abortion, and extrauterine pregnancy (EUP). The aim of the present review is to discuss comprehensively three of these complications: bleeding, pelvic inflammatory disease (PID), and EUP.

Bleeding Vaginal bleeding During ultrasound-guided transvaginal oocyte aspiration, multiple punctures of the vaginal vault, or inappropriate handling and rotation of the ultrasound vaginal probe while inserting an aspiration needle through the vaginal vault, can injure or tear the vaginal mucosa, ovaries, intra-abdominal organs, or blood vessels.1,3–7 Bleeding from the vaginal vault is a common consequence of ovum pick-up (OPU), with a reported incidence of 1.4–18.4%.4 In most cases, vaginal bleeding as a result of OPU stops spontaneously at the end of the procedure.5 In cases in which it does not, the bleeding site needs to be identified by vaginal exploration with a large speculum, followed by application of pressure with a sponge forceps or vaginal packing with a large gauze roll. If this is unsuccessful, or the tear is wide and deep, suturing is necessary.

Intraperitoneal or retroperitoneal bleeding Transvaginal oocyte aspiration can also cause bleeding if intraperitoneal or retroperitoneal pelvic blood vessels are injured or if there is damage to the fine vascular network surrounding the punctured ovarian follicle. The reported incidence of severe intra- or retroperitoneal bleeding varies from 0 to 1.3%;1,5–7 a recent report described one case of intra-abdominal bleeding complicating aspiration of 1000 oocyte donors.8 Intraperitoneal bleeding tends to be severe with acute hemodynamic deterioration, whereas retroperitoneal bleeding usually has a later and more indolent presentation. Yih et al9 studied serial complete blood counts before and after OPU in 93 in vitro fertilization (IVF) cycles and demonstrated a nonsignificant change in hematocrit levels, indicating that a clinically significant blood loss after OPU is actually uncommon. Azem et al10 described a patient who presented to the emergency room 10 hours after OPU with severe lower abdominal pain, vomiting, and tenesmus. Examination revealed a distended abdomen with severe tenderness in the pouch of Douglas; on transvaginal sonography, a minimal, 3–4 cm collection of fluid was noted. Laparoscopy followed by laparotomy, which was performed on the basis of the clinical profile, revealed a retroperitoneal hematoma 7 cm in diameter. After evacuation and hemostasis, active bleeding from the midsacral vein occurred and was controlled by a metal clip. This case demonstrates the indolent course of retroperitoneal bleeding and should alert physicians to the possibility of retroperitoneal hematoma despite an absence of free fluid in the pouch of Douglas. It is noteworthy that a similar case with no significant intraperitoneal fluid collection was recently described that resulted from ureteral injury, with the consequent uroretroperitoneum.11 Intra-abdominal bleeding should be suspected immediately after OPU on the development of signs and symptoms of anemia – specifically, weakness, dizziness, dyspnea, or persistent tachycardia. Early management consists of intense hemodynamic monitoring, together with serial measurement of blood hemoglobin

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concentrations and ultrasonographic evaluation for the presence of intra-abdominal fluid. It should be emphasized, however, that intra-abdominal blood clots or retroperitoneal bleeding might be invisible even to an experienced ultrasound operator. A drop in hemoglobin concentration is an indication for prompt blood transfusion. If hemodynamic deterioration continues or acute abdominal pain develops, diagnostic laparoscopy or exploratory laparotomy with subsequent hemostatis of the bleeding site(s) is required. The clinician must make sure to handle the fragile overhyperstimulated ovaries very cautiously. Our group described three cases of severe intraabdominal bleeding from ovarian puncture sites during OPU, leading to acute abdominal complications.1 In two of the patients, symptoms developed 3 hours after OPU (hemoglobin 9.0 g/100 ml and 8.1 g/100 ml), and laparoscopic drainage and hemostatis were sufficient. The third patient became symptomatic after 4 hours (hemoglobin 7.3 g/100 ml) and required exploratory laparotomy and hemostatis in addition to the transfusion of four units of blood as a life-saving procedure. More recently, Battaglia et al12 reported severe intra-abdominal bleeding from the surface of both ovaries in a patient with coagulation factor XI deficiency. As expected, the patient became symptomatic 3 hours after OPU and required laparotomy, partial resection of stuffed ovaries, and hemostasis. Physicians should be aware of the presence of concomitant coagulopathy and might therefore consider intense coagulation factor replacement before or during abdominal exploration. A description of the intraoperative measures needed to control intra-abdominal hemorrhage is beyond the scope of this text, and the reader is referred elsewhere for a detailed review.13

low-grade fever or vague abdominal pain.17 Therefore, in the absence of alternative diagnoses, the physician should be alert to this unusual complication. OPU can also lead to severe abdominal complications. Our group reported on nine patients (0.24%) with tubo-ovarian or pelvic abscess after transvaginal-guided OPU.16 Three patients required laparotomy and adnexectomy, whereas in six patients, culdocentesis was performed for adequate pelvic abscess drainage. Kelada and Ghani18 have recently described a case of bilateral ovarian abscesses following transvaginal oocyte retrieval, complicated by early signs of consumption coagulopathy. The latter is a serious and life-threatening complication of pelvic infection and sepsis, which should be diagnosed and corrected immediately.

Pelvic inflammatory disease

Effect of acute pelvic infection on IVF–ET outcome

Pelvic inflammatory disease is an infrequent complication of ultrasound-guided transvaginal aspiration of oocytes or ET, with a reported incidence of 0.2–0.5% per cycle.7,14–16 Signs or symptoms of pelvic infection, such as pyrexia, continuous low abdominal pain, dysuria, or offensive vaginal discharge, are infrequent.14 However, this does not exclude occult, subclinical bacterial colonization, which may influence the success of the IVF–ET treatment. Our group evaluated the outcome of all IVF–ET procedures performed in our unit between 1986 and 1992.16 Of the 4771 patients who underwent transvaginal OPU, 28 (0.58%) had symptoms of PID within 1–7 days. The diagnosis was established by a rise in body temperature to 38°C for more than 48 hours, signs of pelvic peritonitis on physical examination, leukocyte count of >12 000 cells/m,3 and elevated erythrocyte sedimentation rate. All patients were admitted to hospital for treatment with intravenous antibiotics. It is noteworthy that an ovarian abscess following oocyte retrieval may manifest late during pregnancy with

The first study of the impact of pelvic infection on IVF–ET outcome was reported by our group in 1994.16 We found that the number of oocytes recovered, fertilized, and cleaved in 28 patients undergoing IVF in whom PID developed was similar to that of a comparison group with mechanical infertility. However, there were no pregnancies in the PID group, as compared with the 23–31% pregnancy rate per transfer in the whole group of patients treated by IVF, indicating that the appearance of PID at the critical time of implantation may cause a failure to conceive. This finding has several possible explanations, as outlined in detail below.

Mechanisms underlying pelvic infection During transvaginal aspiration, accidental needle transport of cervicovaginal flora into ovarian tissue can cause unilateral or bilateral oophoritis, and accidental puncture of a contaminated or sterile hydrosalpinx can cause salpingitis. Some authors have attributed pelvic infection to infected endometriotic cysts or tubo-ovarian abscess after aspiration of endometriomas,19,20 or, rarely, to inadvertent puncture of the bowel. Pelvic infection can occur as a direct consequence of transcervical ET. This is evidenced by reported cases of PID following ET in an agonadal donor-egg recipient,21 or during cryopreserved ET;22 it may also occur as a result of the reaction of a silent or persistent subclinical infection, as seen occasionally after hysterosalpingography. Another possible cause during ET is catheterization of the uterus, which may force bacteria-laden air or fluid into one or both tubes by a piston-like effect.

Endotoxemia Endotoxin-releasing bacteria can be introduced into the peritoneal cavity during transvaginal oocyte recovery, and into the uterine cavity or tubes during ET. Ng et al23 described a case in which human oocytes were degenerated and fragmented, with no evidence of fertilization, in

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the presence of Klebsiella-derived endotoxin. In a study of the effects of endotoxin infusion on the circulating levels of eicosanoids, progesterone, and cortisol, and on abortions, Giri et al24 found that the first-trimester cows were more sensitive to the abortifacient effect of endotoxin than the second- and third-trimester cows. The mechanism of the endotoxin-induced abortion apparently involved the prolonged release of prostaglandin F2α, which has a stimulant effect on uterine smooth-muscle contractions and a luteolytic effect resulting in a gradual decline in the plasma level of progesterone.24 In addition, high endotoxin doses can induce the release of various autocoids, catecholamines, and cortisol, which directly or indirectly lead to metabolic and circulatory failures and, thereby, termination of pregnancy.

Local inflammatory reaction Bacteria trigger a chain of events that lead to the activation, proliferation, and differentiation of lymphocytes, and the production of specific antibodies and various cytokines. This excessive production of cytokines may disrupt the delicate balance between the immune and reproductive systems and result in reproductive failure.25–27

Temperature elevation Apart from their direct role on implantation and early embryonic development, cytokines may mediate temperature elevation and indirectly affect the outcome of IVF–ET. The febrile reaction is an integrated endocrine, autonomic, and behavioral response coordinated by the hypothalamus. The actions of circulating cytokines, such as interleukin (IL)-1 and tumor necrosis factor (TNF), on the central nervous system result in the secretion of prostaglandin E2, which initiates the elevation in body temperature together with corticosteroid secretion,28 also a component of the stress response. Some authors have suggested that fever is essential for amplifying the emergence of T-cell immunity in peripheral tissues.29 In vitro experiments have shown that temperature elevation leads to disintegration of the cytoskeleton30 and may affect the transport of organelles. In pregnancy, maternal heat exposure can cause intracellular embryonic damage31 and inhibit cell mitosis, proliferation, and migration, resulting in cell death. In a study of guinea pig embryos, Edwards et al32 reported cell damage within minutes and cell death within hours after heating. Other mechanisms of heat-induced cell injury are microvascular lesions, placental necrosis, and placental infarction.33

Treatment

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vaginal disinfection.34 Meldrum35 found no case of pelvic infection among 88 transvaginal retrievals with the use of intravenous cefazolin and vaginal preparation with povidone–iodine and saline irrigation; nor did Tsai et al36 in patients with ovarian endometrioma, using only vaginal douching with aqueous povidone–iodine followed by normal saline irrigation. Borlum and Maiggard37 reported on two cases of serious pelvic infection in almost 400 transvaginal aspirations. They used only two vaginal douchings with sterile saline and noted that minimizing the number of repeated vaginal penetrations may have helped in lowering the risk of infection. However, the appropriate type of antibiotic administration, timing or duration of therapy, and the efficacy of therapy have not yet been established.35,38 Indeed, some authors claim that these measures may not only further reduce the incidence of PID after oocyte retrieval but may also even increase the risks of both an adverse reaction and of colonization with resistant organisms. Our experience with vaginal douchings with sterile saline in approximately 1100–1200 OPUs per year revealed a very low rate of PID after OPU. Peters et al39 suggested that only women with a tubal abnormality and a history of pelvic infection should receive prophylactic antibiotics before oocyte aspiration, and also possibly after ET. Others have suggested that such patients may benefit from transabdominal or transvesical rather than transvaginal procedures.40,41 It is also noteworthy that Egbase et al,42 in a study of the effect of prophylactic antibiotics in OPU on the endocervical microbial inoculation of the endometrium at ET, found that prophylactic antibiotics not only reduced the number of positive microbiology cultures of embryo catheter tips but also significantly increased implantation and clinical pregnancy rates. On the other hand, in their prospective randomized study, Peikrishvili et al43 could not demonstrate any beneficial effects of antibiotic prescription (amoxicilline + clavulanic acid 1 g + 125 mg) for 6 days following oocyte retrieval on implantation, pregnancy, or miscarriage rates.

Curative Pelvic inflammatory disease or tubo–ovarian abscess after OPU requires accurate diagnosis and prompt treatment with broad-spectrum antibiotics. In the presence of a pelvic abscess that is >8 cm or unresponsive to medication, transvaginal or percutaneous drainage is the treatment of choice,44 with or without ultrasoundguided intracavitary instillation of a combination of antibiotics.45 Sometimes surgical laparoscopy or laparotomy is needed to evacuate the abscess or remove the infected tubes or adnexae.

The role of prophylactic antibiotics in IVF–ET The potential for intraperitoneal bacterial contamination during transvaginal oocyte recovery is well known and has led to the routine use of prophylactic antibiotics and

Summary The appearance of PID at the critical time of implantation results in failure to conceive. This effect may be

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mediated by bacterial endotoxins, a local inflammatory reaction against bacteria with the involvement of cytokines that affect implantation and early embryonic development, or temperature elevation that directly affects the conceptus. Although the role of prophylactic antibiotics is still controversial, they can be considered in the presence of risk factors for PID; aspiration of hydrosalpinx or endometriomas during OPU might be a risk factor for infection and should be avoided. Furthermore, to prevent total failure, if PID develops before ET, cryopreservation and ET in subsequent cycles should be considered. However, if PID develops after ET, the bacterial infection and fever should be treated rigorously to prevent reproductive failure.

Extrauterine pregnancy EUP is the implantation of a blastocyst anywhere except in the endometrial lining of the uterine cavity. In recent years, EUPs have shown a marked increase in both absolute number and rate of occurrence.46 Already in 1992, almost 2% of all pregnancies in the USA were EUPs, and ectopic pregnancies accounted for 10% of all pregnancy-related deaths.46,47 The rates of abortions, multiple pregnancies, and EUPs are higher in pregnancies resulting from assisted reproduction technologies (ART) than in spontaneous pregnancies. Other factors associated with the development of EUP include previous EUP, salpingitis, previous surgery to the fallopian tube, peritubal adhesions, pelvic lesions that distort the tube, developmental abnormalities of the tube, and altered tubal motility.

EUP after ART The first IVF–ET pregnancy reported was an ectopic pregnancy.48 Today, the incidence of EUPs after IVF ranges from 2.1 to 9.4% of all clinical pregnancies.49,50 In 1996, the Society for Assisted Reproductive Technology (SART)51 reported a decrease in the incidence of EUP to 0.8% of transfers and 1.6% of pregnancies, compared with 0.9% and 2.8%, respectively, in 1995. This finding was attributable to the decrease in the proportion of couples with tubal factor infertility undergoing IVF treatment and a concomitant increase in couples with male factor infertility. Recently, the SART reported the outcome of ART initiated in the USA in 2001.52 The incidence of EUP for all ART procedures was 0.8% per transfer and 1.6% per clinical pregnancy, which compares favorably with the estimated overall incidence of EUP in the USA of 2% per reported pregnancy.46

Risk factors Data on risk factors for EUP after IVF are still unclear. Martinez and Trounson53 failed to identify any risk factors, whereas Karande et al54 pointed to a prior ectopic pregnancy. Verhulst et al55 found a significantly higher

rate of EUP after IVF in patients with tubal disease (3.6%) compared with those with normal tubes (1.2%); this finding was confirmed by several other studies.50,56–58 Cohen et al59 showed that the number of patent tubes at the time of transfer was a risk factor, with a higher EUP rate in patients with zero or two patent tubes than in patients with one. In an analysis of the Bourn Hall Clinic data, Marcus and Brinsden60 noted that the main risk factor was a history of PID. Although they found EUP to be more prevalent in patients with tubal factor infertility, those who received a higher culture medium volume and those with a higher progesterone/estradiol ratio on the day of ET had no associated history of EUP. Finally, Ankum et al61 in a meta-analysis of risk factors for EUP, concluded that the four most significant risk factors were previous EUP, documented tubal pathology, previous tubal surgery, and in utero exposure to diethylstilbestrol. These results were confirmed by Lesny et al,62 who also added one more – a difficult ET on day 2 rather than day 3. Clayton et al63 have recently analyzed the EUP risk among 94 118 patients who conceived with ART procedures: 2009 (2.1%) were ectopic. In comparison with the ectopic rate (2.2%) among pregnancies conceived with IVF (fresh, non-donor cycles), the ectopic rate was significantly increased when zygote intrafallopian transfer (ZIFT) was used (3.6%) and significantly decreased when donor oocytes were used (1.4%) or when a gestational surrogate carried the pregnancy (0.9%). Among fresh nondonor IVF–ET procedures, the risk for ectopic pregnancy was significantly increased among women with tubal factor infertility, endometriosis, and other nontubal female factors of infertility and significantly decreased among women with a previous live birth. Moreover, transfer of high-quality embryos was associated with a decreased ectopic risk when ≤2 embryos were transferred, but not when ≥3 embryos were transferred. There are many theories on the manner by which embryos implant in the fallopian tube following ET: by the hydrostatic force of the transfer medium containing the embryos in the fallopian tube ostia; by the gravitational pull of the embryos to the hanging tubes, which are located lower than the uterine fundus; and by reflux expulsion of the embryo due to embryonic migration to the fallopian tubes, either spontaneously or secondary to uterine contractions.64 The technique of ET itself may also be a culprit in EUP, although this is controversial.65 For example, Yovich et al66 noted a significantly higher rate of EUP when the embryos were placed high near the uterine fundus or into the tube itself, rather than in the lower uterus. The transfer volume of culture media containing embryos may play a role in embryonic migration into the fallopian tubes. While most clinicians contend that more than 80 µl of media are needed for the embryo to reach the fallopian tube,50 Knutzen et al67 using a mock intrauterine ET with 50 µl of radiopaque dye, demonstrated easy passage of all or part of the material in 44% of patients. Lesny et al68 explained

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these findings by the propulsion of the embryo from the uterine fundus into the tubes by the junctionalzone contractions. Therefore, as the likelihood of tubal placement is very high, the development of tubal pregnancy is not due solely to embryos reaching the tubes, but rather to an additional pathological process that prevents their movement back into the uterine cavity. Potential mechanisms may be tubal disease affecting the luminal surface and thereby delaying or blocking embryonic passage into the uterine cavity, external factors that interfere with tubal motility, and abnormal embryos,56 such as those derived from chromosomally abnormal gametes.69 To ameliorate the role of abnormal fallopian tubes in the pathogenesis of EUP after IVF, several authors have recommended that the tubes be occluded at the level of the uterotubal junction.70,71 However, this measure does not prevent the development of an interstitial pregnancy,58 although it certainly prevents the well-known phenomenon of spontaneous pregnancies after IVF treatment, which occurs in 30% of the patients with patent tubes.72 Another potential interfering factor in tubal function and ET is the different hormonal milieux resulting from ovulation-induction protocols, particularly those including clomiphene citrate.55,73 This may result from the effect of the high estradiol levels on tubal peristalsis through the control of tubal smoothmuscle contractility and ciliary activity.66,73 Pygriotis et al58 however, did not demonstrate a difference in estradiol levels on the day of human chorionic gonadotropin (hCG) administration between IVF patients with and without EUP. Furthermore, they found an increased proportion of EUPs in frozen ETs following natural cycles in which the estradiol levels were comparatively low.

Heterotopic pregnancy following ART The general incidence of combined intrauterine and extrauterine (heterotopic) pregnancy is 1:15 000 to 1:30 000, and it increases dramatically to 1:100 in pregnancies following ART or ovulation induction.74–76 Although a distorted pelvic anatomy is responsible for the predisposition to both extrauterine and heterotopic pregnancy,77–79 heterotopic pregnancies are associated with a greater number of embryos transferred, whereas EUP is not. Tummon et al80 reported that when four or more embryos were transferred, the odds ratio for the development of a heterotopic pregnancy vs EUP was 10. The difficult diagnosis of this potentially life-threatening complication is often made during emergency surgery following tubal rupture and hemoperitoneum. In about 70% of cases, the outcome of the intrauterine pregnancy is favorable (live birth) once the extrauterine pregnancy is terminated.81,82 A high index of suspicion and early intervention are mandatory to salvage the viable intrauterine pregnancy and prevent maternal mortality.

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Diagnosis and treatment Noninvasive diagnostic measures using transvaginal ultrasonography combined with serum hCG monitoring have proved to be a reliable tool in the diagnosis of EUP. Since most pregnancies following ART are monitored at an early stage before the onset of symptoms, early diagnosis of the condition and improved management and care have resulted in a decline in the morbidity and mortality of EUP. The diagnosis and treatment of EUP are beyond the scope of this chapter, and readers are referred elsewhere for a detailed review.83,84

References 1. Dicker D, Ashkenazi J, Feldberg D, et al. Severe abdominal complications after transvaginal ultrasonographically-guided retrieval of oocytes for in vitro fertilization and embryo transfer. Fertil Steril 1993; 59: 1313–15. 2. Feldberg D, Goldman JA, Ashkenazi J, et al. Transvaginal oocyte retrieval controlled by vaginal probe for in vitro fertilization: a comparative study. J Ultrasound Med 1988; 7: 726–8. 3. Modder J, Kettel LM, Sakamoto K. Hematuria and clot retention after transvaginal oocyte aspiration: a case report. Fertil Steril 2006; 86: e1–2. 4. Tureck RW, Garcia C, Blasco L, Mastroianni L. Perioperative complications arising after transvaginal oocyte retrieval. Obstet Gynecol 1993; 81: 590–3. 5. Lenz S, Leeton J, Renou P. Transvaginal recovery of oocytes for in vitro fertilization using vaginal ultrasound. J In Vitro Fert Embryo Transf 1987; 4: 51–5. 6. Serour GI, Aboulghar M, Mansour R, et al. Complications of medically assisted conception in 3500 cycles. Fertil Steril 1998; 70: 638–42. 7. Govaert I, Devreker F, Delbaere A, et al. Short-term medical complications of 1500 oocyte retrievals for in vitro fertilization and embryo transfer. Eur J Obstet Gynecol Reprod Biol 1998; 77: 239–43. 8. Sauer MV. Defining the incidence of serious complications experienced by oocyte donors: a review of 1000 cases. Am J Obstet Gynecol 2001; 184: 277–8. 9. Yih MC, Goldschlag D, Davis OK, et al. Complete blood counts (CBC) after oocyte retrieval: what is normal? Fertil Steril 2001; 76: S115–16. 10. Azem F, Wolf Y, Botchan A, et al. Massive retroperitoneal bleeding: a complication of trans-vaginal ultrasonography-guided oocyte retrieval for in vitro fertilization–embryo transfer. Fertil Steril 2000; 74: 405–6. 11. Fiori O, Cornet D, Darai E, Antoine JM, Bazot M. Uro-retroperitoneum after ultrasound-guided transvaginal follicle puncture in an oocyte donor: a case report. Hum Reprod 2006; 21: 2969–71. 12. Battaglia C, Regnani G, Giulini S, et al. Severe intraabdominal bleeding after transvaginal oocyte retrieval for IVF–ET and coagulation factor XI deficiency: a case report. J Assist Reprod Genet 2001; 3: 178–81. 13. Thompson JD, Rock WA. Control of pelvic hemorrhage. In: Rock JA, Thompson JD, eds. Te Linde’s Operative Gynecology, 8th edn. Philadelphia: Lippincott-Raven, 1997: 197–232.

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(IVF/ET). Proc 45th Annual Meeting of the American Fertility Society, San Francisco, CA, November 13–16, 1989. American Fertility Society, Program Supplement, pS152: 299. Lesny P, Killick SR, Tetlow RL, et al. Embryo transfer – can we learn anything new from the observation of junctional zone contraction? Hum Reprod 1998; 13: 1540–6. Job-Spira N, Coste J, Boue J, et al. Chromosomal abnormalities and ectopic pregnancy? New directions for aetiological research. Hum Reprod 1996; 11: 239–43. Svare J, Norup P, Grove Thomsen S, et al. Heterotopic pregnancies after in vitro fertilization and embryo transfer – a Danish survey. Hum Reprod 1993; 8: 116–18. Tucker M, Smith D, Pike I, et al. Ectopic pregnancy following in vitro fertilization and embryo transfer. Lancet 1981; 2: 1278. Ben-Rafael Z, Mashiach S, Dor J, et al. Treatmentindependent pregnancy after in vitro fertilization and embryo transfer trial. Fertil Steril 1986; 45: 564–7. Fernandez H, Coste J, Job-Spira N. Controlled ovarian hyperstimulation as a risk factor for ectopic pregnancy. Obstet Gynecol 1991; 78: 656–9. Ben-Rafael Z, Carp HJ, Mashiach S, et al. The clinical features and incidence of concurrent intra and extra uterine pregnancies. Acta Eur Fertil 1985; 16: 199–202. Dimitry ES, Subak-Sharpe R, Mills M, et al. Nine cases of heterotopic pregnancies in 4 years of in vitro fertilization. Fertil Steril 1990; 53: 107–10. Tal J, Hadad S, Gordon M, et al. Heterotopic pregnancy after ovulation induction and assisted reproduction technologies: a literature review from 1971 to 1993. Fertil Steril 1996; 66: 1–12. Goldman GA, Fisch B, Ovadia J, Tadpir Y. Heterotopic pregnancy after assisted reproductive technologies. Obstet Gynecol Surv 1992; 47: 217–21. Molloy D, Deambrosis W, Keeping D, et al. Multiplesited (heterotopic) pregnancy after in vitro fertilization and gamete intrafallopian transfer. Fertil Steril 1990; 53: 1068–71. Li HP, Balmaceda JP, Zouves C, et al. Heterotopic pregnancy associated with gamete intra-fallopian transfer. Hum Reprod 1992; 7: 131–5. Tummon IS, Whitmore NA, Daniel SAJ, et al. Transferring more embryos increases risk of heterotopic pregnancy. Fertil Steril 1964; 61: 1065–7. Rizk B, Tan SL, Morcos S, et al. Heterotopic pregnancies after in vitro fertilization and embryo transfer. Am J Obstet Gynecol 1991; 164: 161–4. Rojanski N, Schenker JG. Heterotopic pregnancy and assisted reproduction – an update. J Assist Reprod Genet 1996; 13: 594–601. Rock JA, Damario MA. Ectopic pregnancy. In: Rock JA, Thompson JD, eds. Te Linde's Operative Gynecology, 8th edn. Philadelphia: LippincottRaven, 1997: 501–27. Yao M, Tulandi T. Current status of surgical and nonsurgical management of ectopic pregnancy. Fertil Steril 1997; 67: 421–33.

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59 Iatrogenic multiple pregnancy: the risk of ART Isaac Blickstein

Introduction The common denominator of most assisted reproduction technologies (ART) is ovarian (hyper)stimulation. The scheme to expose excess female gametes to abundant sperm intended to increase fertilization may inadvertently produce multiple zygotes. In ovulation induction, the number of fertilized eggs is uncontrolled and unpredicted. By contrast, the number of zygotes transferred in ART has been always under control. Consequently, multiple pregnancies following ART are almost exclusively physicianmade: i.e. iatrogenic multiple pregnancies (IMPs). There are two exceptions to this statement. First, single embryo transfer (ET) may still be associated with an increased risk of monozygotic (MZ) twins since ART augments the rate of zygotic splitting.1,2 Secondly, recent observations from the East Flanders Prospective Twin Survey suggest that a genetic familial trait for spontaneous twins may also be involved in induced conceptions. Hence, women with ‘twins in the family’ undergoing infertility treatment may be at increased risk of having multiples as compared with women without that characteristic (R Derom unpublished work). Regardless of the mechanism involved in IMP, ART undoubtedly increases the risk of multiple birth. A recent survey reports on a 25% twin and 5% triplets frequency following transfer of three embryos.3 Roughly, these reference figures represent a 20- and 50-times increased frequency for iatrogenic twins and triplets, respectively, as compared with naturally occurring multiples. Table 59.1 shows a simple model of an obstetrical service with 4000 live births/year, including 5% following iatrogenic pregnancies.4 In this model, the number of twins is doubled and that of triplets is 3.5-times increased. Importantly, 5% iatrogenic pregnancies will produce an excess of 31.5/1000 multiple pregnancy neonates over the expected rate in spontaneous pregnancies. ART and ovulation induction, the major contributors to the epidemic of multiple pregnancies, did not arise ex vacuo. In a modern society, women rely on efficient modern fertility treatment when deciding on postponing childbirth. It follows that advanced maternal age, by itself an accepted risk factor for natural

multiples, is also a significant risk factor for reduced fecundity and increased need for fertility treatment. Thus, social trends act in concert with available ART to increase the risk of multiple pregnancy. Fig 59.1 shows the ratio of spontaneous to induced twins in East Flanders over the last two decades. Except for the unexplained ‘hump’ in 1980, there is a clear change in the rate of induced twins from 1:46 into 1 in every 2–3 twins.5 This population-based trend might be even more accentuated in hospital-based data. The wide spectrum of issues encompassed in IMP deserves a separate volume.6,7 In this chapter, several risks of multiple pregnancies following ART will be specifically addressed.

The pregnancy It is beyond the scope of this chapter to describe in detail the risks associated with multiple pregnancy.8,9 It is generally accepted that the human female is programmed for mono-ovulation, monofetal development, and nursing only one neonate. Consequently, pregnancies with more than one fetus overwhelm the uterine capacity to adequately nurture the fetuses. Animal and human models have repeatedly demonstrated the reciprocal relationship between birth weight and gestational age at delivery and litter size. Using singleton standards, a significant proportion of twins and all high-order multiple pregnancies (HOMPs) will be delivered preterm and will be small for gestational age. In addition to absolute growth restriction, relative (discordant) growth is common.10 As a result of the limited uterine capacity, natural reduction in fetal number is frequently seen. At the early stages, the embryo may disappear (‘vanishing twin syndrome’) in 1 of every 6–7 twin pregnancies following ART.11,12 The vanishing twin syndrome, considered by many as natural multifetal pregnancy reduction (MFPR), has recently gained special attention when Pharoah and Cooke hypothesized that single embryonic death may be implicated in cerebral palsy in the survivor.13,14 More recently, however, Matias et al15 summarized several case-control studies on plurality-dependent spontaneous embryonic loss rates after ART and found that twin pregnancies have

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Table 59.1 Estimating the contribution of 5% iatrogenic conceptions in an obstetrical service with 4000 deliveries/year (spontaneous, 1.2% twins and 0.1% triplets; iatrogenic, 25% twins, 5% triplets) Singles

Twins

Triplets

Births

Neonates

100% spontaneous

3948

48

4

4000

4056

5% iatrogenic 95% spontaneous

140 3750

50 46

10 4

200 3800

270 3854

3890

96

14

4000

4124

Total Adapted from Blickstein.

4

50

20

45 40

15 Risk factor

Ratio

35 30 25 20

10

5

15 10

0

5

Spontaneous

0 1976 1978 1980 1982 1984 1986 1988 1990 1992 Year

Fig 59.1 Ratio of spontaneous to induced twins. Since the implementation of effective infertility treatment the ratio changed from 1 induced for every 40–50 spontaneous twin pregnancies, to 1 induced for every 2–3 spontaneous twin pregnancies. Adapted from the East Flanders Prospective Twin Survey.4

a two- to five-times lower miscarriage rate of the entire pregnancy compared with singletons. At present, it is unclear if this advantage is a chance event or related to the presence of a higher placental (and hormonal) support of the early pregnancy. Multiples are associated with higher frequencies of malformations of varied etiology. The yet unknown factor(s) that causes zygotic splitting has been implicated in causing structural malformations in MZs. In the subset with monochorionic (MC) placentas, also encountered in HOMPs, twin–twin transfusion syndrome (TTTS) may affect as many as 10–15% of the pairs and may result in major morbidity of one or both twins. Later in pregnancy, single fetal demise associated with MC placentas may result in severe endorgan damage in the survivor. Finally, it has been shown that the risk of cerebral palsy (CP) is 5–6- and 23-fold increased in twins and triplets, respectively, compared with singletons.16 A model based on British data related to transfer of two and three embryos3 and on British data related to CP in multiples17 suggested a significantly lower estimated CP rate (2.7/1000 neonates) after spontaneous pregnancies compared with transfer of three embryos (odds ratio [OR] = 6.3), two embryos (OR = 3.3), and transfer of three embryos in which all triplets have been reduced to twins (OR = 3.8)18 (Fig 59.2).

3 ET

2 ET

3 ET (3−>2)

Fig 59.2 Estimated risk of cerebral palsy following transfer of three and two embryos, and following MFPR of all triplets to twins. A three- to six-fold increased risk of cerebral palsy is expected. Adapted from Blickstein and Weissman.18

Three additional aspects deserve further consideration. First, as mentioned above, there is an increased risk of zygotic splitting following ART. It is not known why MZs are more frequent in conceptions after ART. The most common cause and effect speculation suggests that the exposure of the zona pellucida to biochemical or mechanical trauma leads to herniation of the blastocyst and splitting of the zygote. Zygotic splitting is not only a biologic enigma but is also a major area of clinical importance, primarily because of the confirmed increased morbidity and mortality associated with MZ twinning. Currently, zygotic splitting is inferred when the number of fetuses exceeds that of transferred embryos, or when monoamniotic twins are diagnosed. Evidently, the reported figures underestimate the true incidence, since bichorionic MZs cannot be clinically differentiated from same-sex bichorionic dizygotic (DZ) twins. In addition, previous reports did not mention the number of transferred embryos and/or the method of ART. In an initial study,1 the data indicated that splitting is expected in 4.9% after in vitro fertilization (IVF) without intracytoplasmic sperm injection (ICSI) 12 times higher than the 0.4% rate of MZs in spontaneous conceptions. In a subsequent study,2 of a much larger data set from British IVF centers, Blickstein et al found ‘only’ a six-fold increased splitting rate. Recently, Blickstein and Keith19 postulated that, given the remarkably constant frequency of MZ twins in different populations, there might be splittingprone oocytes after fertilization. Thus, the higher the

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16

12 Relative risk

number of ovulation events (i.e. following ovarian stimulation), the greater the chance of recruiting a splitting-prone oocyte for ovulation, as is indeed the case with all methods of assisted conceptions. In addition, the more fecund patients with a better chance to conceive are significantly more likely to have MZ twins, as seen in those receiving a less ‘aggressive’ regimen such as clomiphene citrate as the sole treatment, compared with other ovulation enhancing agents.20 It follows that the chance of a follicle that contains an oocyte with a propensity to undergo splitting is quasi ‘dose–dependent,’ where the term ‘dose’ refers to the combined effect of the patient’s fecundity and the specific treatment administered. This finding is supported by the possibility that ovarian stimulation – the common denominator of all assisted procreation – may affect oocyte development which could predispose to splitting (Fig 59.3). Secondly, one must also reconsider mortality figures in HOMPs undergoing MFPR. There is little doubt that MFPR is among the ultimate paradoxes of medicine whereby infertile patients undergo intricate treatments and, when at last successful, may have to consider reduction (= termination) of their ‘surplus’ fetuses (= success). At the same time, there is little doubt that MFPR may be the only solution for a potentially successful outcome of a high-order multiple gestation. MFPR, discussed elsewhere in this volume, is indeed associated with improved perinatal outcome, as expected from comparing HOMPs with twins or singletons. However, given that all fetuses have a similar survival potential, it is argued that the reduced fetus(es) should be included in the mortality figures of MFPR.21 Table 59.2 shows the minimal mortality rates associated with various MFPR procedures, which suggest, quite bluntly, that MFPR is, in fact, a lethal iatrogenic sequence of iatrogenic multiples. The third point to consider is the frequently overlooked risk of chromosomal disorders in IMP. Although each of the fetuses in a polyzygotic multiple gestation has the same chance for an aberration, as does a singleton with similar risk variables, there is an increased risk for the mother that one of her multiples will be affected. Recent data have clearly substantiated older calculations that showed that a 31-year-old mother of DZs carries a similar risk of having one twin with Down’s syndrome as a 35-year-old mother of a singleton.22 Given that IMPs are more common in older mothers and that biochemical markers are less useful for twins and unavailable for HOMPs, one must rely on nuchal translucency measurements23,24 or on invasive cytogenetic procedures (amniocentesis or chorionic villus sampling [CVS]). Regrettably, little information exists about the former in HOMPs and the latter carries increased risk for miscarriage in these premium pregnancies. Considering all the risks associated with IMP, one undoubtedly should prefer a singleton to a multiple pregnancy. To minimize risks, no more than a single embryo should be transferred. This policy has been

797

8

8 6

4 1 0 Spontaneous

ART

hMG

Clomiphene

Fig 59.3 Zygotic splitting. Frequency of MZ twins following various methods of assisted reproduction. The accepted 0.4% of spontaneous zygotic splitting was used as reference. hMG, human menopausal gonadotropin. Data from the East Flanders Prospective Twin Survey, adapted from Blickstein et al.1

Table 59.2 Minimal mortality rates in various MFPR combinations MFPR Minimal mortality

4→2 5→2

50%

60%

3→2

4→1

3→1

2→1

33%

75%

66%

50%

implemented in recent years in many countries and a full discussion of the results is beyond the scope of this chapter. In general, the balance between the risk of multiples, the success rate of single embryo transfer, and the potential need for reimbursement of additional cycles has been considered and the net result seems to favor single embryo transfer. However, there are two additional partners to the triangle. IMP following ART is usually achieved after long-standing infertility and is usually the ‘end-stage’ procedure. At this phase of reproductive life, most couples would consider a multiple pregnancy as compensation for their efforts.25 No wonder that most couples will support, or even persuade the physician, to increase the chances of pregnancy by increasing the number of transferred embryos.

The patient The optimism at the beginning of ART changes quite often to severe psychological morbidity. From the outset, couples are faced with dilemmas that they have never faced before. For instance, couples initiating therapy were given questionnaires to determine attitudes regarding multiple pregnancy and MFPR.25,26 The results suggested declining ratings as the number of fetuses increased. Intrauterine insemination (IUI)

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patients felt more favorable than the IVF group toward all gestational outcomes and less favorable toward MFPR. Baor and Blickstein25 suggested that young adults who postpone childbearing may presume that fertility is granted but, when all other measures fail, the use of ART is considered the ultimate salvation for these couples. However, ART is highly stressful and may lead to significant negative psychological consequences (loss of self-esteem, confidence, health, close relationships, security, and hope). The risks of multiple pregnancy is frequently overlooked or underappreciated by infertile couples. Despite the real risks associated with a multiple pregnancy and birth, infertile patients often express a desperate wish to have twins or triplets, thereby accomplishing an instant family. The authors highlighted the need to provide infertile couples with detailed information on the risks of multiple pregnancy and birth. In the next step, couples may confront the dilemma to donate or destroy supernumerary embryos. This seeming impasse was investigated in 200 couples embarking on IVF–ET treatment.27 Couples’ opinions on genetic lineage and education were more determinant in their decision to destroy or to donate their supernumerary embryos than their opinions on the in vitro embryo status. The couples expressed various attitudes toward risks of twins and triplets, whereby twins were much more desired than triplets, which are often refused. The psychological morbidity following MFPR and/or raising high-order multiples has been documented. When confronting the dilemma of potential loss of the entire pregnancy following MFPR, couples may experience considerable emotional distress. Nevertheless, many viewed this option as their ‘least bad’ alternative.28 The French group that followed couples during pregnancy and for 4 years postpartum provided some important clues to understanding this complex situation.29,30 They first studied the effects of MFPR on the mothers’ emotional well-being and the relationship with the children during the 2 years following intervention. Then, at 2 years, they compared mothers who had a reduction with mothers who had not and had delivered triplets. At 1 year, a third of the women in the reduction group reported persistent depressive symptoms related to the reduction, mainly sadness and guilt. The others made medical and rational comments expressing no emotion. At 2 years all but two women seemed to have overcome the emotional pain associated with the reduction. The comparison with mothers of triplets indicated that the mothers’ anxiety and depression, and difficult relationships with the children, were less acute in the reduction group. At 4 years after delivery, all mothers reported emotional distress, mainly fatigue and stress. One-third of the mothers had a high score of depression and used psychotropic medication. The relationships with the children and difficulties in coping with their behavior and conflicts were the main reason for psychological distress. Difficulties had not

decreased over the years to the extent that one-third of the mothers spontaneously expressed regrets about having triplets. A Swedish study found similar results.31 Couples (n = 21) with complete sets of triplets aged 4–6 years old were interviewed about their experiences of being ‘triplet parents.’ The diagnosis of triplets had been a shock for most. All triplets were born prematurely. The first time at home was chaotic for most of the parents. Eventually, ‘triplet parents’ spent more time organizing their lives and less time on emotional care than did parents of singletons. The psychological effects are often superimposed on maternal complications, which are common in multiple pregnancy. The list of serious morbidity associated with twins and HOMPs has not been specified for IMP. However, risks of hypertensive disorders, eclampsia, complications of treatment for premature contractions, prolonged bedrest, prolonged hospitalization, and operative deliveries are significantly higher in multiples than in singletons. Thus, the possibility of serious maternal morbidity associated with IMP should be considered to the same extent that ovarian hyperstimulation syndrome (OHSS) is considered before ART. Since maternal morbidity is undoubtedly increased in multiple gestations, it has been proposed that maternal mortality is also increased.32 However, since a multiple pregnancy is not registered as the direct cause of death, the risk is unknown. For example, eclampsia-, tocolysisand delivery-related deaths were more common in twins.32 Data from the Perinatal Information System, including over 700 Latin America and Caribbean hospitals, have clearly shown that multiple pregnancy increases the risk of significant maternal morbidity in nulliparas and maternal mortality in multiparas.33 It is believed that IMPs are not spared these risks. The epidemic of iatrogenic HOMPs enabled some insight into the increased maternal morbidity in these cases. The most significant morbidities found in triplets were pregnancy-induced hypertension (27– 33%), HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome (9–10.5%), anemia (27– 58.1%), and postpartum hemorrhage (9–12.3%).34,35 Since maternal morbidity clearly increases with plurality, it is expected that maternal morbidity will decrease following MFPR. Skupski et al36 found that severe preeclampsia was more common among IVF triplet pregnancies (26.3%) than among IVF triplets reduced to twins (7.9%). The prevalence of all preeclampsia cases was also higher among the triplet group (44.7%) than among the twin group (15.8%). Since all pregnancies were successfully implanted triplets, this finding suggests that plurality and placental mass are probably more important to the development of preeclampsia than is successful implantation alone. Similar findings were reported on gestational diabetes. 37 Maternal morbidity should also be considered in the context of maternal age. ART has enabled pregnancies

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beyond the range of reproductive years, when underlying diseases are more common and pregnancy complications are expected to be intensified. Data from the United States National Center for Health Statistics and the Center for Disease Control (NCHS/CDC Press release, September 14, 1999) suggest that (1) between 1980–82 and 1995–97 the twin birth rate rose 63% for women aged 40–44 years old and nearly 1000% for women aged 45–49 years old; (2) the HOMP birth rate rose nearly 400% for women in their 30s and more than 1000% for women in their 40s. In 1997 there were more twins born to women aged 45–49 years old than during the whole decade of the 1980s. Obviously, motherhood at or beyond the edge of reproductive age is a new aspect of what clinicians previously referred to as pregnancy in the ‘older gravida.’38 With ART, the boundary between ‘old’ and ‘young’ no longer exists. Generally, the majority of the published studies have been unanimous about the special, and perhaps supercautious attitude, required for the older mother, an approach that translates to higher rates of peripartum interventions. Despite the fact that some complications may occur more frequently in older mothers as a result of accumulated prior diseases, there is no direct evidence that older age, per se, complicates either gestation or parturition. Quite unexpectedly, Keith et al found that older age has an advantage of better perinatal outcome (mainly in terms of birth weight) of twins and triplets.39 It is unclear if this is a result of a better socioeconomic status of older mothers or if it is related to some uterine ‘programming’ effect. By contrast to these sky-rocketing rates, there are few series describing such ‘geriatric gravidas’ and therefore, the true prevalence of various complications may be underestimated. In one study, 4.5 ± 1.1 cleaving embryos were transferred per cycle to 45–59-year-old patients, resulting in 74 delivered pregnancies (34.9%). There were 29 (39.2%) multiple gestations, including 20 twins, seven triplets, and two quadruplets. Two of the triplet and both of the quadruplet pregnancies underwent MFPR to twins. Antenatal complications occurred in 28 women (37.8%), including preterm labor, hypertension, diabetes, preeclampsia, HELLP syndrome, and fetal growth retardation. Cesarean section was done in 64.8%.40 The age-related risk for trisomy, depending on the source of the female gametes, is of primary importance when ART is performed in the elderly. For those who conceive without donor eggs, this risk might be exceptionally high. However, in the case of a polyzyogtic multiple gestation, the risk of pregnancy loss following cytogenetic studies might be unacceptably high. Thus, the timing of these studies becomes pertinent. In countries where fetocide is permitted only before the 24th week of gestation, the only options are first-trimester CVS or second-trimester amniocentesis. In some countries, fetocide is not restricted to gestational age, and late fetocide is a clear option. In such instances, amniocentesis is scheduled during the 30th–32nd week, with the possibility of fetocide at 33–35 weeks. This logical

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scheme eliminates the risk of losing the entire pregnancy at an unsalvageable age. However, this scheme provokes two major problems: first, the patient might deliver during the time interval before the cytogenetic results; secondly, legitimization of third-trimester fetocide is a formidable ethical dilemma and does not imply that physicians will agree to terminate a viable fetus. These intricacies may be settled if preimplantation diagnosis should become a useful option. Surrogate motherhood is a good example how ART may change all we know about IMP: consider the ‘Angela’ case, in which two embryos of unrelated couples were transferred to a surrogate uterus. The newborn twins, whose parentage was confirmed postpartum, were nonsiblings who shared no common genes and, of course, shared nothing with the surrogate mother.41 It goes without saying that the most common and the most risky complication of multiple pregnancies is preterm birth, for which no remedy is available. However, irrespective of plurality, an association between preterm birth and ART has long been suspected and found to be related to causes such as iatrogenic preterm birth (in the so-called ‘premium’ pregnancies), fertility history, and past obstetric performance, and to underlying medical conditions of the female partner.42 Recent data showed that singleton as well as multiple pregnancies resulting from IVF have increased rates of preterm birth compared with naturally conceived pregnancies.42 The most plausible explanation seems to be a more liberal use of elective preterm birth. In any case, the most appropriate endpoint after ART should also include preterm or term birth as a measure of success. Finally, the patient with IMP should also be considered in evolutionary terms. Innumerable studies have shown that, over the millennia, evolutionary forces selected a female prototype for spontaneous twins. Black, fertile, older, taller, and heavily built women are more likely to have twins and the outcome is likely better than in women with other characteristics. Thus, the fact that ART involves no selection (except fertility), and certainly no selection for motherhood of multiples, makes the IMP in many ways an iatrogenic contraevolutionary phenomenon.

The physician Three types of physicians comprise the third part of the IMP triangle: those involved in ART, those caring for maternal–fetal issues, and the pediatricians. Each is in charge of a different phase.

The reproduction phase Since there seems to be a direct relation between the number of transferred embryos and success rate of ART on the one hand and the IMP rate on the other hand, there seems to be an inherent conflict in the reproduction phase. An idea about the anticipated rates of IMP comes from centers in which all available

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embryos were transferred and MFPR is not used (Fig 59.4).43 Before the implementation of the 2004 Italian Reproduction Law,44 the Reggio Emilia (Italy) Center for Reproductive Medicine observed that 34.6% of the clinical pregnancies were multiples, comprising 20% twins and 14.6% HOMPs.43 Interestingly, implementation of the Italian Reproductive Law, which limited the number of fertilized oocytes to three but obliged transfer of all embryos, did not significantly change the incidence of multiples and somewhat improved the overall outcome.44 Some concern exists, however, regarding the group of patients >38 years old. Ethical, legal, religious, and technical (i.e. availability of cryopreservation) constraints that obviate selection and/or disposal of surplus embryos is the easy way for deciding on the number of embryos that should be transferred. The hard way is careful analysis of success (live birth) vs failure (IMP) rates using selected embryos. Genetic and biochemical markers would supplement morphological criteria as normal-appearing embryos may be genetically abnormal. Preimplantation genetic studies may also replace invasive procedures during pregnancy following ART. For the time being, the first step has already been done by implementing elective single embryo transfer in several countries without significantly reducing outcomes. Many of the recommendations have been based on embryo transfer without specifying their quality and their implantation potential. In the meantime, it has become possible to culture embryos to the blastocyst stage, selecting the fittest embryos for transfer and synchronizing the embryonic with the endometrial stages. Blastocyst transfer has been associated with a muchimproved implantation rate than that of 3-day embryos. It is expected that the high ‘take-home baby’ rate following the excellent implantation rates would lead to transfer of one or two blastocysts only, with concomitant reduction of the IMP rate. However, not all embryos will become blastocysts and it is unknown which dividing embryo will become a blastocyst in vitro. Thus, physicians may not wait for the 5-day stage and will first transfer 3-day embryos and then, when blastocysts are successfully cultured, will transfer additional blastocysts, generating iatrogenic superfecundations. To date, there are no data regarding the consequences of such protocols. Logically, mixed-stage embryo transfers will necessarily increase the chance of IMPs by adding the successful implantation of the 5-day to that of the 3-day embryo(s). In addition, we do not know the influence of co-implantation at different embryonic ages on the risk of zygotic splitting. We, among others, have noticed some bizarre complex chorionicity arrangements that have never been seen with the usual IVF–ET protocols (unpublished data, Fig 59.5). It is therefore reasonable to conclude that demands from infertile couples and fertility clinics to maximize success rates conflict with the need to reduce the number of IMPs.

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45 30 15 0 Singles

Twins

Triplets

Quads

Quints

Fig 59.4 Spontaneous loss in IMPs when all available embryos are transferred and MFPR is not used. light bars: percent of clinical pregnancies at 35 days post-transfer; dark bars: percent of disintegrated gestational sacs. Data adapted from La Sala et al.43

Fig 59.5 Complex chorionicity. Sonographic image showing a 7-week quadruplet pregnancy following sequential transfer of two embryos and one blastocyst. This bichorionic quadruplet pregnancy comprises monochorionic triamniotic triplets (upper sac) and a singleton (lower sac). Image courtesy of B Caspi MD.

The pregnancy phase Once pregnant, the woman is not infertile anymore and there should be no difference in the management of spontaneous as compared to iatrogenic pregnancies. However, the past reproductive history continues to follow the patient, albeit her pregnancy may be absolutely normal. When an IMP results, the designation of ‘premium gestation’ seems appropriate, and most reproduction experts may refer the patient to a clinician involved in maternal–fetal medicine (MFM) conducting high-risk pregnancy clinics. Couples frequently create a special attitude towards the ‘producer’ and may feel abandoned when referred to another physician who takes over. Quite often the optimism involved in infertility treatment may change to pessimism or even to criticism.45 Then, the unprepared couples may consider MFPR or risky interventions as hostile suggestions. It follows that the dissociation between the reproductive and the MFM

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physician is by no means simple for any of the parties involved. It is not yet accepted who should treat the IMP. Obviously, many subspecialties are involved: for example, the sonographer who makes the diagnosis may not be the one who will carry out the MFPR, and both may not take care of the preeclamptic patient. This complicated pregnancy follow-up is therefore never a one-man show, and a well-orchestrated teamwork is encouraged. Indeed, it has been shown that special multiple pregnancy clinics do have better results. The extremely varied spectrum of IMPs is superimposed on the special patient–physician relationship. It is beyond the scope of this chapter to discuss in detail follow-up protocols tailored for the diverse presentations of IMP. A 32-year-old patient with premature ovarian failure and a 48-year-old perimenopausal woman may undergo similar egg or embryo donation, but are expected to run different age-related obstetric risks. Likewise, 20-year-old and 40-year-old women may need similar ICSI techniques for severe oligospermia, but differ in respect of anticipated age-related pregnancy complications. The obligations for the fetus as a patient in multiple pregnancy are quite complicated.46 In addition to the physician–mother–fetus relationship, there are feto– fetal relations that must be contemplated. The simplest example is a preterm multiple pregnancy in which fetal distress is suspected in one fetus. The obstetrician is faced with the dilemma to salvage one fetus by conferring risks of prematurity on the non-distressed fetus. A more complicated example is the consideration of MFPR in a bichorionic (BC) triplet pregnancy (i.e. MC twins plus a singleton). Obviously, a three to two reduction will end with an MC twin gestation in which TTTS is a calculated risk. On the other hand, reducing the twins will increase the risk of losing the entire pregnancy. A third example is a single sac, remote from term, with rupture of the membranes in a triplet pregnancy. Should a delayed-interval delivery be performed (increasing the risk of amnionitis) or should the whole pregnancy be terminated? It seems there is never a dull moment in caring for the mother with multiples, exemplified by conflicts between maternal condition and continuation of pregnancy. The lack of effective prophylactic measures against preterm labor and the risks associated with tocolysis is a good example of how the physiologic adaptation for a multiple gestation may complicate treatment with β-mimetic drugs or with MgSO4. Thus, the risk in arresting preterm labor (to the mother) may be as significant as the risk (to the neonate) of delivering premature multiples. It is beyond the scope of this chapter to describe the plethora of inefficient methods to reduce the preterm birth rate in multiple pregnancy. This pessimistic realization was reached by trying to carry multiple pregnancies to term (by singleton standards), whereas medicine is apparently unable to change the inherent inadequacy

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of the uteroplacental unit to accommodate and nurture multiples that long. In this respect, two points should be made. First, ‘term’ in singletons is different than in twins or in HOMPs. Thus, it seems futile to aim for 38 weeks’ gestation in multiples just to conclude that this target is unattainable. Secondly, it follows that a realistic gestational age based on related survival and morbidity rates should be set. For example, obstetricians should aim for 30 weeks’ gestation if their neonatal service provides good outcome for neonates at this age. Thus, it seems reasonable to suggest that if prematurity in multiples is not preventable, efforts should be made to prevent extreme prematurity. Finally, a time comes when the obstetrician and the patient consider the mode of delivery. There is little doubt that a planned (daytime), elective cesarean delivery offers a simple solution in terms of required personnel and safety to mother and neonates. This seems to be intuitively true for HOMPs and for small twins, although there are no prospective studies to support this assumption. For twins weighing at least 1500 g each, either route of delivery seems to be appropriate, irrespective of fetal presentation.47 However, as mentioned above, IMPs are frequently considered as ‘premium,’ high-risk pregnancies, and many will follow the dictum that ‘no high-risk pregnancy should end with a high-risk delivery’ and opt for an elective abdominal birth.

The neonatal phase There is no significant difference between treating three preterm singletons and a preterm triplet pregnancy, as each of these neonates deserves its own special care. However, the epidemic dimensions of IMP create consequential logistical problems that ideally should be separated from the purely medical problems. Regrettably, advances in ART have been much faster than the preparation of sufficient cribs in the neonatal intensive care unit (NICU). As a result, overproduction of preterm neonates overwhelms the capacity of many NICUs, leading to medical problems associated with overcrowded stations. A Canadian study compared the preterm birth rates in two 3-year periods, 1981–83 and 1992–94.48 Preterm birth rate increased by 9% (from 6.3 to 6.8%). Importantly, the rate of preterm birth among live births resulting from multiples increased by 25% compared with 5% in singletons, confirming that the increase in preterm births is largely attributable to the increase in multiple birth rates. HOMP births are at much greater risk than single births. An NCHS report on the final 1996 birth statistics for the USA found that infant mortality rates are 12 times higher for triplets than for singletons, triplets are 12 times more likely to die within the first year of life, the average birth weight of a triplet baby is half that of a singleton, and the gestational duration is, on average, 7 weeks shorter. For 1995, 92% of triplets

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were preterm compared with about 10% of births in single deliveries. Delivery of a multiple pregnancy should be a carefully planned event. A minimal neonatal team for a triplet delivery may include as many as 10 persons, including physicians, assistants, and a supervisor. Obviously, chaos prevails unless teamwork is harmonized. Neonatal transportation should be available if the expected number of neonates exceeds the number of available NICU cribs. Logistic considerations do not end at delivery. Once at the nursery, all the multiples must be given equal opportunity to bond with their parents and, perhaps, according to psychological view, to continue their intrauterine contacts with their siblings. For instance, there is increasing evidence that co-bedding of twins in the NICU improves thermoregulation, feeding, and sleeping parameters.49 Indeed, the special and unique interaction between multiples during childhood and beyond seems to reflect the unique relationship that exists between fetuses that grow together in utero (Appendix). Fig 59.6 shows mortality rates of twins, triplets, and higher-order multiples in England and Wales in 1993 relative to singletons, demonstrating the much increased incidence of stillbirth, perinatal, neonatal, and infant deaths is in multiple pregnancy.50 Thus, parents of a multiple pregnancy are more likely to experience bereavement than those with singletons. The care that parents should receive when all fetuses/babies die is not different from that when a singleton dies. When one baby of a multiple birth dies, the loss is frequently underestimated; however, the loss of parents that are left ‘with something’ is no less painful. The time spent in the nursery may be the only opportunity for the parents to prepare for the future. At home, mothers may find the reality of coping with their multiples more demanding than they had expected. Needless to say, professional help is needed during infancy and childhood to the same extent that it had been needed before and during pregnancy. Finally, it is well accepted that even perfectly normal multiples are a significant financial burden for every family. Many studies have estimated the expenses involved in IMP. Given that costs involved in ART are similar to conceptions ending with a singleton, and given that costs of pregnancy surveillance of multiples are moderately increased as compared with singletons, the major financial impact of IMP evolves from raising premature infants in the expensive environment of the NICU. No mathematical skills are needed to establish the number of NICU days per IMP and to multiply the product by the daily cost of NICU hospitalization. Moreover, lifelong morbidity, which is significantly associated with preterm birth, has further implications on the expenses involved in caring for the handicapped children. Thus, from a financial perspective, IMP must be considered as a syndrome of an affluent society.

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Singleton Twins Triplets Higher-order

50 40 Percentage

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20 10

0 Stillbirth

Perinatal

Neonatal

Postneonatal

Infant

Fig 59.6 Mortality rates of twins, triplets, and higher-order multiples in England and Wales in 1993 relative to singletons. A much increased incidence of stillbirth, perinatal, neonatal, and infant deaths is shown in multiple pregnancy. Adapted from Dunn and MacFarlane.50

Epilogue: re-defining success Every day there are numerous healthy multiples delivered following ART conceptions. Almost every proud reproductive center documents this success in pictures of smiling parents, cute babies, and grinning physicians. The media love it as well and give primetime priority for items related to HOMP births. As a consequence, infertile couples exposed to these encouraging results are bound to push ART to its available limits, irrespective of the untoward outcome of a multiple pregnancy. As stated previously and until proven otherwise, the human female is programmed by nature to have one child at a time. Consequently, success should have only one meaning – a ‘take-home baby’ rate of one infant per pregnancy. Thus, there is an inherent absurdity in considering a HOMP in need of MFPR as a successful outcome, and it is likewise irrational to consider the delivery of triplets at 29 weeks’ gestation as a successful event. Obviously, producing a three- to six-fold increased risk for a lifelong handicap such as cerebral palsy cannot be considered successful. Two of the several solutions proposed to overcome the epidemic of IMP are relevant to ART. First, the dissociation between members of the ‘production line’ should be minimal. Thus, both reproductive experts and their patients should have an accurate perspective of the potential obstetric, neonatal, and lifelong complications associated with IMPs. Secondly, the current changing trends from quantity to quality in ART, by transferring fewer but higher-quality embryos or blastocysts, may be the light at the end of the tunnel. One should consider the international consensus statement on the perinatal care of multiples (Appendix), where many aspects related to ART are discussed. In any case, the apocalyptic views expressed in this chapter will remain pertinent as long as demands for better pregnancy rates by couples undergoing ART are accepted by overzealous reproduction centers without a clear definition of what should be considered successful.

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Appendix

2.

Recommendations and guidelines for perinatal practice The Istanbul international consensus statement on the perinatal care of multiple pregnancy∗

3.

Isaac Blickstein1,∗∗, Birgit Arabin2, Frank A. Chervenak3, Zehra N. Kavak4, Louis G. Keith5, Eric S. Shinwell6, Alin Basgul4 and Yves Ville7 1

Department of Obstetrics and Gynecology, Kaplan Medical Center, Rehovot, Israel 2 Department of Gynecology, Sophia Hospital Zwolle, Zwolle, The Netherlands 3 Department of Obstetrics and Gynecology, New York Weill Cornell Medical Center, USA 4 Department of Obstetrics and Gynecology, Marmara University Hospital, Istanbul, Turkey 5 Department of Obstetrics and Gynecology, Northwestern University Medicine School, Chicago, USA 6 Department of Neonatology, Kaplan Medical Center, Rehovot, Israel 7 Department of Obstetrics and Gynecology, University Versailles St. Quentin, Poissy, France

4.

5.

Consensus statement 1.

∗

Multiples and their families, as any other individuals, have a right to full protection under the law and freedom from discrimination of any kind.

Pregnant women and their multiples have a right to be cared for by professionals who are knowledgeable regarding the management of multiple gestation and/or the lifelong special needs of multiples. Individuals or couples seeking information and/or treatment for infertility have a right to full disclosure about clinically relevant information that might influence the conception of multiples, the associated risks, and the medically reasonable management alternatives for them. This disclosure should be specific to each potential intervention. Thus, if successive interventions are considered, multiple disclosures should be provided and consent obtained for each intervention. When infertility treatment is contemplated, the a priori risks and consequences of having a multiple pregnancy secondary to each infertility treatment should be discussed. In settings where iatrogenic multiple gestations may result, the potential need for multifetal pregnancy reduction (MFPR) and its associated risks should be discussed. Given the increased risk of any multiple pregnancy: (a) Infertility treatment should intend to prevent multiple pregnancies, in particular high order multiples. This implies that a high order multiple gestation after infertility treatment should be considered as a complication. (b) Infertility services should disclose their number of multiple pregnancies, both intentional and unintentional. (c) The economical justification of choosing a multiple pregnancy, especially high order multiple pregnancy, should be discouraged. (d) The use of a multiple pregnancy as a potential cohort from which fetus of either sex can be selected is discouraged. (e) In the special case of inherited disease, pregestational diagnosis (PGD) to select embryos for transfer should be preferred to producing a multiple pregnancy in order to reduce the affected embryos.

Abstract The purpose of this document is to expand the 1995 ISTS/COMBO Declaration of Rights which was initially produced to promote awareness of the special needs of multiple birth infants, children, and adults. It addresses the clinical and ethical dimensions of perinatal care of multiple pregnancy. The ad hoc committee was chaired by Isaac Blickstein. The following individuals were present (in alphabetical order): Birgit Arabin (Zwolle, Netherlands/Berlin, Germany), Isaac Blickstein (Rehovot, Israel), Frank A. Chervenak (NY, USA), Zehra Nese Kavak (Istanbul, Turkey), Louis G. Keith (Chicago, USA), Eric S. Shinwell (Rehovot, Israel) and Yves Ville (Paris, France). Secretary of the meeting was Alin Basgul (Istanbul, Turkey). This statement was endorsed by the International Society of Twin Studies (Ghent, Belgium, June, 2007) and by the World Association of Perinatal Medicine (Florence, Italy, September, 2007).

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6.

MFPR should be considered as a destructive measure for reduced fetuses and a therapeutic measure for the remaining fetuses. It may also be traumatic for one or both parents. Its only role is to potentially promote a better outcome for the remaining embryo(s). It follows that: (a) The decision about MFPR is ultimately the pregnant woman’s to make. The pregnant woman should be provided reliable information about institutional rather than national success rates of MFPR. The patient’s beliefs

Constructed by an ad hoc committee, convened in Istanbul, January 26, 2007. Coordinator of WAPM Multiple Pregnancies Working Group: Isaac Blickstein. ∗∗ J. Perinat. Med. 35 (2007) 465–467. Copyright  by Walter de Gruyter, Berlin, New York. DOI 10.1515/JPM.2007.134.

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and values are determinative in the decisionmaking process regarding whether MFPR is to be performed and, if so, the number of embryos to be reduced. (b) Diagnostic evaluation of fetuses before MFPR is appropriate, as the resulting information is relevant to the pregnant woman’s decisionmaking. (c) MFPR should be considered as a relevant antenatal event and registries should be encouraged so that outcome can be appreciated. 7. Selective reduction of an anomalous fetus should be performed in a manner as to minimize the potential danger to the remaining fetus(es), taking chorionicity into consideration: (a) Screening, diagnosis, and selective reduction should be performed at a timing to optimize the outcome for the remaining fetus(es). (b) Selective reduction should be considered as a relevant antenatal event and registries should be encouraged so that outcomes can be appreciated. 8. A distinction must be made between a multiple pregnancy and a multiple birth. For epidemiological purposes, singleton births that started as a multiple pregnancy should be recorded in order to properly account for spontaneous and iatrogenic embryonic and fetal loss(es). 9. Ultrasound technology is critical for antenatal care in all multiple pregnancies. Chorionicity should be established by ultrasound as accurately and as early as possible in all multiple pregnancies. Information about chorionicity should be provided to the expectant mother along with its clinical significance. When this information is lacking, careful postpartum placental examination should be performed. 10. Zygosity determination should be a prerogative of the parents or of the multiples and not of the careproviders (except for clearly defined research objectives in which informed consent has been given). Zygosity should be respected as any other human trait and deserves the same privacy rules. Involvement in registries of monozygotic twins should be absolutely voluntary on the part of the multiples. 11. Complex cases associated with monochorionic placentation, such as twin-twin transfusion syndrome (TTTS), twin reversed arterial perfusion (TRAP) sequence, and severe discordance, should be evaluated and treated in specialized centers based on scientific and ethical considerations. In the absence of the possibility of referral, consultation should be obtained requesting the best potential therapy in the local setting. 12. Following single perinatal demise within a set of multiples, parents may consider informing the

13.

14.

15.

16.

survivor(s) that he/she/they were a sib of multiple pregnancy. Whenever the clinical circumstance of one twin jeopardizes the other, care should be exercised to select a management plan that would optimize the outcome of both fetuses. The pregnant woman should be involved in such decision-making. Fetal surveillance before and during labor should be carried out on all fetuses. It follows that each fetus should be adequately and appropriately monitored. Delivery considerations should include the welfare of all fetuses. Mode of delivery should be based on medical considerations pertaining to each fetus, as well as on maternal health and preferences. Delivery of multiples should ideally take place: (a) In a center that is equipped with neonatal intensive care facilities available simultaneously for each infant; (b) Where a medical care provider certified in neonatal resuscitation is present for each neonate; and with (c) Facilities for a high-risk delivery (such as availability of an experienced obstetrician, 24-h anesthesia coverage, blood availability, etc.). If this is not possible, in utero transfer may be preferable to postpartum transfer.

17. Governments and private payers should be aware of the financial costs of multiple pregnancy and birth and the future upbringing of the multiples. Whenever possible, direct financial aid should be supplied to parents in proportion to plurality. 18. Any research involving multiples must be conducted using informed consent of the participants or their parents and must comply with accepted international codes of ethics and scientific standards for conducting human subject research. 19. In the instance when one or more of a set of multiples manifests a physical and/or mental handicap, management plans should respect the special needs of the handicapped member(s) in the setting of a multiple pregnancy while also giving attention to the special needs of the non-handicapped sib(s). 20. Public policy should support a set of multiples remaining together in foster care and adoptive families.

Acknowledgments The committee acknowledges with thanks the assistance of Laurence B. McCullough, PhD (Huston, TX) in reviewing the preliminary draft of this document. The committee acknowledges with thanks the unrestricted educational grants provided by Philips Turk, EMA electronic devices, and Abdi Ibrahim Drug Company, Istanbul.

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References 1. Blickstein I, Verhoeven HC, Keith LG. Zygotic splitting after assisted reproduction. N Engl J Med 1999; 340: 738–9. 2. Blickstein I, Jones C, Keith LG. Zygotic-splitting rates after single-embryo transfers in in vitro fertilization. N Engl J Med 2003; 348: 2366–7. 3. Templeton A, Morris JK. Reducing the risk of multiple births by transfer of two embryos after in vitro fertilization. N Engl J Med 1998; 339: 573–7. 4. Blickstein I. Perinatal implications of iatrogenic multiple pregnancies. In: Voto LS, Margulies M, Cosmi EV, eds, 4th World Congress of Perinatal Medicine. Bologna: Monduzzi Editore, 1999: 167–72. 5. Leroy F. Les Jumeaux dans Tous Leurs Etats. Bruxelles: De Boeck-Wesmael, 1995: 87. 6. Blickstein I, Keith LG, eds. Iatrogenic Multiple Pregnancy: Clinical Implications. Lancaster, UK: Parthenon, 2000. 7. Blickstein I, Keith LG, eds. Multiple Pregnancy, 2nd edn. Oxford: Taylor & Francis, 2005. 8. Blickstein I, Smith-Levitin M. Twinning and twins. In: Chervenak FA, Kurjak A, eds. Current Perspectives on the Fetus as a Patient. Lancaster, UK: Parthenon, 1996: 507–25. 9. Blickstein I, Smith-Levitin M. Multifetal pregnancy. In: Petrikovsky BM, ed, Fetal Disorders: Diagnosis and Management. New York: John Wiley and Sons, 1998: 223–47. 10. Blickstein I, Goldman RD, Smith-Levitin-M, et al. The relation between inter-twin birth weight discordance and total twin birth weight. Obstet Gynecol 1999; 93: 113–16. 11. Palermo GD, Cohen J, Alikani M, Adler A, Rosenwaks Z. Intracytoplasmic sperm injection: a novel treatment for all forms of male factor infertility. Fertil Steril 1995; 63: 1231–40. 12. Gavriil P, Jauniaux E, Leroy F. Pathologic examination of placentas from singleton and twin pregnancies obtained after in vitro fertilization and embryo transfer. Pediatr Pathol 1993; 13: 453–62. 13. Pharoah PO, Cooke RW. A hypothesis for the aetiology of spastic cerebral palsy – the vanishing twin. Dev Med Child Neurol 1997; 39: 292–6. 14. Blickstein I. Reflections on the hypothesis for the etiology of spastic cerebral palsy caused by the ‘vanishing twin’ syndrome. Dev Med Child Neurol 1998; 40: 358. 15. Matias A, La Sala GB, Blickstein I. Early loss rates of entire pregnancies after assisted reproduction are lower in twin than in singleton pregnancies. Fertil Steril 2007; 88: 1452–4. 16. Blickstein I. Do multiple gestations raise the risk of cerebral palsy? Clin Perinatol 2004; 31: 395–408. 17. Pharoah PO, Cooke T. Cerebral palsy and multiple births. Arch Dis Child Fetal Neonatal Ed 1996; 75: F174. 18. Blickstein I, Weissman A. Estimating the risk of cerebral palsy after assisted reproduction. N Engl J Med 1999; 341: 1313–14. 19. Blickstein I, Keith LG. On the possible cause of monozygotic twinning: lessons from the 9-banded armadillo and from assisted reproduction. Twin Res Hum Genet 2007; 10: 394–9.

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20. Derom C, Leroy F, Vlietinck R, Fryns JP, Derom R. High frequency of iatrogenic monozygotic twins with administration of clomiphene citrate and a change in chorionicity. Fertil Steril 2006; 85: 755–7. 21. Blickstein I. Should the reduced embryos be considered in outcome calculations of multifetal pregnancy reduction? Am J Obstet Gynecol 1994; 171: 866–7. 22. Meyers C, Adam R, Dungan J, Prenger V. Aneuploidy in twin gestations: when is maternal age advanced? Obstet Gynecol 1997; 89: 248–51. 23. Sebire NJ, Snijders RJ, Hughes K, Sepulveda W, Nicolaides KH. Screening for trisomy 21 in twin pregnancies by maternal age and fetal nuchal translucency thickness at 10–14 weeks of gestation. Br J Obstet Gynaecol 1996; 103: 999–1003. 24. Maymon R, Jauniaux E, Holmes A, et al. Nuchal translucency measurement and pregnancy outcome after assisted conception versus spontaneously conceived twins. Hum Reprod 2001; 16: 1999–2004. 25. Baor L, Blickstein I. En route to an ‘instant family’: psychosocial considerations. Obstet Gynecol Clin North Am 2005; 32: 127–39. 26. Goldfarb J, Kinzer DJ, Boyle M, Kurit D. Attitudes of in vitro fertilization and intrauterine insemination couples toward multiple gestation pregnancy and multifetal pregnancy reduction. Fertil Steril 1996; 65: 815–20. 27. Laruelle C, Englert Y. Psychological study of in vitro fertilization–embryo transfer participants’ attitudes toward the destiny of their supernumerary embryos. Fertil Steril 1995; 63: 1047–50. 28. Berkowitz RL, Lynch L, Stone J, Alvarez M. The current status of multifetal pregnancy reduction. Am J Obstet Gynecol 1996; 174: 1265–72. 29. Garel M, Stark C, Blondel B, et al. Psychological reactions after multifetal pregnancy reduction: a 2-year follow-up study. Hum Reprod 1997; 12: 617–22. 30. Garel M, Salobir C, Blondel B. Psychological consequences of having triplets: a 4-year follow-up study. Fertil Steril 1997; 67: 1162–5. 31. Akerman BA, Hovmoller M, Thomassen PA. The challenges of expecting, delivering and rearing triplets. Acta Genet Med Gemellol Roma 1997; 46: 81–6. 32. Blickstein I. Maternal mortality in twin gestations. J Reprod Med 1997; 42: 680–4. 33. Conde-Agudelo A, Belizan J. Maternal mortality and morbidity associated with multiple pregnancy. Twin Res 1999; 2: S3. 34. Malone FD, Kaufman GE, Chelmow D, et al. Maternal morbidity associated with triplet pregnancy. Am J Perinatol 1998; 15: 73–7. 35. Albrecht JL, Tomich PG. The maternal and neonatal outcome of triplet gestations. Am J Obstet Gynecol 1996; 174: 1551–6. 36. Skupski DW, Nelson S, Kowalik A, et al. Multiple gestations from in vitro fertilization: successful implantation alone is not associated with subsequent preeclampsia. Am J Obstet Gynecol 1996; 175: 1029–32. 37. Sivan E, Maman E, Homko CJ, et al. Impact of fetal reduction on the incidence of gestational diabetes. Obstet Gynecol 2002; 99: 91–4. 38. Blickstein I. Motherhood at or beyond the edge of reproductive age. Int J Fertil Womens Med 2003; 48: 17–24.

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39. Keith LG, Goldman RD, Breborowicz G, Blickstein I. Triplet pregnancies in women aged 40 or older: a matched control study. J Reprod Med 2004; 49: 683–8. 40. Sauer MV, Paulson RJ, Lobo RA. Oocyte donation to women of advanced reproductive age: pregnancy results and obstetrical outcomes in patients 45 years and older. Hum Reprod 1996; 11: 2540–3. 41. Simini B. Italian surrogate twins. Lancet 1997; 350: 1307. 42. Blickstein I. Does assisted reproduction technology, per se, increase the risk of preterm birth? BJOG 2006; 113(Suppl 3): 68–71. 43. La Sala GB, Montanari R, Cantarelli M, et al. Iatrogenic multifetal pregnancies and SPIER. Twin Res 1999; 2: S6. 44. La Sala GB, Vilani MT, Nicoli A, et al. The effect of legislation on outcomes of assisted reproduction technology: lessons from the 2004 Italian law. Fertil Steril 2008; 89: 854–9.

45. Blickstein I. Litigation in multiple pregnancy and birth. Clin Perinatol 2007; 34: 319–27. 46. Chervenak FA, McCullough LB. Ethics in Obstetrics and Gynecology. New York: Oxford University Press, 1994. 47. Blickstein I. Cesarean section for all twins? J Perinat Med 2000; 28: 169–74. 48. Joseph KS, Kramer MS, Marcoux S, et al. Determinants of preterm birth rate in Canada from 1981 through 1983 and from 1992 through 1994. N Engl J Med 1998; 339: 1434–9. 49. Mazela JL, Gdzinowski J. Co-bedding twins and multiples – is there strong clinical evidence? Twin Res 1999; 2: S17. 50. Dunn A, MacFarlane A. Recent trends in the incidence of multiple births and associated mortality in England and Wales. Arch Dis Child 1996; 75: F10–19.

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60 Egg and embryo donation Mark V Sauer, Matthew A Cohen

Introduction Human egg (oocyte) and embryo donation was first introduced in 1983 and has evolved over the past 25 years into a relatively common procedure that addresses a variety of reproductive disorders. This method has provided key insights into the physiology and pathophysiology of reproduction and, like other assisted reproductive technologies (ART), has engendered its share of controversy. Furthermore, techniques introduced by egg donation, such as schemes for adequate hormonal preparation of the uterus for synchronizing embryos with a receptive endometrium, have been successfully applied to other fertility therapies, including the management of patients with cryopreserved embryos for transfer and those requiring in vitro maturation of immature oocytes. The first report of a successful egg donation in a mammalian species involved rabbits. Heape in 1890 described the transfer of rabbit embryos from the uterus of a donor to the uterus of a synchronized recipient, followed by the delivery of healthy offspring.1 During the 1970s, mammalian embryo transfer was applied to cattle in order to improve the reproductive efficiency of prize animals. By 1990 almost 19 000 calves were born annually in the United States as a result of embryo transfer procedures.2 The vast majority of mammalian egg donations resulted from embryos fertilized in vivo, recovered from the donor by uterine lavage, and then transferred to the recipient uterus. Using a modification of this technique, in 1983 researchers at the University of California, Los Angeles, fertilized an oocyte in vivo after the artificial insemination of a human donor and then transferred the recovered embryo into a synchronized recipient.3 A total of 14 insemination cycles resulted in two ongoing pregnancies.4 In 1984 the first delivery of a healthy male infant was reported.5 During this same time period, researchers at Monash University in Melbourne began transferring embryos to infertile recipients as a result of eggs fertilized in vitro from donated oocytes obtained laparoscopically from infertile women.6 In 1984 they reported the first live

birth following egg donation and in vitro fertilization (IVF).7 Synchronization of the recipient and donor was achieved using oral estradiol valerate and intravaginal progesterone pessaries prescribed to the functionally agonadal recipient. Donor uterine lavage was popular in the early 1980s since it was far less invasive than laparoscopy, but by 1987 uterine lavage was discontinued in humans because of the fear of human immunodeficiency virus (HIV) transmission and the inability to prevent occasional retained pregnancies in the embryo donors. Furthermore, around this time the introduction of transvaginal oocyte aspiration using ultrasound guidance enabled oocyte donation to be performed within an office setting, greatly reducing its inconvenience, improving its safety, and lessening its cost. The popularity of egg and embryo donation is evidenced by the rapidly increasing demand for services. In the United States, 13 722 procedures involving fresh or frozen embryos procured through oocyte donation were reported to the Centers for Disease Control in 2004, nearly three times the number reported in 1996.8 This increase is largely due to the rising percentage of women who remain childless past the age of 40 years old, a number that has sharply increased over the past 20 years.9 Many women are marrying later, or are pursuing education and vocation and deliberately delaying childbearing.10 Unfortunately, there is a natural decline in fertility associated with advancing age, and many healthy women later experience difficulties as a result of normal aging.

Indications for egg and embryo donation The indications for egg and embryo donation have expanded since its inception. Originally envisioned as a fertility treatment for women with premature ovarian failure (POF),11 today women with many other reproductive disorders are considered prime candidates for therapy (Table 60.1).

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Table 60.1

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Indications for oocyte donation

Premature ovarian failure Gonadal dysgenesis Repetitive IVF failure Natural menopause Inheritable disorders

Noniatrogenic POF, defined as women <40 years old with persistent amenorrhea and elevated gonadotropins, affects approximately 1% of the female population.12 The majority of cases are idiopathic, but about 20% are suspected of being autoimmune in nature or the result of concomitant glandular autoimmune disease.13 Thus, it is important to ensure that clinical or subclinical failure of the thyroid, parathyroid, and adrenal glands does not coexist, as well as diabetes mellitus and myasthenia gravis. Any of these conditions may adversely affect pregnancy outcome as well as impact upon the general health and well-being of the patient. If POF occurs <30 years old, a karyotype should also be requested to ascertain the presence of Y-chromosome mosaicism. Patients discovered to be mosaic are at risk of gonadal tumors and require extirpation of the abnormal gonad.14 In addition, a bone density evaluation is helpful to identify patients with osteopenia or osteoporosis, which may be present despite hormone replacement therapy.15 Other rare conditions associated with POF include congenital thymic aplasia (e.g. DiGeorge syndrome),16 galactosemia,17 and ataxia-telangiectasia,18 all of which require a more thorough and specific evaluation. Chemotherapy and radiation treatments for cancer may also lead to POF. Gonadotoxicity is age and dose dependent, with younger patients being more resistant to damage.19,20 Removal of the ovaries is often required for treatment of malignancies, but surgical castration more commonly results from noncancerous conditions, including infection, torsion, or overly aggressive removal of intraovarian lesions (e.g. cystic teratomas, endometriomas). Repetitive failure at IVF is common when a poor ovarian response to gonadotropins occurs. Occasionally patients are identified as poor candidates for IVF treatment prior to initiating care, thus sparing them the expense and psychological distress of multiple failed cycles. The first consideration is the age of the patient. It has long been known that natural fertility decreases with age, and this is also true with IVF (Fig 60.1).8 Many IVF centers have a maximum age limit beyond which they will not perform IVF without oocyte donation (45 years old at Columbia University). Women of advanced reproductive age have far greater success with donated oocytes.21 Ovarian reserve is evaluated with serum follicle-stimulating hormone (FSH) levels on day 2 or 3 of the menstrual cycle.22 Values >15 mIU/ml are prognostic for a greatly reduced IVF success rate. Another useful serum marker is day 2 or 3 estradiol.23

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60 50 40 30 20 10 0 25 26 28 00 02 04 05 08 40 42 Art patient’s age (years) Donor eggs

44 45 42

Own eggs

Figure 60.1 Live births per transfer for ART cycles using fresh embryos from own and donor eggs, by ART patient’s age, 2004.8

Values >45 pg/ml are predictive of lower pregnancy rates and, if >75 pg/ml, usually attempts end in failure. It is important that each laboratory determines the threshold values that are useful for their program. Other tests are extant to assess ovarian reserve, but are more cumbersome than day 3 serum FSH and estradiol. The clomiphene challenge test measures serum FSH, luteinizing hormone (LH), and estradiol at baseline and again after 5 days (days 5–9) of 100 mg clomiphene citrate.24 Serum FSH values >15 mIU/ml post clomiphene are predictive of IVF failure. Day 2 or 3 serum inhibin B may also define ovarian reserve,25 but the commercially available assay is currently far more complex and time consuming than assays for FSH and estradiol, and not readily available. In certain cases ovarian stimulation is adequate but fertilization rates are poor and often oocyte quality is marginal. Intracytoplasmic sperm injection (ICSI) may or may not be helpful, but if fertilization failure is persistent then oocyte donation is indicated. Similarly, successful fertilization may be present, but implantation does not occur. Assisted hatching may be helpful in these cases. Both ICSI and assisted hatching are discussed in detail in other chapters, but the belief is that recurrent implantation failure is often secondary to poor gametes and may be overcome by oocyte donation. Less clear is the patient with recurrent pregnancy loss, although at least one report suggests oocyte donation is effective in these cases as well.26 Finally, in rare instances IVF failure may be due to ovaries that are inaccessible to either transvaginal or laparoscopic retrieval, and oocytes can be provided only through donation. Although controversial, oocyte donation to treat infertility in women with physiological menopause is very effective.21 The Ethics Committee of the American Society for Reproductive Medicine (ASRM) stated that because of the physical and psychological risks involved (to both mother and child) oocyte donation in postmenopausal women should be discouraged.27 However,

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data on pregnancy outcome in these women, albeit after careful medical and psychological screening, do not reveal any unreasonable risks.28 Some have argued that postmenopausal pregnancy is ‘unnatural,’ but the same may be said of most ART. Furthermore, denying healthy older women donated oocytes while allowing older men complete access to reproductive care is considered by many to be both prejudicial and sexist.29 Less controversial is the use of egg donation for inheritable conditions such as X-linked or autosomal traits and chromosomal translocations.30 However, with progress in preimplantation diagnosis, this reason for choosing egg donation may ultimately decrease.31

Recipient screening In addition to a complete history and physical exam, the suggested medical screening for recipients is shown in Table 60.2. Most of the tests are requisite standards for expectant mothers and IVF candidates. Patients of advanced maternal age are at higher risk for certain conditions such as diabetes mellitus and heart disease and therefore require additional testing focused on these disorders. Other recipients may warrant more comprehensive evaluations, such as a karyotype and autoimmune screen in patients with POF, or screening for anomalies of the aorta and urological system in patients with gonadal dysgenesis. Psychological screening of recipient couples is also recommended. The stress that infertility places on relationships is well known.32 Furthermore, with respect to oocyte donation, the resulting child will not be genetically related to the mother. Most couples reconcile themselves to this, and research has shown that the desire to be parents is more important for positive parenting than a genetic link with the child.33 However, it remains important to address any grief, anxiety, and depression directly with the couple prior to proceeding. The role of the mental healthcare professional is usually one of support and guidance for the couple struggling with these issues. Occasionally, a couple is found to have greatly disparate ideas of what the pregnancy will accomplish. A pregnancy conceived merely to salvage a marriage or relationship is best deferred until the couple resolves their differences. The presence of endometriosis does not affect the pregnancy rate of patients undergoing oocyte donation.34 However, a hydrosalpinx is probably deleterious, and surgical treatment to relieve the obstruction (tuboplasty) or remove the damaged tube (salpingectomy) is recommended.35 Recipients should have a normal uterine cavity free of adhesions, space-occupying lesions, and pathology. This is best assessed by a precycle sonohysterogram. A mock endometrial preparation cycle and timed endometrial biopsy is performed in many programs to ensure that an adequate response to endometrial priming is present. Glandular/stroma dyssynchrony is

809

Table 60.2 Suggested medical screening of oocyte recipient(s). Oocyte recipient

Male partner

Complete blood count with platelets Blood Rh and type Serum electrolytes, liver, and kidney function Sensitive TSH (thyroid stimulating hormone) Rubella and hepatitis screen VDRL HIV-1, HTLV-1 Urinalysis and culture Cervical cultures for gonorrhea and chlamydia Pap smear Transvaginal ultrasound Uterine cavity evaluation (sonohysterogram or hysterosalpingogram) Electrocardiogram* Chest X-ray* Mammogram* Glucose tolerance test* Cholesterol and lipid profile*

Blood Rh and type Hepatitis screen VDRL HIV-1, HTLV-1 Semen analysis and culture

*If over 39 years of age.

often found during endometrial stimulation,36 but apparently this does not adversely affect pregnancy rates.37 Other studies have evaluated endometrial thickness as a predictor of success with oocyte donation.38–40 An endometrium of <6 mm is associated with poor outcome. Another study showed that all endometrial biopsies were in phase if the thickness was >7 mm.41 The great majority of women will have adequate responses to hormone replacement and, therefore, at Columbia University, we have chosen to forgo the mock cycle except in women in whom a poor response is anticipated, such as patients with prior pelvic radiation.42 Should a recipient have a thin endometrium on a previous attempt at egg donation or during a mock cycle, a trial of low-dose aspirin (81 mg daily) given at the time of the transfer cycle may increase pregnancy rates. Weckstein et al found that in women with a previous endometrial thickness of <8 mm, the addition of low-dose aspirin increased the implantation rate from 9% in the untreated group to 24% in the treated group, despite a lack of increased endometrial thickness.43 It has been argued that women of advanced reproductive age may demonstrate a higher percentage of out-of-phase biopsies,44 but both the biopsy and pregnancy outcome may be corrected with appropriate doses of progesterone.45 Of note, older women can expect pregnancy rates with oocyte donation comparable to younger recipients,46–48 whether or not mock cycles are performed.

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Oocyte donor recruitment Perhaps the greatest obstacle to performing oocyte donation is the recruitment of suitable donors.49 Historically, donor eggs were obtained from women undergoing IVF with ‘excess oocytes.’ Many of these patients had ovarian abnormalities underlying their own infertility, making them imperfect donors. Furthermore, with the advent of increasingly successful embryo cryopreservation, ‘extra oocytes’ became scarce. Obvious sources for oocytes are women undergoing tubal sterilization who might be willing to be hyperstimulated. However, very few of these women are eligible, since most are not willing to undergo fertility drug treatment, and many are >35 years old.50 Known designated donors are yet another option. Typically a family member (e.g. sister, niece) or very close friend is selected. The final sources of donors are women recruited from the general population at large, most often through advertisement. There has been a long-standing debate as to whether it is ethical to pay oocyte donors for their eggs, and if so, how much. Areas of contention include the selling of body parts and exaggerated incentives that may represent an enticement for a procedure that carries risk and no direct medical benefit to the donor. For this reason, many countries do not permit oocyte donation (e.g. Germany, Norway, and Sweden).51 Other countries allow only IVF patients with excess oocytes to donate. Israel, Great Britain, and Canada allow anonymous oocyte donation, but strongly discourage payment to the donor, except for verified expenses. The United States has no current regulation on payments to donors. The payments are construed as reimbursement for time and inconvenience,52 and indeed, without payment it is doubtful any country will recruit sufficient donors to meet demand.53 The amount of payment remains hotly debated.54 Another area of controversy focuses on anonymity and identity disclosure. Most donors express a strong desire not to be identified by the children. In exchange for anonymity they willingly forfeit all legal obligations as parents. There is, however, an opposing view that, similar to adopted children, offspring of egg donation should have the same right to ultimately identify their genetic mother.55 As a result, the United Kingdom now mandates that donor identity be revealed to a child resulting from egg donation once he or she reaches the age of 18 years. In the United States there is little historic precedent for such a change in public policy, but should such legislation be enacted, a deleterious effect on donor recruitment can be expected.56

Oocyte donor screening Oocyte donors need to be provided full and comprehensive informed consent. The risks of participating in oocyte donation are few, and basically no different from

Table 60.3

Suggested medical screening of oocyte donors

Complete blood count with platelets Blood type Hepatitis screen VDRL HIV-1, HTLV-1 Cervical cultures for gonorrhea and chlamydia Pap smear Transvaginal ultrasound of pelvis Appropriate genetic tests

those of standard IVF. Even less risk of severe ovarian hyperstimulation syndrome (OHSS) occurs in donors compared with patients undergoing IVF, since pregnancy does not occur and moderate cases of OHSS are not exacerbated.57 In addition to a complete medical history and physical exam, the suggested medical screening of oocyte donors is shown in Table 60.3. Of utmost importance is the screening for infectious diseases. Unlike sperm, which are amenable to cryopreservation, oocytes cannot be easily frozen for subsequent use. In sperm donation, cryopreservation allows a quarantine period and follow-up testing for infectious diseases. With respect to current practice, this is not easily adapted to oocyte donors. Transvaginal ultrasound examination is performed to detect pelvic pathology and determine ovarian morphology. It is preferable that oocyte donors be under 30 years old, as younger donors appear to have higher pregnancy rates.46,58,59 Pregnancy rates of donors >30 years old are still acceptable, however, and other traits and characteristics (e.g. a close physical match to the recipient or advanced educational degree) may make a particular older donor desirable to a recipient. The prior fertility history of the donor does not appear to affect pregnancy outcomes.58,59 The concept of a ‘proven’ donor is a popular myth, and lacks evidence-based support. Psychological evaluation by a licensed mental health practitioner is recommended for anonymous donors and is mandatory for known donors. Screening should focus on their motivation to donate, as well as their financial status to ensure that their participation is not overly influenced by monetary enticement. An assessment of coping skills and lifestyle are important to predict the donor’s ability to participate in a lengthy and complicated process. Occasionally, a history of psychiatric illness or drug and/or alcohol use in the donor or her family is elicited. These behaviors may have a genetic etiology and as such would exclude the potential donor from participation. Genetic screening begins with a detailed history of the potential donor and her family. A sample history form is presented in Table 60.4.60 The presence of any of the disorders should exclude her from participating. Donors should be <35 years old to reduce the risk of aneuploidy in the offspring. Exceptions can be made in circumstances such as sister-to-sister donation where

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Table 60.4

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Genetic screening form given to oocyte donors

Pregnancy history: (please list all the times you have been pregnant and the outcomes) Family ethnic background: Please indicate all relevant information in the following tables. When the requested information is unknown, please say so. If comments are needed, please make them. Remember that we are interested in your genetic background. If any relevant family member is adopted, please say so. Relation Grandfather (pat) Grandmother (pat) Grandfather (mat) Grandmother (mat) Father Mother Brothers Sisters Family Genetic History Familial Conditions High blood pressure Heart disease Deafness Blindness Severe arthritis Juvenile diabetes Alcoholism Schizophrenia Depression or mania Epilepsy Alzheimer’s disease Other (specify)

Age if living

Self

Age at death

Mother

Father

Cause of death

Siblings

Comments

Malformations Cleft lip or palate Heart defect Clubfoot Spina bifida Other (specify) Mendelian disorders Color blindness Cystic fibrosis Hemophilia Muscular dystrophy Sickle cell anemia Huntington’s disease Polycystic kidneys Glaucoma Tay-Sachs disease Please take the time to explain any other problems or conditions in your family history that you feel could pertain to the health of future generations. Reproduced with permission from ref. 59.

the benefits of shared genetic background may balance the known risks (which can be largely discovered by amniocentesis). Donors should also be tested for disorders common to their ethnic background. These include cystic fibrosis in whites, a sickle cell anemia test for blacks, and a complete blood count and mean corpuscular volume followed by hemoglobin electrophoresis in abnormal results for people of Mediterranean and

Chinese ancestry to assess the risk of beta-thalassemia, and in people of southeast Asian ancestry for alphathalassemia. Jews of eastern European ancestry should be screened for Tay–Sachs disease, Gaucher disease, mucolipidosis IV, Niemann–Pick disease, Bloom syndrome, familial dysautonomia, Fanconi anemia, fragile X syndrome, and Canavan disease. It is important to inform the recipient couple that even with appropriate

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screening 2–3% of babies are born with a major or minor malformation, and many genetic disorders cannot be detected or prevented with current testing methodology.61 Guidelines for gamete and embryo donation have been periodically published and recently updated in an attempt to standardize screening policies and to incorporate recent regulations from the US Food and Drug Administration (FDA).62

Endometrial stimulation and synchronization Endometrial preparation of the recipient is modeled on the natural menstrual cycle, using estrogen and progesterone.21 The initial estrogenic phase is most often maintained using either daily oral estradiol 4–8 mg or transdermal estrogen 0.2–0.4 mg. The initial results of oocyte donation cycles were significantly better than typically seen after standard IVF. The apparent detrimental effect of standard IVF on embryo implantation was felt to be secondary to the supraphysiologic concentrations of estrogen attained after controlled ovarian hyperstimulation.63,64 Transdermal estrogen adequately prepares the endometrium with overall lower serum concentrations of estrogens because of the lack of hepatic first-pass effect. However, the higher concentrations of serum estrogens noted following oral administration are of questionable clinical significance. Krasnow et al found estradiol concentrations 10-fold higher in the oral estrogen group and noted a higher rate of out-of phase endometrial biopsies.65 Others, however, have shown no detriment with high levels of estrogen.66 Most programs continue to prescribe oral estradiol due to its ease of administration, lack of side effects, and long history of clinical success. The length of estrogenic exposure may vary widely with little apparent clinical effect, again mimicking the variable follicular phase found in natural menstrual cycles. Anywhere from 6 to 38 days of prescribed estrogen prior to progesterone appears adequate.36,67,68 Most programs prescribe at least 12– 14 days of estrogen before initiating progesterone, but studies report that if it is necessary to prolong this period, perhaps because of a slow stimulation of the oocyte donor, no adverse effects are expected. Synchronization of the recipient and donor is relatively easy to accomplish. The recipient begins estrogen several days prior to beginning ovarian stimulation in the donor in order to provide approximately 14 days of estradiol prior to progesterone administration. Ovulating recipients typically receive gonadotropin-releasing hormone (GnRH) agonist for down-regulation as in standard IVF cycles (e.g. 1 mg leuprolide acetate daily until suppressed, then 0.5 mg daily thereafter) in order to render them functionally agonadal. Alternatively, ovulating recipients are started on oral estrogen at the beginning of their menstrual cycle and maintained on estrogen until

the day of the donor’s oocyte retrieval when progesterone is begun (Fig 60.2).67 The timing of progesterone administration is more stringent. Navot et al reported the optimal time for embryo transfer was 2–4 days after progesterone initiation for embryos at the 2–12-cell stage.69 This corresponds to days 17–19 of the recipient’s cycle, with day 15 defined as the day of progesterone initiation. No pregnancies were observed before 2 days or after 4 days of progesterone administration. These findings were confirmed by Prapas et al, who further delineated the optimal time for transfer of 4–8-cell embryos to days 18 and 19.70 The dose of progesterone is typically 100 mg IM daily or 100–600 mg transvaginally daily. Many groups prefer the transvaginal approach because lower serum concentrations of progesterone are required to achieve target organ effect. Serum levels are low in these patients, but local tissue levels are high probably because of the absence of the hepatic firstpass effect on clearance. As with estrogen, however, it is not resolved whether the mode of delivery of progesterone or its dose is of clinical significance. Most groups continue estrogen support through the progestational period, although at least one study has shown that continued estrogen use is not actually required.71 Progesterone (and estrogen) administration can be discontinued once the placenta has established adequate steroidogenesis. Devroey et al estimated this to occur at 7–9 weeks of gestation,72 while others have advanced this to the 5th week.73 Clinically, we begin weekly monitoring of serum progesterone concentrations 10 weeks after embryo transfer when a serum level of ≥30 ng/ml typically is attained. At that point, prescribing exogenous steroids is superfluous.

Clinical and obstetric outcomes Recipients of donated eggs experience implantation and pregnancy rates similar to those normally seen in young women undergoing IVF. Thus, the ASRM recommends that no more than two high-quality embryos be transferred to patients to lessen the risk of multiple gestation. Oocyte donation has always been associated with the highest success rate among assisted reproductive technologies, and presently more than 50% of embryo transfers result in live births.8 PGD has also been used to better select embryos for transfer since nearly half of biopsied normal appearing embryos selected from donors in their mid-twenties were aneuploid.74 Transferring the preimplantation genetic diagnosis (PGD)-selected embryos improved delivery rates and lowered the miscarriage rate of recipients. Egg donation has been applied to treat infertility in women of advanced reproductive age (>45 years old) since 1990 and has soared in popularity as a result of its ability to reverse the inevitable loss of fertility in women approaching

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Fig 60.2 Schematic of cycle synchronization using a GnRH agonist in both donor and recipient. GnRH agonists are used to down-regulate the pituitary of recipients with evidence of ovarian activity prior to beginning oral estradiol. Oral estradiol is prescribed to the recipient 4–5 days in advance of the donor starting gonadotropin injections. Progesterone is administered start-ing the day after hCG injection in the donor, and 1 day prior to aspirating oocytes. Embryo transfer is performed 3 days following oocyte retrieval. Serum pregnancy testing occurs 12 days post-transfer. Pregnant patients are maintained on estradiol and progesterone through 12 weeks of gestational age.

menopause.75 However, as demonstrated by a large retrospective review of 3089 cycles from Valencia, recipients >45 years old had lower pregnancy rates (49% vs 44%), lower implantation rates (21% vs 17%), and higher miscarriage rates (17% vs 23%) than younger recipients.76 This trend is similarly apparent in reviewing the CDC/SART data from 20048 (Fig 60.3). Therefore, the ability to totally restore uterine receptivity using estradiol and progesterone replacement remains uncertain in the presence of advancing reproductive age. Several groups have evaluated the obstetric outcome of pregnancies following oocyte donation and concluded that results are favorable.28,77–80 Common to all reports, however, were increases in the incidence of pregnancyinduced hypertension (PIH) and delivery by cesarean section. Soderstrom et al compared 51 oocyte donation deliveries to 97 IVF deliveries and noted a higher rate of PIH (31% vs 14%) and cesarean section (57% vs 37%) with oocyte donation.79 PIH was evaluated by the study of 72 pregnancies from donated gametes with age- and parity-matched controls.81 Pre-eclampsia was noted to be much higher in the donated gamete group (18.1% vs 1.4%), suggesting an autoimmune component to the disorder. The increased risk of PIH appears to occur in younger recipients (<35 years old) as well.82 Two other studies evaluated older oocyte donation patients and found most complications, such as gestational diabetes and preterm labor, were associated with multiple pregnancies.28,77 Another clinical trial showed that 59 children of oocyte donation aged 6 months to 4 years old had growth and development comparable to children from IVF and the general population.83 A review of pregnancy outcomes of 45 women >50 years old who delivered babies following egg donation at the University of

Southern California demonstrated an increase in obstetric complications, with pre-eclampsia occurring in 35%, gestational diabetes in 20%, and multiple births in 35%.84 Antinori et al described a 12-year experience with peri- and postmenopausal women between 45 years old and 63 years old in which 2729 women were screened.85 Only 42% of these women were suitable candidates, as the majority were deemed too high risk for pregnancy due to underlying medical conditions. Overall, 1288 recipient cycles resulted in pregnancy in 38% of transfer events with 28% delivering per transfer. Antenatal complications were common (23.6%) in the ongoing pregnancies and included gestational hypertension, gestational diabetes, and preterm labor. In summary, oocyte donation pregnancies should be considered high risk. However, in well-screened patients, the complications are manageable and parents can reasonably expect healthy children.

Embryo donation Embryo donation has become more common as social attitudes towards single women and assisted reproduction have relaxed and the enhanced efficiency of cryopreservation has led to the banking of a large number of human embryos. The deliberate use of donor gametes, utilizing both sperm and egg, was described in 1995 as a means of ‘preimplantation adoption.’86 A programmed approach for creating embryos using donor gametes in single women of advanced reproductive age was suggested again in 1999 as a highly efficient and costeffective means for establishing pregnancy.87 More often, donated embryos are obtained from couples who have successfully conceived through IVF and now wish to give their cryopreserved supernumerary embryos

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70 60

Percent

50 40 30 20 10 0 24 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 54 ART patient’s age (years) Live births per transfer (donor eggs) Singleton live births per transfer (donor eggs)

Figure 60.3 Live births per transfer and singleton live births per transfer for ART cycles using fresh embryos from donor eggs, by ART patient’s age, 2004.8

to clinical programs for use in infertile women.88 Interestingly, couples and women who did not use frozen embryos after pregnancy with donor gametes were more likely to donate them for use in other women than women who had embryos banked following standard IVF with their own eggs. Original guidelines for embryo donation were published by the ASRM in 1998.89 Recommendations include that the embryos undergo a minimum of 6 months’ quarantine and that all donors are retested for infectious diseases prior to their use. Proper documentation of chain-of-custody of donated embryos and witnessed written relinquishment of embryos is also suggested. Although the program may charge professional fees for the service, embryos cannot be ‘sold,’ and donors cannot receive compensation.

Future directions The next frontier in oocyte donation may include the use of enucleated donor oocytes, which would allow recipients to use their own genetic material. This has been successful in cattle and other mammals, but has not been successful as yet in humans.90 Improvements in oocyte freezing may soon permit ‘egg banks’ to be set up, reducing the need to synchronize patients while allowing for quarantine.91,92 Meanwhile, traditional oocyte donation continues to benefit many infertile women who would not otherwise become biological mothers.

References 1. Heape W. Preliminary note on the transplantation and growth of mammalian ova in a foster mother. Proc R Soc London 1890; 48: 457. 2. Hasler JF. Current status and potential of embryo transfer and reproductive technology in dairy cattle. J Dairy Sci 1992; 75: 2857–79.

3. Buster JE, Bustillo M, Thorneycroft I, et al. Nonsurgical transfer of an in-vivo fertilised donated ovum to an infertility patient. Lancet 1983; 1: 816–17. 4. Buster JE, Bustillo M, Thorneycroft IH, et al. Nonsurgical transfer of in vivo fertilised donated ova to five infertile women: report of two pregnancies. Lancet 1983; 2: 223–4. 5. Bustillo M, Buster JE, Cohen SW, et al. Delivery of a healthy infant following nonsurgical ovum transfer. JAMA 1984; 251: 889. 6. Trounson A, Leeton J, Besanko M, Wood C, Conti A. Pregnancy established in an infertile patient after transfer of a donated embryo fertilised in vitro. Br Med J (Clin Res Ed) 1983; 286: 835–8. 7. Lutjen P, Trounson A, Leeton J, et al. The establishment and maintenance of pregnancy using in vitro fertilization and embryo donation in a patient with primary ovarian failure. Nature 1984; 307: 174–5. 8. Centers for Disease Control and Prevention. 2004 Assisted Reproductive Technology Success Rates. National Summary and Clinic Reports Department of Health and Human Services, December 2006. 9. Ventura SJ. First births to older mothers, 1970–86. Am J Public Health 1989; 79: 1675–7. 10. Hollander D, Breen JL. Pregnancy in the older gravida: how old is old? Obstet Gynecol Surv 1990; 45: 106–12. 11. Bustillo M, Buster JE, Cohen SW, et al. Nonsurgical ovum transfer as a treatment in infertile women. Preliminary experience. JAMA 1984; 251: 1171–3. 12. Coulam CB, Adamson SC, Annegers JF. Incidence of premature ovarian failure. Obstet Gynecol 1986; 67: 604–6. 13. Coulam CB. Premature gonadal failure. Fertil Steril 1982; 38: 645–55. 14. Manuel M, Katayama PK, Jones HW Jr. The age of occurrence of gonadal tumors in intersex patients with a Y chromosome. Am J Obstet Gynecol 1976; 124: 293–300. 15. Cohen MA, Chang PL, Lindheim SR, Sauer MV. Diminished bone density in menopausal women undergoing ovum donation. Annual Meeting of the American Society for Reproductive Medicine, San Francisco, California, 1998 (abstract). 16. Moncayo R, Moncayo HE. Autoimmunity and the ovary. Immunol Today 1992; 13: 255–8. 17. Kaufman FR, Donnell GN, Roe TF, Kogut MD. Gonadal function in patients with galactosaemia. J Inherit Metab Dis 1986; 9: 140–6. 18. Christin-Maitre S, Vasseur C, Portnoi MF, Bouchard P. Genes and premature ovarian failure. Mol Cell Endocrinol 1998; 145: 75–80. 19. Byrne J, Mulvihill JJ, Myers MH, et al. Effects of treatment on fertility in long-term survivors of childhood or adolescent cancer. N Engl J Med 1987; 317: 1315–21. 20. Gradishar WJ, Schilsky RL. Ovarian function following radiation and chemotherapy for cancer. Semin Oncol 1989; 16: 425–36. 21. Sauer MV, Paulson RJ, Lobo RA. A preliminary report on oocyte donation extending reproductive potential to women over 40. N Engl J Med 1990; 323: 1157–60. 22. Scott RT, Toner JP, Muasher SJ, et al. Follicle-stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome. Fertil Steril 1989; 51: 651–4.

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Egg and embryo donation 23. Licciardi FL, Liu HC, Rosenwaks Z. Day 3 estradiol serum concentrations as prognosticators of ovarian stimulation response and pregnancy outcome in patients undergoing in vitro fertilization. Fertil Steril 1995; 64: 991–4. 24. Navot D, Rosenwaks Z, Margalioth EJ. Prognostic assessment of female fecundity. Lancet 1987; 2: 645–7. 25. Seifer DB, Lambert-Messerlian G, Hogan JW, et al. Day 3 serum inhibin-B is predictive of assisted reproductive technologies outcome. Fertil Steril 1997; 67: 110–14. 26. Remohi J, Gallardo E, Levy M, et al. Oocyte donation in women with recurrent pregnancy loss. Hum Reprod 1996; 11: 2048–51. 27. Ethics Committee of the American Society for Reproductive Medicine. Ethical considerations of assisted reproductive technologies. Fertil Steril 1997; 67(Suppl 1): 1S–9S. 28. Sauer MV, Paulson RJ, Lobo RA. Oocyte donation to women of advanced reproductive age: pregnancy results and obstetrical outcomes in patients 45 years and older. Hum Reprod 1996; 11: 2540–3. 29. Paulson RJ, Sauer MV. Pregnancies in postmenopausal women. Oocyte donation to women of advanced reproductive age: ‘how old is too old?’ Hum Reprod 1994; 9: 571–2. 30. Van Voorhis BJ, Williamson RA, Gerard JL, Hammitt DG, Syrop CH. Use of oocytes from anonymous, matched, fertile donors for prevention of heritable genetic diseases. J Med Genet 1992; 29: 398–9. 31. Munné S, Magli C, Bahce M, et al. Preimplantation diagnosis of the aneuploidies most commonly found in spontaneous abortions and live births: XY, 13, 14, 15, 16, 18, 21, 22. Prenat Diagn 1998; 18: 1459–66. 32. Burns L. An overview of the psychology of infertility. Infertil Reprod Med Clin N Am 1993; 3: 433–54. 33. Golombok S, Cook R, Bish A, Murray C. Families created by the new reproductive technologies: quality of parenting and social and emotional development of the children. Child Dev 1995; 66: 285–98. 34. Bustillo M, Krysa LW, Coulam CB. Uterine receptivity in an oocyte donation programme. Hum Reprod 1995; 10: 442–5. 35. Cohen MA, Lindheim SR, Sauer MV. Hydrosalpinges adversely affect implantation in donor oocyte cycles. Hum Reprod 1999; 14: 1087–9. 36. Navot D, Anderson TL, Droesch K, et al. Hormonal manipulation of endometrial maturation. J Clin Endocrinol Metab 1989; 68: 801–7. 37. Navot D, Bergh PA, Williams M, et al. An insight into early reproductive processes through the in vivo model of ovum donation. J Clin Endocrinol Metab 1991; 72: 408–14. 38. Abdalla HI, Brooks AA, Johnson MR, et al. Endometrial thickness: a predictor of implantation in ovum recipients? Hum Reprod 1994; 9: 363–5. 39. Antinori S, Versaci C, Gholami GH, Panci C, Caffa B. Oocyte donation in menopausal women. Hum Reprod 1993; 8: 1487–90. 40. Shapiro H, Cowell C, Casper RF. The use of vaginal ultrasound for monitoring endometrial preparation in a donor oocyte program. Fertil Steril 1993; 59: 1055–8. 41. Hofmann GE, Thie J, Scott RT Jr, Navot D. Endometrial thickness is predictive of histologic endometrial

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maturation in women undergoing hormone replacement for ovum donation. Fertil Steril 1996; 66: 380–3. Li TC, Dockery P, Ramsewak SS, et al. The variation of endometrial response to a standard hormone replacement therapy in women with premature ovarian failure. An ultrasonographic and histological study. Br J Obstet Gynaecol 1991; 98: 656–61. Weckstein LN, Jacobson A, Galen D, Hampton K, Hammel J. Low-dose aspirin for oocyte donation recipients with a thin endometrium: prospective, randomized study. Fertil Steril 1997; 68: 927–30. Potter DA, Witz CA, Burns WN, Brzyski RG, Schenken RS. Endometrial biopsy during hormone replacement cycle in donor oocyte recipients before in vitro fertilization–embryo transfer. Fertil Steril 1998; 70: 219–21. Meldrum D. Female reproductive aging – ovarian and uterine factors. Fertil Steril 1993; 59: 1–5. Balmaceda JP, Bernardini L, Ciuffardi I, et al. Oocyte donation in humans: a model to study the effect of age on embryo implantation rate. Hum Reprod 1994; 9: 2160–3. Abdalla HI, Wren ME, Thomas A, Korea L. Age of the uterus does not affect pregnancy or implantation rates; a study of egg donation in women of different ages sharing oocytes from the same donor. Hum Reprod 1997; 12: 827–9. Stolwijk AM, Zielhuis GA, Sauer MV, Hamilton CJ, Paulson RJ. The impact of the woman’s age on the success of standard and donor in vitro fertilization. Fertil Steril 1997; 67: 702–10. Marina S, Exposito R, Marina F, et al. Oocyte donor selection from 554 candidates. Hum Reprod 1999; 14: 2770–6. Feinman M, Barad D, Szigetvari I, Kaali SG. Availability of donated oocytes from an ambulatory sterilization program. J Reprod Med 1989; 34: 441–3. Gunning JH. Oocyte donation: the legislative framework in Western Europe. Hum Reprod 1998; 13(Suppl 2): 98–104. Sauer MV. Reproductive prohibition: restricting donor payment will lead to medical tourism. Hum Reprod 1997; 12: 1844–5. McLaughlin EA, Day J, Harrison S, et al. Recruitment of gamete donors and payment of expenses. Hum Reprod 1998; 13: 1130–2. Sauer MV. Indecent proposal: $5,000 is not “reasonable compensation” for oocyte donors. Fertil Steril 1999; 71: 7–10. Annas GJ. The shadowlands – secrets, lies, and assisted reproduction. N Engl J Med 1998; 339: 935–9. Sauer MV. Further HFEA restrictions on egg donation in the UK: two strikes and you’re out! Reprod Biomed Online 2005; 10: 431–3. Sauer MV, Paulson RJ, Lobo RA. Rare occurrence of ovarian hyperstimulation syndrome in oocyte donors. Int J Gynaecol Obstet 1996; 52: 259–62. Faber BM, Mercan R, Hamacher P, Muasher SJ, Toner JP. The impact of an egg donor’s age and her prior fertility on recipient pregnancy outcome. Fertil Steril 1997; 68: 370–2. Cohen MA, Lindheim SR, Sauer MV. Donor age is paramount to success in oocyte donation. Hum Reprod 1999; 14: 2755–8.

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60. Brown S. Genetic aspects of donor selection. In: Sauer MV, ed. Principles of Oocyte and Embryo Donation. New York: Springer-Verlag, 1998: 53–63. 61. Baird PA, Anderson TW, Newcombe HB, Lowry RB. Genetic disorders in children and young adults: a population study. Am J Hum Genet 1988; 42: 677–93. 62. The Practice Committee of the American Society for Reproductive Medicine and the Practice Committee of the Society for Assisted Reproductive Technology. 2006 Guidelines for gamete and embryo donation. Fertil Steril 2006; 86(Suppl 4): S38–50. 63. Edwards RG. Why are agonadal and post-amenorrhoeic women so fertile after oocyte donation? Hum Reprod 1992; 7: 733–4. 64. Check JH, Nowroozi K, Chase J, Nazari A, Braithwaite C. Comparison of pregnancy rates following in vitro fertilization–embryo transfer between the donors and the recipients in a donor oocyte program. J Assist Reprod Genet 1992; 9: 248–50. 65. Krasnow JS, Lessey BA, Naus G, et al. Comparison of transdermal versus oral estradiol on endometrial receptivity. Fertil Steril 1996; 65: 332–6. 66. de Ziegler D. Hormonal control of endometrial receptivity. Hum Reprod 1995; 10: 4–7. 67. Serhal PF, Craft IL. Ovum donation – a simplified approach. Fertil Steril 1987; 48: 265–9. 68. Younis JS, Mordel N, Ligovetzky G, et al. The effect of a prolonged artificial follicular phase on endometrial development in an oocyte donation program. J In Vitro Fert Embryo Transf 1991; 8: 84–8. 69. Navot D, Scott RT, Droesch K, et al. The window of embryo transfer and the efficiency of human conception in vitro. Fertil Steril 1991; 55: 114–18. 70. Prapas Y, Prapas N, Jones EE, et al. The window for embryo transfer in oocyte donation cycles depends on the duration of progesterone therapy. Hum Reprod 1998; 13: 720–3. 71. Lewin A, Benshushan A, Mezker E, et al. The role of estrogen support during the luteal phase of in vitro fertilization–embryo transplant cycles: a comparative study between progesterone alone and estrogen and progesterone support. Fertil Steril 1994; 62: 121–5. 72. Devroey P, Camus M, Palermo G, et al. Placental production of estradiol and progesterone after oocyte donation in patients with primary ovarian failure. Am J Obstet Gynecol 1990; 162: 66–70. 73. Scott R, Navot D, Liu HC, Rosenwaks Z. A human in vivo model for the luteoplacental shift. Fertil Steril 1991; 56: 481–4. 74. Reis Soares S, Rubio C, Rodrigo L, et al. Does preimplantation genetic diagnosis improve the outcome of egg donation cycles? Presented at the 19th Annual Meeting of ESHRE, 2003, P-568, xviii, 189. 75. Sauer MV, Kavic SM. Oocyte and embryo donation 2006: reviewing two decades of innovation and controversy. Reprod Biomed Online 2006; 12: 153–62. 76. Reis Soares S, Troncoso C, Bosch E, et al. Age and uterine receptiveness: predicting the outcome of oocyte donation cycles. J Clin Endocrinol Metab 2005; 90: 4399–404.

77. Pados G, Camus M, Van Steirteghem A, Bonduelle M, Devroey P. The evolution and outcome of pregnancies from oocyte donation. Hum Reprod 1994; 9: 538–42. 78. Wolff KM, McMahon MJ, Kuller JA, Walmer DK, Meyer WR. Advanced maternal age and perinatal outcome: oocyte recipiency versus natural conception. Obstet Gynecol 1997; 89: 519–23. 79. Soderstrom-Anttila V, Tiitinen A, Foudila T, Hovatta O. Obstetric and perinatal outcome after oocyte donation: comparison with in-vitro fertilization pregnancies. Hum Reprod 1998; 13: 483–90. 80. Abdalla HI, Billett A, Kan AK, et al. Obstetric outcome in 232 ovum donation pregnancies. Br J Obstet Gynaecol 1998; 105: 332–7. 81. Salha O, Sharma V, Dada T, et al. The influence of donated gametes on the incidence of hypertensive disorders of pregnancy. Hum Reprod 1999; 14: 2268–73. 82. Keegan DA, Krey LC, Chang HC, Noyes N. Increased risk of pregnancy-induced hypertension in young recipients of donated oocytes. Fertil Steril 2007; 87: 776–81. 83. Soderstrom-Anttila V, Sajaniemi N, Tiitinen A, Hovatta O. Health and development of children born after oocyte donation compared with that of those born after in-vitro fertilization, and parents’ attitudes regarding secrecy. Hum Reprod 1998; 13: 2009–15. 84. Paulson RJ, Boostanfar R, Saadat P, et al. Pregnancy in the sixth decade of life. Obstetric outcomes in women of advanced reproductive age. JAMA 2002; 288: 2320–3. 85. Antinori S, Gholami GH, Versaci C, et al. Obstetric and prenatal outcome in menopausal women: a 12year clinical study. Reprod Biomed Online 2002; 6: 257–61. 86. Sauer MV, Paulson RJ, Francis MM, Macaso TM, Lobo RA. Preimplantation adoption: establishing pregnancy using donated oocytes and spermatozoa. Hum Reprod 1995; 10: 1419–22. 87. Lindheim SR, Sauer MV. Embryo donation: a programmed approach. Fertil Steril 1999; 72: 940–1. 88. Sehnert B, Chetkowski RJ. Secondary donation of frozen embryos is more common after pregnancy initiation with donated eggs than after in vitro fertilization–embryo transfer and gamete intrafallopian transfer. Fertil Steril 1998; 69: 350–2. 89. The American Society for Reproductive Medicine. Guidelines for Gamete and Embryo Donation. Guidelines for embryo donation. Fertil Steril 1998; 70(Suppl 3): 7S–8S. 90. Dominko T, Mitalipova M, Haley B, et al. Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuceli from various mammallian species. Biol Reprod 1999; 60: 1496–502. 91. Tucker MJ, Morton PC, Wright G, Sweitzer CL, Massey JB. Clinical application of human egg cryopreservation. Hum Reprod 1998; 13: 3156–9. 92. Kuleshova L, Gianaroli L, Magli C, Ferraretti A, Trounson A. Birth following vitrification of a small number of human oocytes: case report. Hum Reprod 1999; 14: 3077–9.

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61 Gestational surrogacy Peter R Brinsden

Overview Surrogacy has been practiced as a means of helping women who are unable to bear children for centuries. The earliest mention is in the Old Testament of the Bible.1 Before the advent of modern assisted conception techniques, ‘natural surrogacy’ was the only means of helping certain barren women to have babies. Before the introduction of artificial insemination, babies were conceived the ‘natural way,’ as practiced by Abraham.1 Later, as artificial insemination techniques were introduced, it became more socially acceptable to use these than ‘natural means.’ Later still, when assisted conception techniques, such as in vitro fertilization (IVF) were introduced, embryos created entirely from the gametes of the ‘genetic’ or ‘commissioning couple’ could be transferred to the ‘surrogate host,’ who therefore provided no genetic contribution to any child that resulted from the arrangement. She bore the child and handed it over to the full ‘genetic parents.’ ‘Gestational surrogacy,’ otherwise known as ‘IVF surrogacy’ or ‘full surrogacy’ is now generally accepted in many countries as a treatment option for infertile women with certain clearly defined medical problems. The first report of a baby being born by gestational surrogacy was from the USA in 1985.2 Gestational surrogacy is now accepted in the UK as a treatment option for infertile women, provided there are clearly defined medical indications. A report commissioned by the British Medical Association (BMA) in 19903 provided the first evidence that surrogacy was formally accepted as a legitimate treatment option. However, in 1984 the Warnock Committee4 had recommended to the UK Government that surrogacy should be prohibited. Opinions then started to change in 1985, when the annual representative meeting of the BMA passed a resolution – ‘This meeting agrees with the principle of surrogate births in selected cases with careful controls.’5 However, the BMA then published a further report in 1987,6 which stated that surrogacy was not an acceptable form of treatment and, at the Annual General Meeting later that year, the concept of surrogacy was further rejected, in spite of the 1985 resolution. The 1987 report made it clear that doctors ‘Should not participate in any surrogacy arrangements.’6 However, a working party which subsequently reported to the BMA in 1990,3

stated that – ‘It would not be possible or desirable to seek to prevent all involvement of doctors in surrogacy arrangements, especially as the Government does not intend to make the practice illegal.’ This report proposed guidelines for doctors, making it clear that only after intensive investigation and counseling, and very much as a last resort option, should IVF surrogacy be used as a treatment to overcome a couple’s infertility problem. In the same year, the Human Fertilisation and Embryology Act (1990)7 was passed through the UK Parliament and did not ban surrogacy. The most recent report of the BMA8 states that ‘surrogacy is an acceptable option of last resort in cases where it is impossible or highly undesirable for medical reasons for the intended mother to carry a child herself.’ During the years of this protracted debate in the UK, most other European countries had decided to ban the practice of surrogacy of any kind. The largest experience of both natural and gestational surrogacy is in the USA, where commercial surrogacy arrangements are allowed. Relatively few publications of experience with gestational surrogacy appeared in the literature in the early years, and there were few long-term follow-up studies of the babies or of the couples involved in surrogacy arrangements9–12 in spite of strong recommendations to do so.13,14 However, more recently, a number of studies have been published on couples’ long-term experiences and their children, which are reassuring, and which are further discussed in the Results section of this chapter. In 1986 at Bourn Hall Clinic, despite opposition from the BMA and the recommendation of the Warnock report, Mr Patrick Steptoe and Professor Robert Edwards, the pioneers of IVF, first proposed treating a patient by IVF surrogacy. After extensive discussions with the independent Ethics Committee to the Clinic, they undertook treatment of the first couple in the United Kingdom.15 Following an IVF treatment cycle, embryos from the ‘genetic couple’ were transferred to the sister of the woman and a child was born to them in 1989. In the same year, the Ethics Committee to Bourn Hall drew up guidelines for the treatment of women by IVF surrogacy and the full program was formalized in 1990. The outcomes of the treatment of 49 genetic couples treated at Bourn Hall since then are detailed later in this chapter, together with a review of the results of clinics in the USA, where there is the largest experience of gestational surrogacy.

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Methods

Table 61.1

Indications for treatment by gestational surrogacy

Definitions of terms There has always been confusion among patients, practitioners, and even different countries on the definition of the different forms of surrogacy. It is common practice to use the term ‘surrogate mother’ or ‘surrogate’ for the woman who carries and delivers a baby. Others would argue, however, that it is the woman who rears the child, rather than the one who gives birth, who is the surrogate mother, and the woman who gives birth is the mother not the surrogate. Since the woman who gives birth is initially the legal mother of that child, further confusion is added. ‘Gestational surrogacy,’ ‘full surrogacy,’ or ‘IVF surrogacy’ is defined as treatment by which the gametes of the ‘genetic couple,’ ‘commissioning couple,’ or ‘intended parents’ in a surrogacy arrangement are used to produce embryos that are subsequently transferred to a woman who agrees to act as a host for these embryos. The ‘surrogate host’ is therefore genetically unrelated to any offspring that may be born as a result of this arrangement. With ‘natural surrogacy’ or ‘partial surrogacy’ the intended host is inseminated with the semen of the husband of the ‘genetic couple.’ Any resulting child is therefore genetically related to the host. In this chapter, only treatment by ‘gestational surrogacy’ is considered and the couple who initiate the surrogacy arrangement and whose gametes are used will be known as the ‘genetic couple’ and the woman who subsequently carries the child will be known as the ‘surrogate host.’

Indications for ‘gestational surrogacy’ The principal indications for treatment by ‘gestational surrogacy’ in our practice at Bourn Hall are shown in Table 61.1. Absence of a uterus following hysterectomy for uterine or cervical carcinoma, or following hemorrhage, or congenital absence are the main indications. Other women who have suffered repeated miscarriages and are deemed to have little or no chance of carrying a child to term are considered for treatment. Repeated failure of treatment by IVF is also an indication for treatment, but it has only been used for women who have never shown any signs of implanting normal embryos in an apparently normal uterus after at least 6–8 IVF–ET (embryo transfer) cycles. There are certain medical conditions which would threaten the life of a woman were she to become pregnant, such as severe heart disease or renal disease, which are also indications. Discussion is always held with the specialist looking after the medical problems of these women, and the Ethics Committee require evidence that the female partner of the ‘genetic couple’ will be able to look after a child adequately and that her life expectancy is reasonable. Women are not considered for treatment who request it purely for career or social reasons.

• Congenital absence of the uterus • Following hysterectomy for cancer, postpartum hemorrhage, or menorrhagia • Repeated failure of IVF treatment • Recurrent abortion • Severe medical conditions incompatible with pregnancy

Because the indications for treatment are relatively limited, the actual need for treatment by gestational surrogacy is also limited. In our own practice, treatment by surrogacy accounts for <1% of the total annual throughput of cases, out of a total of about 1400 IVF and frozen embryo replacement cycles. It is practiced in only a limited number of IVF centers both in the UK16 and in the USA. In a worldwide survey on behalf of the International Federation of Fertility Societies (IFFS), Jones HW et al17 reported that, of the 57 countries surveyed, 20 allowed and/or practiced surrogacy.

Selection of patients for treatment In our own practice, all ‘genetic couples,’ are referred by their general practitioners or gynecologists and are therefore already selected as probably being suitable for treatment. The ‘genetic couple’ are seen alone in the first instance and in-depth consultation and counseling on all medical aspects of the treatment are carried out. If they are considered to be medically suitable for treatment and fall within the guidelines laid down by the independent Ethics Committee to Bourn Hall Clinic and they comply with the Code of Practice of the Human Fertilisation and Embryology Authority (HFEA),18 particularly with regard to the welfare of any child born as a result of treatment, the couple are informed that they are required by law in the UK19 to find a host for themselves. They are told that the host may be a member of the ‘genetic couples’ family, or a close friend, or that they may be able to find a suitable host through one of the patient infertility support groups in the UK set up to help couples seeking hosts and for potential hosts seeking couples to help. Other groups have also reported using sisters,20 mothers,21 and support groups or other agencies.10,22 All groups practicing gestational surrogacy are adamant about the need for in-depth medical and psychological screening of all ‘genetic couples,’ the surrogate hosts, and other members of the family, especially any existing children of the surrogate host, and possibly also the parents of the hosts and genetic couples.22,23 In our own practice, when a suitable host has been found, she and her partner are interviewed at length and a full explanation of the implications of acting as a surrogate host explained to them. If the host is thought to be suitable, then both the genetic and host couples are counseled in depth. If this process is satisfactory and there are

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no obvious reasons why the arrangement should not be allowed to proceed from a medical and counseling standpoint, a report is prepared and submitted to the independent Ethics Committee to this Clinic. The Committee then either approves the arrangement, holds it over for further information and discussion, or rejects it. In every case, the Clinic has acted in accordance with the recommendations of the Ethics Committee. It must be stressed that, in all surrogacy arrangements, the welfare of any child born as a result of treatment and of any existing children of a family is given the utmost importance. This is in accordance with the Code of Practice of the HFEA18 drawn up as a result of the Act of Parliament passed by the UK government in 1990.8

Counseling In-depth counseling of all parties engaged in surrogacy arrangements is of paramount importance and aims to prepare all parties contemplating this treatment of last resort to consider all the facts which will have an influence on the future lives of each of them. They must be confident and comfortable with their decisions and have trust in each other, so that no one party is felt to be taking advantage of the other. The BMA in its 1990 report3 produced a most useful statement: ‘The aggregate of foreseeable hazards should not be so great as to place unacceptable burdens on any of the parties – including the future child.’ Many issues must be discussed with both the genetic couple and the proposed host surrogate, including:

For the genetic couples:8 • • • • • • • • • • •

• •

A review of all alternative treatment options The need for in-depth counseling The need to find their own host (UK) The practical difficulty and cost of treatment by gestational surrogacy The medical and psychological risks of surrogacy Potential psychological risk to the child The chances of having a multiple pregnancy The degree of involvement that the host may wish to have with the child The possibility that a child may be born with a handicap The risks to the baby of the host smoking and drinking during a pregnancy The possibility that the host may wish to retain the child after birth and the fact that surrogacy contracts in the UK are not enforceable The importance of obtaining legal advice The genetic couple are advised to take out insurance cover for the surrogate host

For the host:8 • •

The full implications of undergoing treatment by IVF surrogacy The possibility of multiple pregnancy

• • • • •

• •

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The possibility of family and friends being against such treatment The need to abstain from unprotected sexual intercourse during and just before the treatment The normal medical risks associated with pregnancy and the possibility of cesarean section Implications and feelings of guilt on both sides if the host should spontaneously abort a pregnancy The possibility that the host will feel a sense of bereavement when she gives the baby to the genetic couple The possibility that the child may be born with a handicap The fact that hosts in the UK are expected to only claim ‘reasonable expenses’

Other issues that must be discussed with both parties to a surrogacy arrangement include in-depth discussions on whether and what both parties will tell the children born as a result of treatment in the future about their origins and also what the host mother will tell any children she has. There is an increasing willingness of all couples involved with treatment by assisted reproductive technologies (ART) to be more open about their treatment, whether this be by IVF, the use of donor gametes, or surrogacy. It is felt by most workers in the area that it is better for couples to be open with their children about their origins rather than to try and cover them up. Another issue that is often raised in counseling is whether the genetic mother may be able to breastfeed her baby when it is given to her by the host surrogate. There is a belief that the genetic mother may be able to provide some breast milk, which will almost certainly require bottle supplementation, if she puts the child to the breast regularly. It has been proposed that the genetic mother who receives the baby should prepare for the possibility of breastfeeding by stimulating secretion of milk manually, or with a breast pump, in the few weeks leading up to delivery of her child. If there is an enthusiasm to breastfeed, then it is worth an attempt, but there is a strong possibility of disappointment.

Patient management Management of the genetic mother The majority of ‘genetic mothers’ treated in this Clinic are fully assessed by their gynecologist before referral. The work-up usually includes a laparoscopy, if there are congenital anomalies, but it is not necessary after hysterectomy. Evidence of ovarian function can often be obtained from a history of cyclical premenstrual symptoms or symptoms of ovulation. This can be confirmed by one or more estimations of serum folliclestimulating hormone (FSH) and luteinizing hormone (LH), and possibly timed progesterone levels in the estimated luteal phase. The blood groups of the genetic parents are requested in case the host is rhesus negative

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and both the genetic parents are tested for hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus (HIV) status. Ultrasound scanning of the ovaries is carried out on some patients to confirm the presence of one or both ovaries, their position, and possible evidence of their activity. Other investigations are carried out as necessary on an individual basis. On completion of the full medical assessment, the counseling process, and when the approval of the Ethics Committee has been obtained, treatment of the genetic couple is started, provided the host has already been identified, fully counseled, and approved. Since most women requesting treatment by gestational surrogacy are perfectly normal with regard to their ovarian function, the management of their IVF treatment cycles is straightforward. Ovarian follicular stimulation, monitoring, and oocyte recovery methods as practiced in this Clinic have previously been described.24–26 Oocytes are collected by the standard transvaginal ultrasound-guided technique originally described by Wikland et al in 1985,27 and by our own group.28 In all treatment cycles in the UK, the embryos obtained from the genetic couple must be frozen for a 6-month ‘quarantine’ period for HIV status prior to their transfer to the uterus of the surrogate host. However, where a delay in treatment is expected, the semen of the husband of the genetic couple may be frozen for ≥6 months and, after a further test of HIV status, the embryos are then transferred ‘fresh’ to the host. This policy is in line with the regulation of the HFEA that the sperm used in surrogacy cases should be treated in the same way as donor sperm, which by law must be frozen and quarantined for 6 months before it can be used.26

Table 61.2 Relationship of genetic mothers to surrogate hosts in the Bourn Hall gestational surrogacy program, with proportions in each group Relations: – Sister-to-sister – Sister-to-sister-in-law – Stepdaughter to stepmother

35% 20% 5%

• Friend-to-friend

15%

• Through an organization (e.g. COTS)

25%

COTS, Childlessness Overcome Through Surrogacy.

Embryo transfer to the surrogate host may either be carried out in a natural menstrual cycle or in a cycle controlled with exogenous hormone treatment. The latter is recommended if the menstrual cycles of the host are irregular, if they are found not to be ovulating normally, or if luteal phase insufficiency is suspected. In the early days of our own program, all fertile hosts who relied on barrier methods of contraception were placed on a luteinizing hormone-releasing hormone (LH-RH) analog regimen combined with hormone replacement therapy (HRT) in order to prevent any chance of natural conception. More recently, however, with proper advice on barrier contraception and an awareness of the strong motivation of hosts, this method has largely been discontinued. The management of the hormone-controlled cycles for the transfer of frozen–thawed embryos has been described previously.30,31

Management of the surrogate host

Results

In the UK, the recruitment of a host surrogate must be carried out by the genetic couple themselves. In our own Unit, only normal fit women who are ≤38 years old and who have had at least one child are considered. Other groups will consider any woman <45 years old who is willing to act as host.29 The relationships between the surrogate hosts and the genetic mothers in our own series are shown in Table 61.2. The Ethics Committee have recommended that hosts should be married or in a stable heterosexual relationship and that the husband or partner should be made fully aware during the counseling process of the implications of his partner acting as a surrogate host. Fertility investigations of the proposed host have not been necessary. All hosts and their partners are tested for HBV, HCV, and HIV status before the embryo transfer is carried out, and the HIV status of the ‘genetic couple’ is retested. If the surrogate host is taking the oral contraceptive pill, it is discontinued one cycle before the replacement cycle and barrier methods of contraception or abstinence from intercourse are strongly recommended.

Treatment by gestational surrogacy generally achieves satisfactory pregnancy and delivered baby rates per genetic couple and per surrogate host. In our own series live birth rates of between 37% and 43% per genetic or commissioning couple and 34% and 39% per host surrogate have been achieved with a mean of two embryos transferred.26,32 Another UK series in which all the female partners in the ‘genetic couple’ had had an hysterectomy achieved a pregnancy rate of 37.5% per surrogate host and 27.3% (6/22) per cycle of treatment begun.33 In the original series reported by Utian and colleagues,9 they achieved a clinical pregnancy rate of 11.9% (7/59) per cycle initiated and 23% clinical pregnancy rate per embryo transfer. Other more recently reported series from the USA have shown ongoing or delivered pregnancy rates of 36% (172 of 484 surrogate hosts),34 with a mean of 5 ± 1.3 embryos transferred. Corson and colleagues reported a clinical pregnancy rate of 58% per commissioning couple and 33.2% per embryo transfer in women where the genetic women were <40 years old.35 What has recently become apparent is that very little investigation of the immediate and long-term outcome

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of the babies born as a result of gestational surrogacy has been carried out. However, Parkinson et al36 have reviewed the perinatal outcome of pregnancies from IVF surrogacy and compared them to the outcome of pregnancies resulting from standard IVF. As would be expected, the surrogate hosts who carried twin and triplet gestations delivered substantially earlier than those who gestated singleton pregnancies, and the twin newborns were significantly lighter than singleton infants born through IVF surrogacy. Interestingly, the occurrence of pregnancy-induced hypertension and bleeding in the third trimester of pregnancy was up to 5 times lower in the surrogate hosts than in the standard IVF patient controls. Apart from birth weights and prematurity, little other information is given about the outcome of the babies. There have been very few long-term follow-up studies of women who have acted as surrogate hosts, but there is little to suggest any long-term harm or regret among them.12,13,37 The most recent studies of hosts and commissioning couples show reassuring data and positive outcomes, particularly for the hosts.38–41

•

•

•

Complications Problems encountered with gestational surrogacy The major problems that have been reported with surrogacy arrangements have almost entirely arisen from ‘natural surrogacy’ arrangements. The major problems have been legal, and mostly revolve around the ‘ownership’ and rights of both the ‘genetic couple’ and the birth mothers. These are not further considered in this chapter but they are well documented in a number of papers published on the subject.3,13,14,42–45 The main reason these problems have arisen is that the majority of the arrangements were largely unsupervised and did not involve careful clinical and psychological assessment, counseling, and discussion with lawyers. With gestational surrogacy, professionals in all of these areas are invariably involved and, as a consequence, the number of complications arising out of these treatments are very few. In the past 18 years of our own experience, no serious clinical, ethical, or legal problems have been encountered. The major ethical and practical problems that might be encountered with IVF surrogacy include: •

•

The host may wish to keep the child. This is the complication that all practitioners in this area worry about most, but, with proper counseling and legal advice, it has not occurred in our own series. The cases that have come to light have invariably involved ‘natural surrogacy.’ An abnormal child may be rejected by both the genetic and host parents. This is of course a major concern, but has not yet occurred in our own experience nor has any other group published on the occurrence of this complication.

821

The question of whether it is ethical to pay hosts and, if so, how much has always caused concern. In the USA, payment is ‘up front’ and revealed, whereas in the UK and most of Europe altruistic surrogacy is what everyone aspires to, but it is in effect impractical and payment is often hidden as ‘reasonable expenses.’ Many also consider it unethical not to pay hosts for the sacrifices that they make to help other couples. The European Society for Human Reproduction & Embryology (ESHRE) Task Force on Ethics and Law (2005)29 states that ‘payment for [surrogacy] services is unacceptable….’ While the IFFS Surveillance Report 200717 states only that ‘The payment to the surrogate raises special concerns.’ The long-term effects on the children born as a result of gestational surrogacy are not known. The American Society for Reproductive Medicine (ASRM)13 and BMA8 strongly recommend longterm follow-up studies. However, these issues have been addressed more recently and, to date, the outcomes are reassuring.37–41 The long-term psychological effect on both the ‘genetic couple’ and ‘host surrogates’ is not known, nor is the effect on the hosts existing children. Again long-term studies do need to be carried out and are also strongly advocated by the ASRM14 and BMA.9

In our own series, a number of relatively minor complications have occurred: •

•

A few of our ‘genetic women’ have responded poorly to follicular stimulation and achieved relatively small numbers of oocytes. The mean number of oocytes recovered following the stimulation cycle has been 10, but the range has been 2–24. In the series of Meniru and Craft,33 three of their 11 patients failed to respond to ovulation induction and two other patients produced only very few oocytes, which failed to fertilize. In the posthysterectomy cases, this reduced follicular response may be due to reduced vascular supply to the ovaries.46 The follicular responses of women with the Rokitansky–Küster–Hauser (RKH) syndrome were remarkably good. Four women with RKH syndrome underwent 10 stimulation cycles in the program of Ben Raphael and colleagues47 with human menopausal gonadotropin (HMG) 2–3 ampules per day. A mean of 14.6 oocytes (range 8–24) was collected and the fertilization rate was 71%. Of considerable interest and reassurance for this particular group of young women, has been a study to follow up the children born to women with congenital absence of the uterus and vagina (RKH syndrome). Petrozza and colleagues48 sent questionnaires to all treatment centers performing surrogacy procedures and asked them to follow up the frequency of congenital abnormalities among the progeny born to RKH syndrome

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women. Results of 162 IVF cycles produced 34 live born children, half of whom were female. No congenital anomalies were found amongst these females. These results appear to suggest that congenital absence of the uterus and vagina, if it is genetically transmitted, is not inherited commonly in a dominant fashion. In a survey of all licensed clinics performing surrogacy in the UK,16 29 of 113 licensed clinics perform or have performed surrogacy. In general, very few problems were reported, the most significant of which were: •

•

• • • •

•

There was one report of a surrogate who failed to surrender the baby after the birth, but did so subsequently One surrogate asked for more money from the ‘genetic couple’ once she had achieved a pregnancy One couple separated just before treatment started There was unwelcome newspaper publicity in one case A number of couples pulled out of treatment during the counseling phase Poor response rates to follicular stimulation were noted in several clinics, particularly after Wertheim’s hysterectomy One patient changed her mind during the treatment and actively attempted not to get pregnant. She did not conceive and this led to friction within the family, despite many hours of counseling

When questioned for this survey, most clinics felt that there should be greater control of surrogacy, particularly of natural surrogacy, and that it should be performed within licensed clinics where appropriate health screening and counseling may be provided.

Future directions and controversies In the UK and the USA, the public generally accepts that treatment by surrogacy, particularly gestational surrogacy, is a reasonable treatment option if there are good clinical indications. Because there are a number of countries, particularly in Europe, where surrogacy is not permitted, and as the ease of travel around the world increases, there are concerns that couples will travel the world for treatment which is unavailable in their own countries. The concern is that these practices may lead to disputes and exploitation of desperate couples seeking this particular treatment.8 As an example of these concerns, there have been press reports of women from Eastern Europe taken and exploited as surrogate hosts in wealthier countries where gestational surrogacy is allowed. As a result of this, a number of countries have completely banned surrogacy. Existing controls by the HFEA and proposed changes which may be instituted, which are

discussed later in this chapter, should prevent such exploitation in the UK. There is evidence, certainly in the UK, that there is an increasing level of sympathy and support for the proper use of treatment by ‘gestational surrogacy’ from the media and general public.49 With increasing education and awareness, the public has been able to better judge the benefits of this treatment when there are proper indications. Similarly, in the USA there is much greater acceptance of ‘gestational surrogacy,’ especially now that it is superseding ‘natural surrogacy’ as the treatment option of choice for most couples. In the surrogacy program at Bourn Hall, the main principle by which we have been guided is consideration of the welfare of any child that may be born as the result of treatment and of the existing children. This is enshrined in the Code of Practice18 of the Human Fertilisation and Embryology Act 19907 and also followed by the independent Ethics Committee to Bourn Hall. If the best interests of the child are considered at all times as the priority, then the other issues will invariably fall into place. For instance, the fitness and welfare of the proposed host to go through with the treatment, the age, physical, and psychological ‘fitness’ of the female partner in the commissioning couple, and the seeking of proper legal advice will nearly always become apparent if due consideration is given to the welfare of the child.

Legal issues The majority of the legal problems that have arisen as a result of surrogacy have been associated with cases of ‘natural surrogacy.’ There have been two cases that have received particular publicity – the ‘baby M case’50,51 and also the case of Smith vs Jones.52,53 In the ‘baby M case,’ the final decision was that the genetic couple would have precedence for custody of the child over the birth mother. In the case of Smith vs Jones, which involved ‘gestational surrogacy,’ the District Court recognized the genetic parents to be the legal parents and gave them the right to put their names on the birth certificate of the baby.52,53 In the USA, a number of States have specific regulations regarding surrogate motherhood, but some are more specific than others about the rights of the ‘genetic mother’ over those of the ‘birth mother.’ The complex differences between States have been well summarized by Schuster.44,45 Similarly, in the case of Johnson vs Calvert in the California Superior Court, where Johnson was the ‘gestational surrogate,’ the Calverts, the ‘genetic parents’ of the child, were ruled to be the natural parents of the child.54 Following a widely reported case in 1997 of a natural surrogacy arrangement which experienced severe difficulties, UK health ministers decided to seek views on certain aspects of the existing legislation relating to surrogacy and to ‘take stock and reassess

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the adequacy of existing law in this difficult area.’55 A review body was appointed and was asked specifically to address the following issues: •

•

•

to consider whether payments, including expenses, to surrogate mothers should continue to be allowed, and if so on what basis to examine whether there is a case for the regulation of surrogacy arrangements through a recognized body or bodies; and if so to advise on the scope and operation of such arrangements in the light of the above, to advise whether changes are needed to the Surrogacy Arrangements Act 198519 and/or Section 30 of the Human Fertilisation and Embryology Act 1990.7

The Minister for Public Health at that time stated: ‘my aim is to ensure that the government provides a sensible and sensitive way forward, within a framework that inspires public confidence, in an area of personal life where feelings are inevitably raw and highly charged for those involved.’55 The review panel comprised three Professors – of Law, Psychology, and Ethics. In response to the health minister’s request, the report of the Surrogacy Review Team56 was presented to the UK Parliament and published in October 1998. The following is a summary of their recommendations: 1.

2.

3.

Payments to surrogate mothers should cover only genuine expenses which should be supported with documentary evidence. Additional payments should be prohibited in order to prevent surrogacy arrangements being entered into for financial benefit. Agencies involved in surrogacy arrangements should be registered by the UK Health Department and operate in accordance with a Code of Practice to be prepared for record keeping, and the reporting of specified statistics on surrogacy and guidelines on research should be established by the health departments. The existing Surrogacy Arrangements Act 1985 and Section 30 of the Human Fertilisation and Embryology Act 1990 should be replaced with a new Surrogacy Act which would address in one statute the main legal principles governing surrogacy arrangements in the UK: a. To continue the current provision relating to non-enforceability of surrogacy contracts b. The continuation of current provisions prohibiting commercial agencies from assisting in the creation of surrogacy arrangements and prohibiting advertisements in relation to surrogacy c. New statutory provisions defining and limiting lawful payments to surrogate mothers d. Provision for promulgation of a Code of Practice governing surrogacy arrangements generally

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e. Provision for the registration of non-profit making surrogacy agencies would be required to comply with the department’s Code of Practice on surrogacy arrangements to prohibit the operation of unregistered agencies g. To make new provisions for the granting of parental orders to commissioning couples. 4.

5.

Parental orders should only be obtained in the High Court and judges should be able to order DNA tests and guardians ad liteum should be able to check criminal records. In order for a parental order to be granted the commissioning couple should be habitually resident in the UK, the Channel Islands, or the Isle of Man for a period of 12 months immediately preceding the application for a parental order.

Still, at the time of writing in 2007, these recommendations by the Surrogacy Review Team have not been implemented. Treatment by ‘gestational surrogacy’ is already fully regulated in the UK, since it can only be practiced in centers licensed by the HFEA. This should be sufficient to ensure that proper clinical and scientific services, counseling, and legal advice are provided to both commissioning couples and host surrogates. If ‘gestational surrogacy’ is to be allowed to continue, and there is a general consensus that it should be,49 then the existing regulations are probably sufficient. However ‘natural surrogacy’ is completely unregulated in the UK and we believe that it should be brought under the control of a regulatory body, in the UK, probably the HFEA. As in the USA, Australian States have different regulations. Surrogacy is freely available in New South Wales, Western Australia, and the Australia Capital Territory. In Tasmania, South Australia, and Victoria, it is not illegal, but very strict controls on payment and the lack of binding legal arrangements make it almost impossible to carry out.57 There is a tendency, therefore, for couples seeking surrogacy arrangements to move from state to state.58 The most recent issue concerning the provision of treatment by surrogacy is the ethical question of whether or not it is acceptable to provide this treatment, hitherto only recommended for heterosexual couples, to gay and lesbian couples. The Ethics Committee Report of the ASRM in 2006 carefully considered ‘the changing nature of reproduction and the family.’ They concluded that ‘… there is no sound basis for denying to single persons and gays and lesbians the same rights to reproduce that other individuals have,’ and they finally state: ‘As a matter of ethics, we believe that the ethical duty to treat persons with equal respect requires that fertility programs treat single persons and gay and lesbian couples equally with married couples in determining which services to provide.’59

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Religious issues Religious attitudes towards surrogacy differ widely: •

•

•

The Catholic Church is strongly against all forms of assisted conception, particularly those which involve gamete donation and surrogacy.60 The Anglican Church is less rigid in its view on surrogacy and has not condemned it. The Jewish religion, which is very much family orientated and puts a duty on Jewish couples to have children, does not forbid the practice of gestational surrogacy.61 From the religious point of view, a child born through gestational surrogacy to a Jewish couple will belong to the father who gave the sperm and to the woman who gave birth.62–64 The Islamic view appears absolute and, in the same way that the use of donor gametes is strictly forbidden, so surrogacy is not allowed. It is suggested that it may be permissible between wives in the same marriage, but the debate continues.65

Conclusion It is now more than 20 years since the birth of the first child following a gestational surrogacy arrangement in the USA.2 In the 18 years of our own experience at Bourn Hall, we have shown that the treatment of young women with very specific indications is successful and relatively free of complications. The practice of gestational surrogacy is almost entirely confined to the UK, where it can only be carried out in clinics licensed by the HFEA, a very few countries in Europe, and in the USA. The indications for treatment by gestational surrogacy are limited to a small group of women who have no uterus, suffer recurrent abortions, or suffer from certain medical conditions which would threaten the life of a woman were she to become pregnant. Times are changing, however, and recently gestational surrogacy has been used to help gay couples who wish to have families. The treatment process in itself is straightforward. The woman from the ‘genetic couple’ undergoes a normal stimulated IVF cycle and, unless the sperm of her partner has previously been frozen for 6 months, any embryos which are retrieved are frozen and later transferred to a selected surrogate host. The difficult aspects of the treatment concern the extreme care with which the surrogate host must be selected by the genetic couple to ensure complete compatibility and also the in-depth counseling that is required, both in the short and the long term, on all aspects of the treatment. The support and advice of an independent counselor and lawyer are absolutely essential and we believe the advice of an independent Ethics Committee is also essential in assessing the suitability of individual cases. As clinicians and counselors, we are inclined to become so deeply involved in the problems of individual couples that some of the more obvious pitfalls in the social,

religious, or ethical aspects of treating a particular couple may easily be overlooked. During the past 18 years of our experience, no serious clinical, ethical, or legal problems have been encountered. In one sister-to-sister arrangement, failure of the treatment caused some disagreement and unhappiness between the sisters and support counseling was necessary for more than 3 years. Another more minor problem encountered has been that both parties very often have unreasonably high expectations of the success of treatment, in spite of very frank explanations and counseling being provided to them. Because the host is fit, young, and known to be fertile, she and the genetic parents invariably expect success and they feel badly let down if this is not achieved. An interesting problem that has arisen is that the miscarriage rate has been higher than expected: ⬄40% of the pregnancies have aborted spontaneously26,32 in our own series, an outcome which obviously causes severe stress to both parties. The host feels guilt that she has lost the genetic couple’s hard won pregnancy and the genetic couple feel guilt that the host has been through the stress of a miscarriage and possible curettage. Full support counseling for both couples is essential when this occurs. At Bourn Hall we believe that a gestational surrogacy service should be part of a comprehensive infertility treatment program that most larger centers should offer now that it is an ethically accepted form of treatment in the UK. In our own practice it accounts for less than 1% of all the assisted conception cycles that we carry out. With a policy of careful selection and screening of both genetic and host couples, together with independent counseling, good success rates can be achieved. Long-term follow-up of the babies born as a result of this treatment is being carried out at present, as is the long-term follow-up of the genetic parents and hosts of these arrangements.

Acknowledgments I would like to thank Reverend Dr Tim Appleton for his help and support as an independent counselor with the surrogacy program at Bourn Hall over the past 18 years, and more recently Mrs Linda Koncewicz. Sincere thanks also go to my medical and nursing colleagues who, through their dedication in caring for couples going through the surrogacy program, have ensured its success and the happiness of many deserving couples.

References 1. Holy Bible. The Book of Genesis. 16: 1–15; 17: 15–19; 21: 1–4. 2. Utian WH, Sheean LA, Goldfarb JM, Kiwi R. Successful pregnancy after in vitro fertilisation and embryo transfer from an infertile woman to a surrogate. N Engl J Med 1985; 313: 1351–2. 3. British Medical Association. Surrogacy: Ethical Considerations. Report of the Working Party on Human Infertility Services. London: BMA Publications, 1990.

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Gestational surrogacy 4. Report of the Committee of Inquiry into Human Fertilisation and Embryology. London: HMSO, 1984. 5. British Medical Association. Annual Representative Meeting Report, 1985. 6. British Medical Association. Surrogate Motherhood. Report of the Board of Science and Education. London: BMA Publications, 1987. 7. Human Fertilisation and Embryology Act 1990. London: HMSO, 1990. 8. British Medical Association Report. Changing Conceptions of Motherhood. The Practice of Surrogacy in Britain. London: BMA Publications, 1996. 9. Utian WF, Goldfarb JM, Kiwi R, et al. Preliminary experience with in vitro fertilization-surrogate gestational pregnancy. Fertil Steril 1989; 52: 633–8. 10. Marrs RP, Ringler GE, Stein AL, Vargyas JM, Stone BA. The use of surrogate gestational carriers for assisted reproductive technologies. Am J Obstet Gynecol 1993; 168: 1858–63. 11. Fisher S, Gillman I. Surrogate motherhood: attachment, attitudes and social support. Psychiatry 1991; 54: 13–20. 12. Blyth E. Interviews with surrogate mothers in Britain. J Reprod Infert Psychol 1994; 12: 189–98. 13. Ethics Committee of the American Fertility Society. Ethical considerations in the New Reproductive Technologies. Fertil Steril 1986; 46 (Suppl 1): 62–8. 14. American College of Obstetricians and Gynecologists. Committee on Ethics: Ethical Issues in Surrogate Motherhood. Washington, DC: American College of Obstetricians and Gynecologists, 1990. 15. Steptoe P. Surrogacy. Br Med J (Clin Res Ed) 1987; 294: 1688–9. 16. Balen AH, Hadyn CA. British Fertility Society Survey of all Licensed Clinics that perform surrogacy in the UK. Hum Fert 1998; 1: 6–9. 17. Jones HJ, Cohen J, Cooke I, Kempers R. IFFS Surveillance 07. Fertil Steril 2007; 87 (Suppl 1): S50–1. 18. Code of Practice for Clinics Licensed by the Human Fertilisation and Embryology Authority. Edition 7.0. London: Human Fertilisation and Embryology Authority, 2007. 19. Surrogacy Arrangements Act 1985. London: HMSO, 1985. 20. Leeton J, King C, Harman J. Sister–sister in vitro fertilisation surrogate pregnancy with donor sperm: the case for surrogate gestational pregnancy. J In Vitro Fert Embryo Transf 1988; 5: 245–98. 21. Michello MC, Bernstein K, Jacobsen MJ, et al. Mother– daughter in vitro fertilisation triplet surrogate pregnancy. J In Vitro Fert Embryo Transf 1988; 5: 31–4. 22. Sheean LA, Goldfarb JM, Kiwi R, Utian WH. In vitro fertilisation (IVF) – surrogacy: application of IVF to women without functional uteri. J In Vitro Fert Embryo Transf 1989; 6: 134–7. 23. Ethics Committee of the American Fertility Society. Surrogate gestational mothers: women who gestate a genetically unrelated embryo. Fertil Steril 1990; 53: 64S–7. 24. Marcus SF, Brinsden PR, Macnamee MC, et al. Comparative trial between an ultra-short and long protocol of luteinizing hormone-releasing hormone agonist for ovarian stimulation in in-vitro fertilization. Hum Reprod 1993; 8: 238–43.

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25. Brinsden PR. Superovulation strategies in assisted conception. In: Brinsden PR, ed. A Textbook of In Vitro Fertilization and Assisted Reproduction. London: Taylor & Francis, 2005: 177–88. 26. Brinsden PR. IVF surrogacy. In: Brinsden PR, ed. A Textbook of In Vitro Fertilization and Assisted Reproduction. London: Taylor & Francis, 2005: 393–404. 27. Wikland M, Enk L, Hamberger L. Transvesical and transvaginal approaches for the aspiration of follicles by the use of ultrasound. Ann NY Acad Sci 1985; 442: 683–9. 28. Brinsden PR. Oocyte recovery and embryo transfer techniques for in vitro fertilization. In: Brinsden PR, ed. A Textbook of In Vitro Fertilization and Assisted Reproduction. London: Taylor & Francis, 2005: 271–86. 29. Shenfield F, Pennings G, Cohen J, et al. ESHRE Task Force on Ethics and Law. ESHRE Task Force on Ethics and Law surrogacy. Hum Reprod 2005; 20: 2705–7. 30. Sathanandan M, Macnamee M, Rainsbury P, et al. Frozen–thawed embryo replacement in artificial and natural cycles; a prospective study. Hum Reprod 1991; 5: 1025–8. 31. Marcus SF, Brinsden PR. Oocyte donation. In: Brinsden PR, ed. A Textbook of In Vitro Fertilization and Assisted Reproduction. Carnforth and New York: Parthenon Publishing. 1999: 343–54. 32. Brinsden PR, Appleton TC, Murray E, et al. Treatment by in vitro fertilisation with surrogacy – The experience of a single centre in the United Kingdom. Br Med J 2000; 320: 924–8. 33. Meniru GI, Craft IL. Experience with gestational surrogacy as a treatment for sterility resulting from hysterectomy. Hum Reprod 1997; 12(1): 51–4. 34. Batzofin J, Nelson J, Wilcox J, et al. Gestational Surrogacy: It is Time to Include it as Part of ART? ASRM 1999 Programme Supplement; P–017 (Abst). 35. Corson SL, Kelly M, Braverman A, English ME. Gestational carrier pregnancy. Fertil Steril 1998; 69: 670–4. 36. Parkinson J, Tran C, Tan T, et al. Peri-natal outcome after in vitro fertilization – surrogacy. Hum Reprod 1999; 14(3): 671–6. 37. Van den Akker OBA. Organisational selection and assessment of women entering a surrogacy agreement in the UK. Hum Reprod 1999; 14(1): 262–6. 38. Jadva V, Murray C, Lycett EJ, et al. Surrogacy: the experiences of surrogate mothers. Hum Reprod 2003; 18: 2196–2024. 39. Kleinpeter, CB. Surrogacy: the parents’ story. Psychol Rep 2002; 91: 201–19. 40. Golombok S, Murray C, Jadva V, et al. Families created through a surrogacy arrangement: parent–child relationships in the 1st year of life. Dev Psychol 2004; 40: 400–11. 41. MacCallum F, Lycett E, Murray C, et al. Surrogacy: the experience of commissioning couples. Hum Reprod 2003; 18: 1334–42. 42. Cohen B, Friend TL. Legal and ethical implications of surrogacy mother contracts. Clin Perinatal 1987; 14: 281–92. 43. Brazier M, Golombok S, Campbell A. Surrogacy: Review for the UK Health Minister of Current

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44. 45.

46.

47.

48.

49. 50.

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52. 53.

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Textbook of Assisted Reproductive Technologies Arrangements for Payments and Regulation. Report of the Review Team. London: Department of Health, 1998. Shuster E. Non-genetic surrogacy: no cure but problems for infertility? Hum Reprod 1991; 6: 1176–80. Shuster E. When genes determine motherhood: problems in gestational surrogacy. Hum Reprod 1992; 7(7): 1029–33. Siddle N, Sarrel P, Whitehead M. The effect of hysterectomy on the age at ovarian failure: identification of a subgroup of women with premature loss of ovarian function and literature review. Fertil Steril 1987; 47: 94–100. Ben-Raphael Z, Barr-Hava I, Levy T, Orvieto R. Simplifying ovulation induction for surrogacy in women with Mayer–Rokitansky–Küster–Hauser syndrome. Hum Reprod 1998; 13(6): 1470–1. Petrozza JC, Gray MR, Davies AJ, Reindollar RH. Congenital absence of the uterus and vagina is not commonly transmitted as a dominant genetic trait: outcomes of surrogate pregnancies. Fertil Steril 1997; 67(2): 387–9. Bromham DR. Surrogacy: the evolution of opinion. Br J Hosp Med 1992; 47(10): 767–72. Rothenberg KH, Baby M. The surrogacy contract, and the healthcare professional: unanswered questions. Law Med Health Care 1988; 16: 113–20. Andrews LB. The stork market: the Law of the new reproductive technologies. Am Bar Assoc J 1984; 78: 50–6. Annas G. Using genes to define motherhood: the California solution. N Engl J Med 1992; 326: 417–20. Smith vs Jones. Los Angeles Superior Court, Los Angeles County. June 9, 1987. No CF 025653.

54. Oxman RB. California’s experiment in surrogacy. Lancet 1993; 341: 1468–9. 55. Warden J. Surrogacy to be reviewed in United Kingdom. Br Med J 1997; 314: 1782. 56. Surrogacy. Review for the UK Health Ministers of current arrangements for payments and regulation. Consultation Document. London: Department of Health, 1997. 57. Leeton J. The current status of IVF surrogacy in Australia. Aust NZ J Obstet Gynaecol 1991; 31: 260–2. 58. Johnson I. Regulation of assisted reproductive technology: the Australian experience. In: Brinsden PR, ed. A Textbook of In Vitro Fertilization and Assisted Reproduction. Carnforth and New York: Parthenon Publishing, 1999: 424–7. 59. The Ethics Committee of the American Society for Reproductive Medicine. Access to treatment by gays, lesbians, and unmarried persons. Fertil Steril 2006; 86: 1333–5. 60. McCormick RA. Surrogacy: a Catholic perspective. Creighton Law Rev 1992; 25: 1617–25. 61. Schenker JG. Assisted reproduction practice in Europe: legal and ethical aspects. Hum Reprod Update 1997; 3: 173–84. 62. Hirsh AV. Infertility in Jewish couples, biblical and rabbinic law. Hum Fertil (Camb) 1998; 1: 14–19. 63. Schenker JG. Infertility evaluation and treatment according to Jewish law. Eur J Obstet Gynaecol Reprod Biol 1997; 71: 113–21. 64. Benshushan A, Schenker JG. Legitimizing surrogacy in Israel. Hum Reprod 1997; 12: 1832–4. 65. Hussain FA. Reproductive issues from the Islamic perspective. Hum Fertil 2000; 3: 124–8.

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62 Human embryonic stem cells Rachel Eiges, Benjamin Reubinoff

Introduction A pluripotent stem cell is an undifferentiated cell that has the potential to develop into virtually any cell type in the body. Pluripotent stem cells are transiently present during embryogenesis, in preimplantation embryos and fetal gonads. They can also be maintained as established cell lines, derived either from preimplantation embryos, primordial germ cells, or germ cell tumors. Embryonic stem (ES) cell lines are certain types of pluripotent stem cell lines that have been derived by the isolation and propagation of inner cell mass (ICM) cells of blastocyst-stage embryos. These unique cell lines can develop into a wide range of cell types in vitro and in vivo. In addition, they are immortal. They can be grown continuously in culture without loosing their properties or their wide developmental potential. These two features, pluripotency and unlimited self-renewal, have made ES cells extremely interesting and important to basic and applied research, especially to cell-based therapy and the study of early embryonic development. The derivation of ES cell lines in mammals was first demonstrated in mice1,2 in which basic methods for their isolation, propagation, and genetic manipulation were established. The accumulated experience in the mouse has allowed scientists to better define the properties of ES cells, which are: • derived from ICM/epiblast of blastocysts • are capable of undergoing an unlimited number of symmetrical cell divisions without differentiating • maintain a normal karyotype • can give rise to differentiated cells of ectoderm, mesoderm, and endoderm origin in vitro, and in vivo within teratoma/teratocarcinoma tumors following engraftment into immunodeficient mice • can colonize all fetal tissues, including the germ line, during embryonic development following their injection into host blastocysts • are clonogenic, with each single cell giving rise to many other genetically identical cells that share the same properties and potentials as the original

• specifically express the transcription factor Oct-4, a regulatory molecule characteristic of pluripotent cells. Based on the accumulated experience both with mouse ES cells and with human embryonal carcinoma (EC) cells,3 which are pluripotent and resemble ES cells in many respects, ES cell lines were successfully derived from nonhuman primates (common marmoset and rhesus monkeys).4,5 These studies set the stage for the derivation of human ES cells (hESCs) in humans first by J. Thomson (1998)6 and Benjamin Reubinoff (2000),7 and later by other groups. The described cell lines were derived from ICM cells of normal surplus blastocysts donated by couples undergoing IVF. The hESCs proliferate for extended periods in vitro, maintain a normal karyotype, differentiate spontaneously into somatic cell lineages of all three primary germ layers, and form teratomas when injected into immunodeficient mice. Moreover, they express a panel of markers that are typical to nonhuman primate ES cells as well as to other types of human pluripotent stem cell lines, EC cells and embryonic germ (EG) cells (reviewed by Pera et al).8 As hESCs research advances, scientists and clinicians better appreciate the far-reaching potential of these cells. Therefore, it is not surprising that many of the IVF clinics worldwide are now aiming to set the required system and skills for the establishment of new ES cell lines from human embryos.

Origin of embryos The increasing use of in vitro fertilization (IVF) for the treatment of infertility has led to the development of improved methods for handling and growing human embryos in culture. The availability of such embryos, combined with the skills obtained in the derivation of ES cells in nonhuman primates, has set the ground for the success in the establishment of hESC lines from blastocyst-stage embryos. However, the use of human embryos for research purposes has always been controversial. In general, most people consider the creation of

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Fig 62.1

Origin and derivation of pluripotent stem cell lines.

embryos solely for research purposes immoral. Yet, harvesting stem cells from surplus embryos that were created during IVF treatments and are no longer required for assisted reproduction is also controversial. Consequently, during the past few years several methods for hESC derivation have been developed (Fig 62.1). Some provide an alternative source to the methods that are based on conventional IVF, and therefore may be more acceptable to those individuals who are opposed to the destruction of living embryos for the benefit of hESC derivation.

Conventional IVF High-quality embryos Preimplantation-stage embryos (blastocyst and early cleavage) In some countries it is permitted to use surplus human embryos for research. In such cases potential providers of high-quality diploid embryos would be couples that have completed their IVF treatments and family planning. It is possible to obtain high-quality embryos for hESC derivation, under appropriate informed consent and ethical approval. By optimizing culture conditions and applying the method of sequential media, high-quality blastocysts can be obtained on day 5 post-fertilization at a fairly

reasonable rate. Such embryos, which were originally created for assisted reproduction, have been used for the derivation of hESC lines, first by Thomson (1998)6 and later by many others. Although the most commonly used method for hESC derivation employs high-quality blastocysts, successful derivation from other stages of preimplantation development, such as morulas, have also been employed.9 There is an enormous amount of surplus human embryos in long-term storage in fertility clinics. These cryopreserved embryos pose hard dilemmas for couples that have completed IVF treatment and do not want any more children. They are also hard on the fertility clinics, which are short in storage place for unclaimed embryos. These surplus embryos provide ample supply of biological material for hESC derivation. Yet, in practice, few couples donate their surplus embryos for research. Nevertheless, studies indicate that tailored education and counseling will encourage potential donors to provide more embryos for hESC derivation.10

Single blastomere An alternative approach to the commonly used methods for creating new stem cell lines that might be ethically more acceptable relies on the ability to propagate in culture a single cell removed from an early cleavage stage embryo. This technique, which is

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based on using single-cell biopsies similar to that used in preimplantation genetic diagnosis, does not interfere with the embryo’s developmental potential. The derivation of ES cells from single blastomere biopsy (SBB) was originally developed in mouse, where multiple pluripotent stem cell lines were established.11 A proof-of principle experiment for this strategy was reported in human, where 19 ES-cell-like outgrowths and two stable hESC lines were obtained.12 The latter maintained undifferentiated growth in culture for more than 8 months, had a normal karyotype, differentiated to embryoid bodies (EBs) in vitro and teratomas in vivo, and expressed a panel of markers that are typical to hESCs.

Low-quality embryos A different source for early-cleavage stage human embryos may be low-quality ones. Such embryos, which show over 50% fragmentation or have less than four blastomeres by day 3 in culture, are considered by many as unsuitable for transfer or freezing. They are usually discarded and therefore may be less controversial for use. Yet, in rare cases these low-quality embryos may develop into blastocysts if allowed to remain in culture.13 Moreover, since low-quality embryos commonly contain several viable cells, they can be utilized for SBB (see above). Thus, embryos of low quality may serve as an alternative source of embryonic cells for the derivation of hESC lines.14,15

Genetically aberrant embryos A potential cell source for pluripotent cell lines are genetically abnormal embryos that are routinely put aside by the IVF clinicians. Such embryos are obtained as part of preimplantation genetic screening (PGS) programs. PGS is a tailor-made assisted reproduction technique especially designed for infertile couples experiencing recurrent miscarriages or IVF failure. It involves aneuploidy screening by a fluorescent in situ hybridization (FISH) analysis of single blastomeres, biopsied from IVF-derived embryos. As such, it allows selective transfer of embryos with a normal karyotype and occasionally results in discarding embryos with numerical chromosomal abnormalities, such as lethal trisomies and nullisomies. Yet, several reports indicate that chromosome self-correction occurs in a significant proportion of embryos, as they continue to grow in culture.16 These embryos may serve as a potential source of cells for hESC derivation, as was demonstrated by Peura et al.17 Moreover, it is possible to establish hESC lines that naturally carry specific gene defects. Such mutant pluripotent cell lines can be established directly from genetically affected embryos, obtained through preimplantation genetic diagnosis (PGD). PGD is an assisted reproduction technique that benefits couples at high risk of transmitting a genetic defect. In PGD, embryos diagnosed to be free of the disease are selectively transferred for implantation while the

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affected ones are discarded. The genetically aberrant embryos can be used for the establishment of novel ES cell lines with naturally inherited mutations associated with particular genetic disorders. To date, PGD-derived cell lines have been established for various disorders.17–21 These type of cell lines have great significance as they serve as cellular models for the study of distinct disorders, especially for those in which no good animal or cellular models are available (see Potential applications and future goals section below).

Parthenogenesis Parthenogenesis is a process by which an oocyte is stimulated to divide and develop into an embryo without being fertilized. It can be induced by triggering the oocyte to resume meiosis without undergoing cell division. The resulting embryos contain only maternal chromosomes and are unviable. They are usually lost at the peri-implantation stage, suffering from poor development of extraembryonic tissues. However, they can easily develop into blastocysts, and in some cases even reach the 25-somite stage (in mice).22 It is possible to establish from parthenogenetic blastocysts ES cell lines. Pathenogenetic ES cell lines are now available in several mammalian species, including mice,23 macaque monkeys,24 as well as humans.25,26 These cell lines display the typical characteristic of normal hESC lines. They are immortal, express the typical markers of undifferentiated cells, differentiate into many different cell types in vitro, and form teratomas in severe combined immunodeficient (SCID) mice. The great advantage of generating parthenogenetic hESCs is that it involves egg manipulation, rather than embryo destruction. Moreover, they may be more acceptable for the production of new cell lines, since they naturally lack a full potential for embryo development. Another advantage of parthenogenetic hESC lines over conventional cell lines is that they have two identical sets of chromosomes: i.e. they are predominantly homozygous. Homozygosity reduces the complexity of tissue matching of the cells and therefore may be a favorable cell source for hESCs when considered for cell-based therapy.25 Yet, it remains to be shown if these cells are indistinguishable from wild-type cells in terms of their function and safety due to their unusual epigenetic status, which reflects the status of the chromosomes in the oocyte, lacking paternal imprints.

Somatic cell reprogramming Nuclear transfer-derived embryos It might be possible to obtain ES cell lines from blastocysts which have been obtained by somatic cell nuclear transfer (SCNT). In this method, a nucleus from a somatic cell of an adult is introduced into an enucleated oocyte, resulting in a cloned embryo. The SCNT-derived

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blastocyst may be used for the establishment of an ES cell line perfectly matched to the donor of the nucleus, which can serve as an unlimited source of cells for autologous transplantation. In addition, this approach is especially advantageous for the derivation of affected hESC lines for a wide range of multifactorial disorders, such as heart disorders, diabetes, and cancer, which result from mutations in multiple genes. The complicated bases of these diseases make them especially difficult to model, study, and treat. Although sophisticated, the procedure of therapeutic cloning is not unreasonable, as it has been previously demonstrated to be feasible in mice27,28 and primates.29 Yet, before therapeutic cloning can be considered for clinical use, safety concerns should be carefully addressed and it should be determined how well a somatic nucleus can be reprogrammed without being transmitted through the germ line.30 Moreover, the problem of egg supply must be resolved to make it practical for clinical application.

Transcription factor-induced pluripotent stem cells If an efficient method for de-differentiating somatic cells directly in culture was available, the technical and ethical complications associated with embryo destruction, egg donation for research purposes, and cloning would have been resolved. Indeed, recent studies in mice show that differentiated cells can be induced to reprogram to an ES-like pluripotent state by overexpression of only few defined transcription factors (Oct4, Sox2, Nanog, c-Myc, and Klf4,).31–34 The induced pluripotent stem (iPS) cells display all the characteristics typical of ES cells, including cell morphology, selfrenewal ability, developmental potential (teratomas and germ-line transmitting chimeras), as well as gene expression pattern. Induced pluripotent stem cells were recently also derived from human somatic cells.32,35,36 If these cells prove to be indistinguishable from hESCs in terms of their biological potential and epigenetic state, and safety issues related to the use of viral vectors to transmit and express the transcription factors for reprogramming are resolved, then this approach would bypass the technical and ethical difficulties that are involved with hESC derivation that impede their use in therapeutic applications.

Derivation of human ES cells The same methodologies developed for the derivation of mouse ES cell lines were used for the initial derivation of human lines, with some modifications. According to these methodologies, human embryos are cultured to the expanded blastocyst stage by using the standard commercially available sequential media. The importance of blastocyst quality for the successful derivation of ES cell lines has not been studied in a systematic manner. Based on data from the first groups that derived fully characterized hESC lines, a success rate of 43% was documented (10 lines from 23 embryos).6,8,14

The zona pellucida of the blastocyst is first removed by either enzymatic7 or chemical digestion.14 To isolate the ICM, the outer trophectoderm layer is removed, most commonly by immunosurgery,6,7 although gentle mechanical (using 27G needles)14 or laser-assisted37 removal is also possible. The ICM is then plated on mitotically inactivated feeders that support the proliferation and prevent the differentiation of the stem cells. In an alternative approach, isolation of the ICM is not performed and the intact blastocyst is plated on feeders.38 While successful derivation of hESC lines has been reported following plating of intact blastocysts, further studies are required to compare the efficiencies of deriving new lines from isolated ICMs vs intact blastocysts. Thus far, mouse embryonic fibroblasts (MEFs) have been most commonly used in the derivation of human ES cells, although STO cells,39 human fibroblasts derived from fetal muscle,40 placenta,41 foreskin,42 and from hESCs themselves have also been utilized. Derivation of the most commonly used MEFs follows the methods that were originally described for the mouse ES cell system,3 and the fibroblasts are maintained in culture according to standard tissue culture techniques. Similar to the mouse ES cell system, in order to maintain the potential of the fibroblasts to support undifferentiated proliferation of hESCs, it is important to avoid overcrowded cultures.3 In addition, minimizing the digestion by trypsin during routine subculturing may also improve the efficiency of the fibroblasts as feeders. A relatively low concentration of trypsin (0.05%) is recommended. Only low passage cells (up to passage 5) are used to prepare feeder layers within gelatin-treated tissue culture dishes (Robertson). Human feeders are commercially available (CRL2429; ATCC, Manassas, VA).42,43 Mitotic inactivation of feeders may be accomplished either by irradiation6 or by treatment with mitomycin C.7 There may be a significant variability between various batches of MEFs, which were derived according to the same protocol, with respect to their capability of supporting undifferentiated proliferation of hESCs. To overcome this problem, the competence of various batches of MEFs to support undifferentiated cultures of established mouse or primate ES cell lines may be tested prior to their use in the derivation of new hESC lines. It has been suggested that human foreskin feeders are more potent than mouse feeders in preventing spontaneous differentiation.42 The composition of the culture medium that was initially used for the derivation of hESC (Appendix C) was similar to the traditional serum-containing composition that was developed for the mouse ES cell system. High-quality water and serum with low endotoxin levels, are required for successful derivation and propagation of hESCs. Batch-to-batch variability in the competence of serum to support undifferentiated proliferation may be remarkable. Clonal assays with mouse ES cells (Appendix C) may be used to test

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Fig 62.2 Human ES cells – derivation, propagation and in vitro differentiation. Phase contrast images of 6-day-old human blastocysts undergoing immunosurgery. Note lysis of trophectodermal cells (a). Isolated ICM near the remnants of the trophectodermal cells (b). An ICM 3 days after plating on MEFs (c) (with permission of Nature Publishing Group). Human ES colonies 4 days after plating on MEFs (d). 20-day-old cystic EBs (e). Figures a and b with the courtesy of A Bongso and CY Fong, Figures d and e with the courtesy of N Benvenisty, and Figure c with the permission of Nature Publishing Group.8

the quality of water and serum batches before their utilization for the derivation of hESCs. Serum-free media are now more commonly used to derive new hESC lines.44 Batch-to-batch variability in the competence of a serum replacement preparations to promote undifferentiated propagation of hESCs may also exist and comparing the competence of a few batches as described above to select the most competent may be considered. Within several days following plating of ICMs or intact blastocysts on feeders, groups of small, tightly packed cells may be identified proliferating from the ICMs. Seven to eight days after plating, clumps of these small cells may be mechanically isolated from outgrowths of differentiated cells by using the sharp

edge of a glass micropipette. Following replating on fresh feeders, they give rise to round flat colonies of cells with well-defined borders (Fig 62.2). The cells within the colonies have distinct borders, a large nucleus, a high nuclear/cytoplasmatic ratio, and a prominent nucleoli (Fig 62.2). The colonies are further propagated about every 7 days. To exploit the potential of hESCs for regenerative medicine, they should preferably be derived and propagated in an animal-free defined culture system. hESCs were derived in a culture system that did not include animal products though it included undefined components such as feeders and human serum.40,42 To avoid these undefined components, in another report hESC lines were derived in a serum-free culture system on

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extracellular-matrix-coated plates that could be easily sterilized. However, animal-derived reagents were used in this report.45 In a more recent report hESCs were derived in defined feeder-free culture conditions that included only recombinant reagents or protein components derived from human material. Still, animal products were used to isolate the ICMs in this report and further validation of the genetic stability of the hESCs derived under these defined culture conditions is required.46,47 Derivation of hESCs raises moral concerns due to the destruction of the ex utero preimplantation early human embryos. To avoid these moral issues, methodology to derive hESCs from single blastomeres – which may be removed from the cleavage-stage embryo by biopsy, as performed during PGD, without interfering with the embryo’s developmental potential – was established. Following initial overnight culture and proliferation of the single blastomere, the resulting cells may serve for both PGD and stem cell derivation. Thus, the procedure does not interfere with the process of PGD or the developmental competence of the embryo. It addresses the ethical concerns of many and allows the generation of matched tissue for children born from transferred PGD embryos.12,48

Maintenance of human ES cells in culture Human ES cell cultures usually include a variable level of background spontaneous differentiation. To minimize this process, selective propagation of predominantly undifferentiated colonies or of undifferentiated areas (usually in the periphery of the colonies) may be required to maintain the culture at an undifferentiated state. Human ES cells are highly sociable cells and the survival of single cells is low; therefore, propagation of clumps of 50 cells is most commonly used (Appendix D). In addition to the traditional serum-containing culture system, an alternative serum-free culture system has been developed and is commonly used (Appendix C). A commercially available supplement is used to replace the serum, and basic fibroblast growth factor (FGF2) is required to promote undifferentiated proliferation.49 The serum-free culture system is more effective in supporting the survival of single hESCs. While the cloning efficiency is extremely low in the presence of serum, it is improved to 0.83% in the serum-free culture system.49 Clonal derivation of pluripotent hESCs may be accomplished by using this system. It should be noted that under optimal culture conditions, when the level of background differentiation is low, nonselective propagation of the cultures in bulk by using gentle enzymatic digestion (with collagenase IV or trypsin) is possible. However, it should be noted that changing the culture conditions and methods of propagation may be associated with the acquirement of genetic abnormalities by the cells. Therefore, monitoring

the genetic stability of the cells by routine repeated cytogenetic analyses is recommended (see below). In addition to MEFs, as mentioned above, STO cells39 and feeders from various human adult and fetal tissues, including fetal muscle,40 foreskin,50,51 and marrow cells,52 can also support the derivation and/or propagation of hESCs. The cytokine LIF can replace the requirement of a feeder layer and support the derivation and propagation of germ line-competent ES cell lines in the mouse.53,54 Leukemia inhibitory factor (LIF) cannot support undifferentiated proliferation of hESCs in the absence of a feeder layer.6,7 However, the requirement for feeders may be eliminated by culturing the stem cells on laminin or Matrigel-coated plastic surfaces in the presence of mouse embryonic fibroblast-conditioned medium.55 The development of feeder-independent culture systems improves the capability of growing the hESCs on a large scale and manipulating them in vitro. Great efforts by many research groups have been directed to identify the growth factors that will support the maintenance of hESCs in the absence of feeders or their conditioned medium. Wnt,56 FGF2,57 transforming growth factor (TGF-β1),58,59 insulin growth factor (IGF) signaling,60 as well as blocking of bone morphogenetic protein (BMP) signaling,57 have been shown to have a role in maintaining pluripotency of hESCs without feeders. New insight into the niche that regulates self-renewal and pluripotency of hESCs was recently reported.60 It has been suggested that the niche is composed of autologous fibroblast-like cells, which originate from the hESCs themselves. In response to FGF signaling the hESCderived fibroblasts secrete IGF-II, which acts directly on hESCs through the IGF-I receptor and promotes selfrenewal. While the expression of IGF-I receptor is restricted to the hESCs, the fibroblasts exclusively express FGF receptor. Activation of FGF signaling in the fibroblasts also induces the secretion of TGF-β, which prevents differentiation of hESCs. Thus, it appears that FGF factors do not act directly on hESCs but rather through the autologously derived fibroblasts which secrete IGF-II and TGF-β factors. In line with these data, it has been shown that IGF-II can promote the undifferentiated propagation of hESCs in the absence of feeders.60 The suggested lack of direct effect of FGF factors on hESCs needs further confirmation by additional studies. Other combinations of growth factors, including FGF2, noggin, TGF-β1, activin A, pipecolic acid, γaminobutyric acid (GABA), LiCl with various media and culturing the cells on various extracellular matrices, was also shown to promote feeder-free cultures of undifferentiated hESCs.46,59,61–65 Further studies are required to identify the ideal chemically defined, feeder and animal reagent-free culture system that will support prolonged self-renewal of genetically stable pluripotent hESCs. hESCs may be cryopreserved by the conventional slow-rate freezing and rapid thawing method (using hESC serum-free culture medium supplemented with

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10% dimethylsulfoxide (DMSO) and 30% serum replacement).55 However, given the relatively low effectiveness of this approach,66 a large number of cells should be frozen to achieve successful and reliable thawing. In the initial steps of derivation of new hESC lines, or when the culture conditions are not ideal, large numbers of undifferentiated hESCs may not be available. Under these circumstances, vitrification with the opened-pulled straw (OPS) method, which was shown to be highly effective in the cryopreservation of hESC clumps, allows reliable cryopreservation.66 The cells retain their key properties following thawing.66

Characterization of human ES cells An international scientific consensus regarding the exact uniform criteria and standards that should be used to characterize and define hESCs has not been established.67 Establishing this consensus would be extremely useful in comparing the characteristics of different hESC lines. So far, the hESC lines that were derived by a number of groups were characterized by demonstrating the key properties of ES cells (as above) that were applicable to the human system. Given the potential unlimited selfrenewal capability of hESCs, an important part of the characterization process is to repeatedly demonstrate the key properties during prolonged propagation of the cells in culture. Unfortunately, the majority of cell lines that were reported to date (http://stemcells.nih.gov/registry) have not been available for sufficient time and have not been fully characterized. Here, we will summarize the properties that were most commonly used to define the reported hESC lines in the literature. Colonies of hESCs are flat with well-defined borders distinct from the surrounding fibroblasts. In the presence of serum, undifferentiated cells have distinct borders, a large nucleus with prominent nucleoli, and a high nuclear/cytoplasmatic ratio (Fig 62.2).6,8 In serum free-culture conditions, the colonies tend to become more tightly packed, with less distinct borders between the cells.49 The International Stem Cell Initiative (ISCI) characterized 59 hESC lines from a large number of laboratories, worldwide.68 Despite diverse genotypes and the different techniques that were used for derivation and maintenance, all lines exhibited remarkably similar phenotypes. All cell lines strongly expressed the glycolipid antigens SSEA3 and SSEA4, the keratin sulfate antigens TRA1-60, TRA-1-81, GCTM2, and GCT343, and the protein antigens CD9 and Thy1 (CD90). In addition, they were all positive for tissue-nonspecific alkaline phosphatase and class 1 HLA, and strongly expressed the developmentally regulated genes NANOG, OCT4 (POU5F1), TDGF, DNMT3B, GABRB3, and GDF3. The characterization of hESCs further includes the demonstration of key properties of ES cells. Standard cytogenetic analysis methods are used to show that the stem cells retain a normal karyotype along propagation

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in culture.6,7,69 Although most hESC lines have a normal diploid karyotype during their initial growth period, accumulated data suggest that the cells tend to acquire chromosomal abnormalities during prolonged culture. The most frequent karyotypic changes observed were gains of chromosomes 12 and 17, and to a lesser extent chromosome X, aberrations which are typical to testicular germ cell tumors.70,71 The nonrandom nature of the observed changes in the hESCs culture strongly points towards a selection pressure that drives to the appearance of these specific chromosomal abnormalities. These changes most probably reflect the progressive adaptation of self-renewing cells to their culture conditions, evidenced by increased growth rate, enhanced cloning efficiency, reduced tendency for apoptosis, and decreased differentiation capacity in high passage cultures. Suboptimal culture conditions, including cell harvesting technique (manual as opposed to bulk), cell density, type of growth media, supporting feeder layer, and rounds of thawing, appear to favor a drift towards chromosomally abnormal cells, which may overtake the culture. Therefore, regardless of what are the selection driving factors, it is absolutely essential to regularly monitor the karyotype of the cells at frequent intervals. The pluripotent potential of hESCs is demonstrated by showing the potential of the cells to differentiate into progeny representing the three germ layers both in vitro and in vivo within teratoma tumors. Induction of differentiation in vitro is described in details in the following section. Teratoma tumors are generated following engraftment (intramuscular6 or under the testicle or kidney capsule7) of undifferentiated cells into SCID mice (4–6 weeks old49) (Figure 62.3). The utilization of variable amounts of undifferentiated cells (103–5 × 106)51,55 was reported to produce teratomas within 6–16 weeks following engraftment. Histological analysis of the tumors reveals a variety of differentiated tissues, including gut-like, primitive bronchus (endoderm) bone, cartilage, striated muscle and fetal glomeruli (mesoderm) squamous epithelium, and primitive neural tissue (ectoderm) (Fig 62.3).6,7,72 Clonal expansion of a pluripotent cell population from a single cell is required to verify that the cultures are not mixtures of early progenitors of multiple lineages but truly include pluripotent cells.

In vitro differentiation Spontaneous differentiation Spontaneous differentiation by cell aggregation It is possible to trigger the differentiation of hESCs in vitro by growing them in suspension culture. In suspension, the cells tend to aggregate, forming multicellular structures termed embryoid bodies.73 As these cell structures form, they undergo spontaneous differentiation to produce terminally differentiated cells of mesoderm, ectoderm, and endoderm origin. The formation of EBs is

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Fig 62.3 Histology of differentiated tissues within teratoma tumors. Shown are cartilage and squamous epithelium (a – mesoderm and ectoderm), neuronal rosettes (b – ectoderm), and ciliated columnar epithelium (c – endoderm).

a gradual process and is accompanied by morphological changes. It begins with the formation of small bodies of densely packed cells (simple EBs), which by day 7 begin to cavitate (cavitated EBs) and eventually accumulate fluid within cysts. By day 20, the cystic EBs, which are a product of spontaneous and disorganized differentiation, are considered to be mature and are composed of various terminally differentiated cell types, including nerve,74–76 blood,77 endothelial,78 heart,79–81 and pancreatic82 cells. Some have even been shown to be functional, as in the case of nerve cells.74 The EBs can easily be obtained by growing the cells under conditions that prevent their adherence to the culture dish. This is performed by growing the cells in bacterial Petri dishes in the absence of feeders, thereby promoting their aggregation (see Appendix E). However, this method entails large variations in size and shape of the EBs and, therefore, also in the differentiation status of the cells. In order to obtain a more homogeneous cell culture, the ‘hanging drop’ method may be applied (see Appendix E).

Spontaneous differentiation by prolonged culture on feeders An alternative method for inducing spontaneous differentiation is to obtain high-density cultures. The hESCs are cultivated to high-density for extended periods (4 weeks) in the presence of serum, without replacement of the feeder layer. High-density cultures lead to the piling up of cells, forming three-dimensional multicellular and vesicular structures.7 Progeny representing the three embryonic germ layers, as well as differentiated cells from the extraembryonic lineages, are generated within high-density cultures.7,72 While, in general, differentiation within high-density cultures is disorganized, areas within these cultures that are composed predominantly from one committed progenitor cell type may be identified. By dissecting these areas out from the cultures, it is possible to isolate and develop highly enriched cultures of early progenitors from a specific lineage such as the neural one.72

Induced differentiation Spontaneous differentiation of ES cells in vitro is a stochastic process that results in the production of heterogeneous cell populations. However, the development of a highly purified population of a specific cell type is required for most of the scientific and therapeutic applications of hESCs. Thus, it is necessary to direct the differentiation of the cells in vitro and/or to combine it with a lineage-based selection approach. There are several strategies that can be utilized for this purpose.

Growth factors Exogenic factors can augment the process of differentiation towards a specific cell fate.75 For example, it has been well established that the addition of retinoic acid (RA) induces the differentiation of ES cells into neurons,75 and that BMP4 can direct their differentiation into trophoblast cells.83 The growth- or differentiation-inducing factors can be supplemented continuously or sequentially to the media, according to the requested cell type and protocol. They may be used to promote differentiation into a specific fate within EBs or in flat feeder-dependent84 or feeder-free65 cultures. Since the cultures that are obtained following treatments with differentiationinducing factors are still relatively heterogeneous, at present, this approach should be combined with additional strategies such as lineage selection, selective culture conditions, and overexpression of key transcription factors.

Lineage selection The lineage selection approach allows a highly purified population of cells to be obtained by performing selection for or against a specific cell type. Cells of a specific type may be sorted from heterogeneous populations of differentiated cells based on the expression of lineage-specific cell surface markers, or by genetic selection. The latter approach is based on the genetic introduction of a selectable marker gene under the

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regulation of a tissue-specific promoter. The marker gene may either be a selectable reporter, such as GFP, which can be selected for by fluorescence-activated cell sorter (FACS),85,86 or the insertion of a drug resistance gene such as the neomycin resistance gene, which allows the direct isolation of the desired cells by the presence of G418 in the media.87

Overexpression of key regulator genes It is possible to force the differentiation of ES cells into specific lineages by overexpressing transcription factors which play major roles in early commitment of cells into specific lineages. This has been previously demonstrated to be feasible in the mouse ES cell system, where overexpression of MyoD resulted in the induction of skeletal myocytes, which fused to create multinuclear contractile myotubes.88 Similar experiments demonstrated the effect of HNF on the generation of hepatocytes,89 and of Nurr1 in the production of dopaminergic neurons.90

Potential applications and future goals Cell source for transplantation Although the establishment of ES cell lines has been achieved in many mammalian species since the first derivation of such cells in mice, none has drawn as much attention as the human-derived cells, owing to their enormous biomedical potential. Since hESCs can be grown indefinitely in culture without losing their basic properties, and have the potential to develop into practically any cell type in vitro, they may be used as an unlimited cell source for cell transplantation. When efficient protocols for induced differentiation are established, it will be possible to generate specific cell types in large numbers for the repair of degenerating or damaged tissues in humans. This will reduce the current supply problems of tissues available for transplantation. Indeed, it has been demonstrated in mice, and to a certain extent in humans, that ES cell-derived progeny can proliferate and integrate following their transplantation into adult animals.72,76 Moreover, in the mouse ES cell system, transplanted progeny were shown to be functional and could improve behavioral deficits in animal models of diseases. Mouse ES cell-derived cardiomyocytes were able to form stable functioning intracardiac grafts,87 and glial precursor derivatives formed myelinating transplants in the brain and spinal cords of myelin-deficient rats.91 Also insulin-secreting cells derived from ES cells normalized glycemia in streptozotocin-induced diabetic mice92 and, in addition, transplanted functional dopaminergic neurons corrected motor asymmetry following transplantation into the animal model of Parkinson’s disease.90 While these results are promising, many more experiments are required to test the functionality and safety of transplanted hESC-differentiated derivatives

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in animal models before they can be considered appropriate for clinical use. In addition, there is a need to overcome the difficulty of graft rejection as a result of the immune response.93 There are several possibilities that can be applied to minimize graft rejection of ES cell derivatives. One possibility is to establish a bank that will include a large number of ES cell lines that differ in their MHC expressed molecules, thus allowing major histocompatibility complex (MHC) matching between the donor cell line and the recipient. Alternatively, it may be possible to generate a ‘universal’ donor cell line by ‘knocking out’ the genes that are responsible for graft rejection. Finally, it might be feasible in the future to generate genetically identical ES cell lines, either by somatic cell nuclear transfer or by de-differentiating somatic cells directly in culture through genetic manipulation, as described earlier in this chapter, to provide the patients with autologous grafts.

Gene therapy One of the great advantages of ES cells over other cell types is their accessibility for genetic manipulation. They can easily be induced to genetic modifications and can be selectively propagated, allowing the clonal expansion of genetically altered cells in culture. Genetic manipulation of hESCs can therefore be utilized for monitoring, selecting, and even directing the differentiation of the cells into specific lineages. Moreover, the hESC-derived progenitors may be used as delivery vectors for the regulated release of drugs and therapeutic proteins at the site of the damaged tissue. Such a cell-based delivery system will permit the production of a therapeutic agent at a steady-state level and in consistent physiological concentrations, overcoming current limitations caused by incomplete drug accessibility. Furthermore, it may be possible sometime in the future to repair the genetic defect by replacing the aberrant gene with an intact sequence. This can be accomplished by coupling embryo cloning with gene therapy, so that the genetic manipulation will be carried out on the genome of SCNTderived isogenic hESC lines obtained from patients. Using this technique, it is possible to cure the disease by providing the patients with genetically engineered autologous grafts, overcoming the difficulties in graft rejection as a result of the immune response. This approach of combining gene therapy with therapeutic cloning has been previously shown to be feasible in mice, where immune-defficient Rag2-/- mutant mice were used as nuclear donors for generating blastocysts from which an isogenic ES cell line was isolated.28 The mutant ES cells were corrected for Rag2 gene activity by homologous recombination and used as a source for hematopoietic committed cells. The genetically engineered blood cells were then grafted into the Rag-/- mutant mice, resulting in the rescue of the immune-deficient phenotype.28

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Drug screening and toxicology Much effort is invested in the development of new drugs by trying to understand the pathology of a disease and its underlying treatment. This approach, which is diseaseoriented, requires model systems that are based on human cells, rather than on animals, which, in many cases, are inadequate. Obviously, the best model system would be specific cell types of humans that display the appropriate phenotype. However, the currently available cellular models in humans are limited in their potential by the lack of relevant and validated cell types. Most are suboptimal, since they are based on the use of abnormal cancer cell lines or on primary cell cultures. Human ES cells, however, may be exceptionally useful for drug screening and development, as they have the capacity to self-renew in culture and can potentially differentiate into all cell types in the body. Provided that efficient protocols for induced differentiation of hESCs are established, it will be possible to generate specific cell types in large numbers so that the targeted tissues will be accessible for large-scale drug screening and development. Moreover, hESCs can be genetically modified relatively easy. This should allow the introduction of reporter genes, under appropriate regulation, that will facilitate the analysis of examined compounds. In addition, establishing mutant cell lines will allow specific terminally differentiated impaired cells to be generated in large numbers so that diseased tissues of patients will be accessible for research. Finally, since differentiating hESCs can recapitulate, to some extent, early human embryogenesis, they may have great value in assessing the potential toxicity of new drug candidates and their teratogenic effect.

Embryo development The study of early human development is restricted by ethical constraints on research of human embryos. Moreover, apart from the early stages of preimplantation development, human embryos are inaccessible for research. The use of animal models, specifically the mouse, has allowed us to overcome these obstacles, taking advantage of their well-defined genetics and reproductive characteristics. Yet, despite the strong conservation throughout evolution, there are still major differences in critical developmental events as a product of genetic variation across species. The limitation of the currently available models has emphasized the need for an alternative system that would better mimic certain aspects of early human embryo development. hESCs may have great value for basic research as well, since they recapitulate early events in embryo development, as they are induced to spontaneously differentiate in vitro. The concept of using differentiating ES cells as an in vitro model system to study developmentally regulated biological phenomena has been originally practiced in the mouse, where growing EBs were used for studying X-chromosome inactivation94 and

globin gene switching.95 In humans, gene expression macroarray studies have shown that spontaneously differentiating hESCs, mimic, in terms of gene transcription, developmental stages in the embryo which are otherwise unattainable for research.96 Moreover, gene profiling has allowed recovering known molecular pathways during human EBs formation, as they occur in the embryo. Hence, there is accumulating evidence to suggest that hESCs can serve as a model to study early human development, at least at the cellular level.

Genetic disorders in humans The limitations of the currently available animal and cellular models, as described above, have emphasized the need for an alternative system that will better mimic certain genetic anomalies. The availability of mutant hESCs that harbor a specific mutation at a discrete site should be most valuable for the study of some pathologies in man. Basically, there are two strategies by which mutant hESCs can be established. One is to artificially induce a specific modification in the DNA of a preexisting cell line by homologous recombination. Using this approach, a cellular model system for Lesch– Nyhan syndrome was generated by targeting the HPRT gene in XY wild-type hESCs.97,98 The other approach is to establish an hESC line directly from a genetically affected embryo so that the resulting cell lines will naturally inherit the genetic defect (see origin of embryos and genetically aberrant embryos sections). The great advantage of this approach, as compared to the former, is that it does not require genetic engineering of the cells. In addition, it enables genetic modifications that are otherwise unobtainable to be introduced, such as triplet repeat expansions as well as numerical and structural chromosomal abnormalities. To date, mutant hESC lines have been established for various disorders, including cystic fibrosis,19 myotonic dystrophy, Huntington disease,18 Duchenne muscular dystrophy, thalassemia,20 fragile X syndrome,21,37 hemophilia A,37 as well as trisomies 16 and 5.17 These types of cell lines have great importance by serving as cellular models for the study of distinct disorders, especially for those in which no good animal or cellular models are available. It will enable the generation of large amounts of the desired cells that are directly impaired by the genetic lesion. Availability of such cells will allow abnormal phenotype at the cellular and molecular levels to be studied, providing greater understanding of the pathology of the disease. Moreover, there are some disorders that are developmentally regulated and their genetic abnormality exerts its effect during the early stage of embryogenesis. In such cases, mutant hESCs will have an extremely important role in investigating the underlying mechanism that leads to the manifestation of disease. Using this approach, a fragile X-affected hESC line was used to investigate the molecular events that are involved in the pathogenesis of this disease, demonstrating that the inactivation of the associated

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gene is a multistep process that is developmentally regulated and is triggered by cell differentiation.21 Finally, the availability of mutant hESC lines will allow disease-oriented drug screenings and development to be carried out and may serve as a powerful tool for gene manipulation and therapy, as described above.

Conclusions The derivation of ES cell lines from human embryos has initiated a new era in the fields of biotechnology, pharmacology, basic scientific research, and regenerative medicine. It is now well established that hESC lines can be readily and reproducibly derived. Yet, there is still a need to increase the number of cell lines that are available to the research community and to generate more lines with a broader genetic and ethnic background. New lines from genetically abnormal embryos are also required, as well as lines suitable for clinical purposes. Much more research and development is required to exploit the remarkable potential of hESCs. Appropriate public support and adequate legislation are crucial for the realization of the far-reaching applications of hESCs.

Protocols Appendix A: immunosurgery 1. Reconstitute anti-human serum (Sigma, St. Louis, MO; Cat number H-3383) with 2 ml DDW. Dilute 1:5 in blastocyst culture medium. 2. Reconstitute guinea pig complement (Invitrogen, Rockville, MD) with 5 ml of PBS. Dilute 1:5 in blastocyst culture medium. 3. Prepare a 4-well dish with a 10 µl drop of the antihuman serum under pre-equilibrated sterile mineral oil. Place 0.5 ml of blastocyst culture medium in the other wells. 4. Transfer the blastocyst to the anti-human serum drop for 30 minutes incubation in 5% CO2. 5. Prepare a 4-well dish with a 50 µl drop of the complement solution under pre-equilibrated mineral oil. Place 0.5 ml of blastocyst culture media in other wells. Incubate 10–15 min in 37°C, 5% CO2. 6. Transfer the embryo after 3 washes in blastocyst medium into the complement drop and incubate 30 minutes. 7. Wash three times in blastocyst medium. 8. Remove the damaged trophectodermal cells by pipetting the blastocyst through a small-bore glass pipette. 9. Plate the resulting ICM clump on an MEF feeder layer that was plated the previous day and cultured with the culture medium for hESCs. 10. If there are indications of low ICM viability (low plating efficiency and/or low colony formation), all washes should be extended to 5 minutes.

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Appendix B: preparation of MEF cells Isolation of MEFs 1. Collect 13.5-day-old fetuses from pregnant mice using sterile equipment: sacrifice pregnant mice and dissect the embryos by removing the uterus and transferring it into a sterile PBS-containing Petri dish. 2. Rinse twice in PBS and relocate all work to laminar flow hood. 3. Using sterile tweezers and scissors, remove the fetuses from the uterus, separate them from extraembryonic tissues (amniotic and yolk sacs) and transfer them to a clean Petri dish with PBS. 4. Count the number of collected fetuses and prepare, for later use, one 10 cm tissue culture dish for every three fetuses. 6. Remove head and internal parts (liver, heart, kidney, lung, and intestine) with sterile tweezers under a stereomicroscope. 7. Cut the remaining tissues into small pieces in a minimal volume of PBS (1–2 ml) and transfer into a sterile 50-ml Falcon tube. 8. Disaggregate the cell clumps obtained by cutting them into tiny pieces with sterile knife scalpels. 9. Add MEF media to reach 10 ml per three embryos, distribute cell suspension evenly into 10-cm tissue culture dishes, and incubate. 10. Change media the following day. When plates are confluent (2–3 days after dissection) split 1:3 by trypsinization. 11. Change media (10 ml) every 2 days. When cell density reaches confluence, trypsinize the cells and freeze each 10-cm plate in one cryovial, store in liquid nitrogen.

Mitomycin C inactivation of MEFs 1. Thaw contents of one cryotube into three 10 cm culture dishes. 2. Grow the cells to confluence by changing the media every other day. 3. Further propagate the cells by splitting them twice at a 1:3 dilution (sums to 27 plates). 4. To inactivate the cells, add 40 µl of mitomycin C stock solution (1 mg/ml) to 5 ml of culture media (final concentration of 8 µg/ml) and incubate at 37°C, 5% CO2, for 3 hours. 5. Aspirate the mitomycin-containing medium and wash the plates twice with 6 ml PBS. 6. Tripsinize cells by adding 1 ml of trypsin-EDTA and incubate at 37°C, 5% CO2, for 5 minutes. 7. Add 5 ml of medium and suspend the cells by vigorous pipetting. 8. Collect cell suspension into a 50 ml Falcon tube. 9. Centrifuge mitomycin-treated cell pool at 1000g for 5 minutes. 10. Aspirate supernatant and add fresh medium to reach a final cell concentration of 4 × 106 cells/10-cm

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dish. Feeder plates can be stored in the incubator for 3–4 days, but should be examined under the microscope before use. 11. It is possible to freeze mitomycin C-treated MEFs and keep them for later use. For this purpose, freeze 1.5–7 × 106 cells in each cryotube and later thaw and plate to give 1–5 × 106 cm dishes, respectively.

Appendix C: composition of media Composition of serum-free medium for undifferentiated growth of hES cells49 • • • •

KnockOut Dulbecco’s Modified Eagle’s Medium – 80% (Invitrogen) Knockout serum replacement (SR) – 20% (Invitrogen) Basic fibroblast growth factor (bFGF) – 4 ng/ml (Invitrogen) β-Mercaptoethanol, NEAA, L-glutamine, penicillin, and streptomycin, as above.

Composition of serum-containing medium for undifferentiated growth of hES cells49 •

• • • • •

Dulbecco’s Modified Eagle’s Medium (DMEM) – 80% (Invitrogen), without sodium pyruvate, with glucose 4500 mg/l) Defined fetal bovine serum – 20% (Hyclone, Logan, Utah) β-mercaptoethanol – 0.1 mmol/l (Invitrogen, keep in original bottle at 4°C) Nonessential amino acids – 1% (Invitrogen) L-Glutamine 2 mmol/l (Invitrogen) Penicillin – 50 µg/ml, streptomycin 50 µg/ml (Invitrogen)

Mouse ES cells clonal assays to test competence and quality of serum batches 1. An established culture of mouse ES cells is used, as previously described (Robertson). 2. All medium components should be those that will be used to culture the hESCs. 3. The culture medium is supplemented with 10% of the tested batch of FBS (instead of 20%) and mouse recombinant LIF at 1000 µ/ml. 4. Trypsinize the mouse ES cells (Robertson)52 and plate individual cells in pre-gelatinized 6 cm Petri culture dishes at a low density (1000 cell per plate). 5. Culture either with the medium that was in current use or the new tested medium at 37°C in a 5% CO2 atmosphere. Change medium once on day 5 after plating. 6. At the day 7, rinse the cultures with PBS and stain for 5 minutes with 0.15% Leishman’s fix and stain (Leishman’s stain, BDH, Poole, England; in 100% methanol).

7. Wash the stained cultures with water and let them dry in air. 8. Compare the number of colonies per plate as well as the size and degree of differentiation. 9. Select the batch of serum with the best performance compared to the batch in use.

Appendix D: selective propagation of clumps of undifferentiated hES cells 1. Use a dark field stereomicroscope in a laminar flow hood. 2. Identification of the morphology of areas within colonies (usually at the periphery of colonies) that are predominantly undifferentiated is easily learned by comparing the morphology of colonies between phase contrast and stereomicroscopy. 3. Replace the culture medium with PBS containing Ca2+ and Mg2+. 4. Slice the colonies into small areas containing about 50 undifferentiated cells by using the sharp edge of a micropipette. 5. Replace the PBS with the regular pre-equilibrated stem cell medium containing Dispase (Invitrogen, 10 mg/ml). 6. Incubate the dish for approximately 5 minutes at 37°C in a humidified atmosphere containing 5% CO2. 7. As soon as the sliced clumps of undifferentiated cells detach from the culture dish, pick them up with a wide-bore micropipette, wash them twice in PBS containing Ca2+ and Mg2+, and plate them onto a fresh fibroblast feeder layer.

Appendix E: EB formation In order to obtain cystic EBs, it is essential to minimize the disruption of the cells as they aggregate and expand. They should be gently manipulated using wide pipettes and kept in an unchanged position in the incubator as they grow.

Mass culture in suspension 1. Passage human ES cells once on gelatin-coated plates in order to avoid the presence of residual MEF cells in the EBs. 2. Harvest cells using trypsin, centrifuge, and resuspend in EB medium (human ES cells serum-free media in the absence of bFGF). 3. Place 107 cells into a UV-irradiated 10 cm2 Petri dish. 4. Incubate the cells for 2 days without moving the plates. 5. After 2 days, small cell clumps are formed. It is essential that these aggregates are not disturbed. At this stage, change media once in 2–3 days, according to its color. This is achieved by placing the plate at an angle, allowing the growing EBs to concentrate at

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the bottom end of the plate. Gently aspirate as much of the media as possible and replace it with the same amount of fresh media. Alternatively, it is possible to use a wide-bore pipette to collect and transfer the EBs in the media into a conical tube. The EBs tend to concentrate at the bottom of the tube without centrifugation, allowing for the careful aspiration of the media and the addition of fresh media. Following resuspension, the cells are transferred gently back to the dish using a wide-bore pipette.

Hanging drops 1. Passage human ES cells once on gelatin-coated plates in order to avoid the presence of residual MEF cells in the EBs. 2. Harvest cells using trypsin, centrifuge, and resuspend in EB medium (see above). 3. Count and resuspend cells to 1–10 × 106/ml (400– 4000 cells/40 µl). 4. Place 40 µl drops on the inner side of a cover of a 35 mm tissue culture dish (no more than 25 drops per lid). 5. Put 10 ml PBS in the dish to avoid the evaporation of the drops. 6. Place lid back on the plate so that the drops are hanging downwards from the cover of the dish. 7. Place in the incubator and do not touch or move for the next 2 days. 8. By day 3, collect all drops very gently with a wide 1 ml cut tip and place in a Petri dish with 10 ml media. 9. Change media every 2–3 days as described above.

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63 Microfluidics in ART: current progress and future directions Jason E Swain, Thomas B Pool, Shuichi Takayama, Gary D Smith

Optimized production of preimplantation embryos for use in assisted reproductive technologies (ART) has been a central goal of reproductive scientists since the inception of the field, and, subsequently, methodologies have continually been refined to aid in this endeavor. For example, skilled technicians meticulously handle gametes and embryos in prescribed manners, extensive research has refined culture media formulations to cater to the changing metabolic needs of gametes and embryos, and commercial manufacturers have even produced specialized equipment to meet the specific needs of cells in ART. Although approached from different perspectives, the commonality between these advancements is the pursuit to minimize external stresses imposed upon gametes and embryos due to artificial manipulation within the in vitro fertilization (IVF) laboratory. Environmental and intracellular factors influenced by these manipulations, such as osmotic imbalances, shifts in temperature, and pH fluctuations, can all have devastating effects on embryo quality. However, even with these tremendous improvements, relatively little attention has been paid to the platform on which gametes and embryos are cultured and manipulated. In regard to culture platform, clinical embryology laboratories have historically selected between polystyrene test tubes, Petri dishes, organ culture wells, or four-well plates to accommodate varying number of cells and volumes of media used. Recently, embryospecific culture dishes have even been produced offering certain ‘ease-of-use’ benefits unique to embryology laboratories.1 Although each of these approaches offers certain advantages, it remains evident that environmental conditions offered by all of these culture platforms are in extreme contrast to what is observed in vivo. In the quest to optimize embryo development in vitro, a ‘back to nature’ ideology has been adopted by some to formulate embryo culture media.2 To the best of its ability, this approach attempts to base culture media formations on composition of fluids in the female reproductive tract to chemically manipulate embryo development in

vitro. Similarly, this same naturalist approach may also be applied to culture platforms. Exploration of the differential effects of physical and structural environment experienced by gametes and embryos in vitro vs in vivo may provide a means to further improve clinical IVF. In vivo, gametes and embryos are exposed to the constricted ‘moist’ environment of the female reproductive tract, surrounded by various oriented glycoproteins as they are moved via ciliated epithelium to their destination. This is in stark contrast to the expansive static environment gametes and embryos are exposed to in vitro, resting on inert synthetic polymers, bathed in a relative ocean of media. Microfluidic technology offers a platform on which to further manipulate culture conditions in vitro by phenomimicking physiologic conditions in vivo, in the hopes of creating an environment more suitable to gamete and embryo development and function.

Basis and benefits of microfluidics The term ‘microfluidic’ refers to technology utilizing characteristics of fluid movement in a micro- or nanoenvironment. These characteristics, discussed in depth elsewhere,3 rely on variables such as fluid density, viscosity, velocity, and size/geometry of the environment. Taken together, these variables are used to calculate Reynolds number. At the macro level, fluid flow results in chaotic particle movement within the fluid stream, resulting in turbulence, as indicated by a high Reynolds number. In contrast, in the decreasing dimensions of microfluidic channels, Reynolds number decreases and fluid is imparted with streamlined and predictable flow patterns. These predictable flow patterns conferred by microfluidic devices impose laminar flow upon fluids, allowing parallel movement of multiple streams of media through the same microchannel with no mixing, except by diffusion across the fluid–fluid interface (Fig 63.1). It is at these extremely small scales that fluid viscosity and surface tension become increasingly important considerations for fluid flow.

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(a)

(b)

Fig 63.1 (a) Turbulent vs laminar flow. Laminar flow is one of the inherent properties of microfluidic platforms which allows for unique applications in ART. (b) Microfluidic device demonstrating laminar flow. Fluid from channels 1, 2, and 3 flows in parallel with no mixing, except by simple diffusion.

Characteristic fluid flow patterns in microfluidic devices are amenable to applications requiring precise fluid sampling or manipulations, including examination of cellular behavior and interactions. Thus, microfluidics carries immense potential for improving clinical ART by offering the ability to seamlessly adjust composition of media flowing to developing gametes and embryos, without the external stresses currently experienced through manual manipulation as required with current sequential culture systems. In addition, microfluidic technology utilizes minimal amounts of media in a constrictive environment, similar to that experienced by gametes and embryos in vivo. Thus, a microfluidic platform allows clinical IVF to pursue physiologic mimicry through both chemical and physical manipulations to the in vitro culture environment. Furthermore, and perhaps more importantly, the scale of microfluidic platforms grants the ability to implement multiple procedural steps of IVF on the same device. This ‘labon-a-chip’ would not only save space but also time, as it allows for automation of processes as well as inclusion of diagnostic assays aimed at non-invasively identifying the healthiest cells for subsequent use.

Fabrication of microfluidic devices for ART Fabrication of microfluidic devices for use in ART has been covered elsewhere, discussing biocompatible materials, as well as manufacturing approaches.4–6 A practical guide of considerations in regard to fabricating perfusion-based microfluidic platforms for adherent cell culture also exists,7 and key issues that must be

addressed for functional application also largely apply to applications in ART. To summarize, various materials have been found to be adequate for gamete and embryo culture, and currently tested devices are composed of polydimethylsiloxane (PDMS), silicone, borosilicate glass, Pyrex, quartz, or combinations of these materials.8–17 Materials selected for construction of microfluidic channels may differ from material selected for cell substrate, or the actual surface on which cells lie. Although the cell substrate surface is a critical consideration for adherent cell culture to insure cell attachment, it is not as crucial a factor for culture of nonadherent oocytes or embryos. Manufacturing techniques for microchannels involve molding, photolithography, and chemically or mechanically etching channels into suitable materials. The most commonly used material for microchannel fabrication is the polymer PDMS, which is selected due to inherent use and fabrication advantages, such as flexibility, ease of soft-lithography patterning, and low autofluoresence for use with microscopy.7 Subsequently, compatible microchannel components are bonded to companion components/ platforms with non-toxic adhesives or epoxies. The benefits of these materials and manufacturing processes include rapid, repeatable, precise, and inexpensive production, a nec-essity for use in clinical ART, as devices should be disposable to ensure sterility.

Media flow/cellular manipulation in ART microfluidic devices As mentioned, one benefit of microfluidics for use in ART is the ability to achieve dynamic media flow on a culture platform. Although the approach of dynamic

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media flow in embryo culture is not new, prior attempts at perfusion systems on the macro scale for a variety of species have proved inefficient and subsequently not been implemented on a large scale.18–20 Fortunately, the unique nature of microfluidic platforms allows for alternate approaches to accomplish media movement that are more amenable for widespread use. These methods are often dictated by constraints of platform design, which is dependent upon whether perfusion systems are recirculating or non-recirculating. Furthermore, ability of perfusion systems to operate over long periods of time with minimal manipulation is essential. In adherent cell systems, perfusion devices have been operated successfully for over 1 week.21,22 Early devices for ART applications have employed gravity-driven passive flow, with hydrostatic pressure in media reservoirs as an important variable to drive media flow down microchannels.10,11,14,17,23,24 While simplicity of the approach is advantageous, it is difficult to regulate flow speed or volume changes, especially over time when height of media columns diminishes. Others have utilized manually applied pressure via externally attached syringes to input/output ports to cause media flow through microfluidic devices.25 Again, the simplicity of this approach is attractive; however, it is difficult to regulate pressure precisely and manual methods to regulate flow are not feasible for use over the long periods of time required for embryo culture. A variation of the syringe-driven flow approach has been further adapted through use of Hamilton syringes attached to a programmable infusion pump.16 Although more precise and feasible for use over time, the external tubing and machinery required is problematic for use within a closed incubator environment. Finally, a Braille pumping system using tiny piezoelectric actuators has been used successfully to peristalsically move media along microchannels during embryo culture.26–28 This approach not only allows for precise computerized regulation of speed and flow patterns but also the devices are compact enough to fit multiple units within a single incubator. It should be noted, however, that the Braille system is an electronic device; thus, special precautions must be taken to account for the humid incubator environment. Other possible approaches yet to be explored for ART to regulate fluid flow in microfluidic devices include pneumatics or magnetic gates/actuators. In addition, perhaps the most intriguing concept for regulating media flow within a microfluidic device entails use of expansive characteristics of a three-dimensional matrix, hydrogel, to regulate flow through channels.29 This technology offers the ability to control media flow through mechanical responses in the hydrogel due to external stimuli such as temperature, light, pH, and biological cues. Regardless of the perfusion approach adopted, a culture environment enabling dynamic media flow will allow chemical manipulation of cells by permitting gradual change of media flowing toward the gamete/embryo. One could imagine a progressive

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sequential or multistep culture media supplemented with varying chemical treatments or energy substrates being deployed to cells at prescribed time points, in a gradually changing manner, without the need for an embryologist to physically move embryos to new culture dishes. In addition, dynamic media flow permits physical manipulation of gametes/embryos, either transporting cells to desired locations or imparting physical changes. Indeed, both chemical and physical manipulation of oocytes/embryos have been achieved in microfluidic devices through manipulations of flowing media. Removal of cumulus cells has been achieved in a microfluidic device through suction applied via an attached syringe, drawing cumulus cells away from the oocyte down a small adjacent channel.30 Syringe-driven flow has also been used to position oocytes to allow removal of zona pellucida by flowing a bolus of acidified media over the cell.31 Finally, a microfluidic device has been designed that utilizes suction and electorotation to isolate and manipulate a single oocyte in a manner that may be suitable for intracytoplasmic sperm injection (ICSI).32 These integrated activities further serve to demonstrate potential for microfluidic technology for ART.

Requirements of microfluidics for ART Although theoretical advantages for microfluidic platforms in ART should be now readily apparent, it is important to recognize that several potential issues must be addressed in order to achieve widespread acceptance. Many of these issues exist regardless of the cell system cultured within microfluidic devices; however, reproductive applications of microfluidic platforms also carry unique considerations. These considerations can be grouped into four major categories (Table 63.1): • • • •

Material/design biocompatibility Device operation/failure Manipulation/handling of embryos IVF laboratory compatibility.

Biocompatibility As with other embryo culture dishes, it must be shown that microfluidic devices are nontoxic and pass quality control assays. Although initial testing has identified suitable fabrication materials, in-house testing must also verify biocompatibility of individual lots, ensuring contaminants were not introduced during production. Paramount in assuring biocompatibility is the ability to sterilize devices following fabrication. Current studies have utilized devices treated with UV light, ethanol, or ethylene oxide, or autoclaving.16,17 Although these approaches do not appear to affect properties of PDMS,7,33 high heat or chemical sterilization can warp or change biochemical properties of other polymers. Future exploration of more traditional sterilization methods, such as gamma irradiation, should be explored.

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Table 63.1 Practical considerations which must be addressed in order for microfluidic devices to gain widespread acceptance into IVF laboratories Biocompatibility

Operation

Manipulation of cells

Lab compatability

• Non-toxic fabrication materials • Sterilization of device

•

Perfusion system (recirculating vs non) Ability to be used over 5–6 days

• Easy visualization

• Fit in conventional incubators

Detection of failure (air bubbles) Easy rectification of failure

• Rapid loading/unloading

• User-friendly external apparatus

• Ability to isolate/ monitor individual cells

• Short set-up time

• Packaging to pass ‘in-house’ QC • Disposable

• • •

• Design issues (volume, embryo #)

Fabrication materials used in current microfluidic devices display unique properties that must also be addressed before implementation into IVF laboratories. Although these properties may be conducive for fabrication purposes, materials such as PDMS are absorptive and can alter media characteristics, including media flow and osmolarity, thereby impeding subsequent embryo development.34 Fortunately, surface modification to fabrication materials, including parylene coating or bonding with polyethylene glycol–methacrylate (PEG–MA), may alleviate some of these concerns.35,36 Furthermore, unique properties of microfluidics may require development of specialized culture media to accommodate the technology and optimize embryo production. Owing to extremely small channel dimensions, media viscosity is an essential variable to maintain laminar flow. Therefore, protein amounts or sources should be explored for compatibility. Additionally, small volumes of media utilized in microfluidics may result in rapid and damaging shifts in pH. This is extremely important when considering use of microfluidic platforms with oocytes, as oocytes appear to lack porters/ antiporters to regulate their intracellular pH.37–40 Thus, specialized culture media with increased buffering capacity may also prove beneficial. Finally, determination of optimal media volumes, as well as optimal numbers of gametes and embryos cultured in each microchannel device, is very important, as depletion of factors or build-up of wastes is potentially problematic at such a small scale, a problem more pronounced if media flow is not present. These would appear to be crucial recommendations that should accompany devices if/when they are commercially available. Future examination of these factors, and others, is essential to demonstrate efficacy of microfluidic devices in ART.

Device operation/failure Microfluidic devices employing dynamic culture must incorporate reliable mechanisms imparting media flow. This requires a safe and effective interface

between the microchannel platform and the perfusion regulatory device. Furthermore, a key consideration in regard to operation of dynamic flow is the ability to avoid bubble formation within microchannels. Construction of a bubble trap along the microfluidic channel may help alleviate this concern.41 Fortunately, if devices fail and media flow halts, the result is the current default static system. However, it is essential that devices possess methods to easily determine if flow has been interrupted and, if so, easy correction or repair should be available.

Manipulation/removal of embryos Perhaps the most difficult criterion to address in regard to construction and implementation of microfluidic technology in the IVF laboratory is the ability to easily manipulate or remove gametes/embryos, a limitation not often considered with devices currently used for adherent somatic cell culture or diagnostic assays. Furthermore, daily observation and manipulation of embryos cannot be a labor-intensive process, as increased time to view and adding or removing embryos will adversely stress blastomeres. Detriments with prolonged manipulation are exacerbated by the inherent problems that exist when dealing with extremely small volumes of liquid in microfluidics, such as rapid evaporation, resulting osmolarity shifts, as well as rapid shifts in pH and temperature. Thus, the design must incorporate a user-friendly interface through which the embryologist can access cells. A current approach that seems to work well is to culture cells within funnels. Furthermore, paramount in minimizing risks with prolonged handling is the ability to easily visualize cells within the device. Therefore, materials used must possess optical properties to visualize and track individual cells within the device for grading purposes. Optical properties should also be compatible with emerging technologies such as polarized microscopy. PDMS appears to be adequate for these purposes. Finally, device designs should accommodate different laboratory practices, perhaps offering

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(c)

50 µm

847

Media flow

2 mm

3 mm

Inlets

Inlets

Outlets

Outlets Motile sperm Non-motile sperm Cellular debris

(b)

Semen sample

Non-motile sperm/ debris 100 µm

(d)

500 µm

300 µm

Fresh media

Motile sperm

Fig 63.2 A microfluidic sperm sorter: (a) top view; (b) side view. (c) Demonstration of how laminar flow allows a motile sperm to swim away from a nonmotile sperm and debris to collect in a separate reservoir. (d) A sperm sorter device composed of PDMS.

various constructions and adjusting design to allow both individual or group culture.

IVF laboratory/embryologist compatible It is important that to gain widespread acceptance in clinical IVF, microfluidic devices need to be compatible initially with current IVF laboratory set-up and practices. Thus, devices must fit into contemporary incubators without external or bulky tubing or apparatus. Although much of the prospective power of microfluidic technology resides in more advanced analytical and automated processes that can be resident on the platform, these advancements cannot be realized without strong initial acceptance by the practicing embryologist.

Microfluidics in andrology Although future refinement is still required, progress addressing key issues of microfluidics has moved forward enough to begin to take advantage of unique applications offered by microfluidic platforms in areas of reproduction such as andrology.42 As early as 1993, microchannel devices made of silicone were used to evaluate sperm function via interactions with cervical mucus, hyaluronan, spermacide, and antisperm antibody beads.43 The same group later published a report demonstrating sperm count and motility assessment performed on etched glass microchannel devices.44 The first peer-reviewed publication on the use of microfluidic technology for separation of motile sperm from semen samples utilized a PDMS passive gravitydriven device where the hydrostatic pressure of two

separate inlet reservoirs drove media flow down a converging microfluidic channel (Fig 63.2).23 The principle of the device took advantage of the fact that only motile sperm can traverse the border that separates the parallel streams of diluted semen and fresh medium. Thus, the laminar flow properties exhibited by media in microchannels allowed motile sperm to swim away from nonmotile sperm, debris, and seminal plasma and collect in a separate outlet reservoir. Follow-up experiments demonstrated that this microfluidic device design was not only biocompatible with human sperm but also that it could isolate motile, morphologically normal cells.10 Furthermore, surface modification of PDMS microfluidic sperm sorter devices to increase hydrophilicy with coatings such as PEGMA may further improve this technology.35 Although limitations exist with the current device – including inability to process large sample sizes, clogging, and sample viscosity issues – this novel approach provides a feasible alternative to isolate sperm from oligozoospermic patients for use in ICSI. In another use of microfluidic technology for andrology, a PDMS/glass device has been constructed that directs sperm flow within oriented microchannels to separate, align, and orient sperm of mouse, bull, and human45 (Fig 63.3), providing potential applications for ICSI. Utilizing the fact that motile sperm orient themselves against media flow within these devices, and that motile sperm can swim against media flow of a certain velocity, a series of three reservoirs and four microfluidic channels allow processing of sperm via hydrostatic media flow. Although this device requires precise regulation of media volumes to regulate hydrostatic pressure, the authors have designed devices with the future intent of exploring

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(a) Reservoir 1 (media)

(b) Reservoir 2 (sample)

Reservoir 2 (collection)

25 µm PDMS

Glass

Junction (c)

Media flow from Reservoir 1 Junction 205 µm 100 µm

80 µm

Reservoir 2

Reservoir 3 205 µm

Media flow from Reservoir 1

implementation of a means to sort X- and Y-bearing sperm, and the ability to add a ‘laser cutting’ component to separate sperm heads from tails for ICSI. Owing to the limitations inherent in a microfluidic sperm-sorting device, utilization of the technology must provide some added benefit over conventional processing methods. Conventional sperm preparation methods such as serial centrifugation, density gradient separation, or swim-up are reported to induce sperm DNA damage, perhaps by exposure to reactive oxygen species (ROS).46–48 Preliminary data indicate that sperm isolated using a microfluidic sperm-sorting device had significantly lower levels of DNA damage and higher motility compared with these more conventional approaches.24 Thus, microfluidic sperm sorting may allow for selection of higher-quality sperm, potentially leading to improved embryo quality. Utilizing these advantages of a microfluidic device for sperm isolation, implementation of a microfluidic sperm sorter manufactured out of quartz has begun in clinical IVF. A preliminary report indicates human semen can be processed rapidly and isolated motile sperm can be used to successfully fertilize human oocytes following ICSI.11 Continued research will determine if this technology results in improved embryo development and/or implantation rates.

Microfluidics in embryology As mentioned, employing microfluidic technology for embryology applications requires considerations unique from microfluidic devices utilizing adherent cells, including ability to isolate or manipulate individual cells. In addition, sensitivity of reproductive

Fig 63.3 (a–c) A microfluidic device for separation, orientation, and alignment of motile sperm from bull, mouse, and human. Fresh media is loaded into Reservoir 1, while semen samples are loaded into Reservoir 2. Hydrostatic pressure differences, due to differences in height of media columns in the three reservoirs, cause media flow in various indicated directions. These media streams converge at a point known as the ‘junction.’ Motile spermatozoa orient themselves against direction of media flow and are able to swim through the current to collect in Reservoir 3. (a) Top view, (b) side view, (c) dimensions and flow patterns.

cells demands specialized precautions. Thus, progress implementing devices has been slow. However, as with andrology applications, refinement of microfluidic designs has resolved several of these issues, and platforms have begun to receive initial testing in all aspects of embryology, including in vitro oocyte maturation (IVM), IVF, and embryo culture.

In vitro maturation In vitro oocyte maturation is an especially appealing approach for human ART and offers tremendous advantages, including reduced cost and reduced health risk to disorders such as ovarian hyperstimulation syndrome (OHSS).49 However, IVM is still an inefficient practice. Fortunately, microfluidic approaches offer the potential to improve current IVM success rates. Preliminary data suggest that while porcine oocytes do not mature efficiently in silicone devices (2% metaphase stage II [MII]), they can indeed be matured successfully in PDMS microchannels (2% vs 71% development to MII).12 Oocytes matured in 200 µm wide PDMS microchannels, containing approximately 8 µl of media, displayed comparable development to MII as control oocytes matured in 8 µl or 500 µl drops. However, oocyte maturation consists of two components; nuclear and cytoplasmic maturation. Interestingly, cumulus cell expansion was noticeably diminished in oocytes matured in microchannels and 8 µl microdrops. This observation may be indicative of quality oocyte cytoplasmic maturation, as oocytes regulate cumulus cell development and function.50 Thus, size and/or volume limitations of microchannels may have some limiting effect on porcine oocyte cytoplasmic properties, or physically restrict

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1000 µm

6.6 cm

10 mm

2.2 cm

38 mm

(b)

Inlet/funnel

Outlet/luer

10 mm

20 µm

250 µm

Hydrostatic pressure-driven flow Borosilicate glass

PDMS

Fig 63.4 A PDMS/borosilicate microfluidic device used to perform IVF ‘on-chip’: (a) top view; (b) side view.

cumulus expansion. In contrast, subsequent follow-up experiments by Walters and coworkers from the same research group suggest oocyte cytoplasmic maturation may actually be enhanced in static microchannels. Pig oocytes matured in 250 µm wide PDMS/borosilicate glass microchannels produced significantly higher numbers of two-cell embryos following IVF and embryo culture in microdrops compared to oocytes matured in 500 µl drops (67 vs 49%).15 Unfortunately, pronuclear formation, embryo development past the maternal–zygotic transition, or blastocyst cell numbers were not reported. Additionally, it should be noted that chips utilized in these studies were not engineered with active media flow. Although some passive gravity-driven media flow may have been present, this parameter was neither measured nor recorded in experiments. Interestingly, preliminary studies indicate that bovine oocytes matured in dynamic microfluidic devices with Braille-pinregulated media flow actually yield improved blastocyst development following IVF compared with static matured oocytes (G Smith, pers comm). Despite these preliminary findings, the field awaits peer-reviewed publications that examine the effects of fluid flow in microfluidic devices on more informative markers of oocyte cytoplasmic maturation, including genomic, proteomic, or metabolomic profiles.

In vitro fertilization Another demonstration of microfluidic application in ART can be seen in IVF performed ‘on chip.’ The first attempt at this procedural step was performed using porcine oocytes placed into PDMS/borosilicate microchannels. Sperm were added in a manner such that pressure differences created from volume of media added resulted in gravity-driven flow of sperm past oocytes (Fig 63.4). It was shown that oocyte numbers

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used in experiments had no affect on sperm penetration. Furthermore, fertilization in microchannels resulted in significantly lower rates of polyspermic penetration compared with fertilization in control microdrops.14 Reduced polyspermic penetration rates were attributed to the physical characteristics of the microfluidic device, mimicking the environment in utero. It is thought that microfluidic devices serve to limit time of oocyte exposure to sperm, as sperm were not confined to the vicinity of the oocytes, as in microdrop culture, but allowed to flow past the eggs along the length of the microchannel. In a similar study, utilizing cleavage of mouse embryos to two-cell embryos as an indicator of successful fertilization, Suh and colleagues17 demonstrated that fertilization of mouse oocytes can occur in a microfluidic device (Fig 63.5). Although initial experiments utilizing high concentrations of sperm revealed that overall fertilization rates were decreased on the microfluidic device compared with controls, subsequent experiments demonstrated that, by lowering sperm concentration, fertilization rates in microfluidic devices were actually higher than fertilization in control drops. Fertilization rates obtained at these lower sperm concentrations in microfluidic devices were comparable to rates obtained with higher sperm concentrations in control microdrops. These results appear to be the result of chip design, as the authors observed an increased concentration of sperm in the vicinity of the oocyte in microfluidic devices. This increased concentration may not only explain increased rates of fertilization at reduced sperm concentration but also reduced fertilization rates at high concentrations of sperm. Increased sperm concentration may potentially result in decreased availability of local metabolic substrates, or result in detrimental shifts in culture conditions, such as pH or localized build up of metabolic byproducts. Finally, Clark and coworkers presented preliminary data demonstrating both IVM and IVF of porcine oocytes could be performed on the same microfluidic device without removal of the cells between procedures.13 Media within devices were changed and sperm added via manual pipetting without disturbing oocytes. Although there were no observable benefits achieving cleavage to two-cell embryos compared with control treatments (49% vs 51%), this was the first demonstration of multiple tasks of in vitro embryo production performed upon the same microfluidic platform. Such a realization has tremendous potential for performing multiple sequential steps ‘on chip’ and minimizing stress to gametes/embryos. In vitro fertilization not only entails mixing of sperm and eggs but also requires manipulation of presumptive zygotes to remove cumulus cells to visualize pronuclei. As previously mentioned, cumulus cells,30,51 and even zona pellucidae,31 can be removed using a microfluidic device with flow driven manually via attached syringes, exposing oocytes to chemical and physical manipulation.

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Fig 63.5 Microfluidic device composed of PDMS utilized to perform mouse IVF ‘on chip.’

(a)

Inlet/loading

Outlet/suction

(b)

Silicon

PDMS

Borosilicate glass

Fig 63.6 The first microfluidic device created to culture preimplantation mouse embryos: (a) top view and (b) side view of devices made out of differing materials.

Inclusion of these abilities on the same device where IVF is performed would be extremely advantageous in minimizing stress imposed upon the embryo. Nevertheless, it is evident that chip design is extremely important to the efficacy of the fertilization process, and refinement with further testing of various designs is essential to advance IVF on microfluidic devices. Additionally, subsequent assessment of developmental competence, implantation, and live birth rates of microfluidic-derived IVF embryos is required to determine the efficacy of this approach. These efforts, though, are important, as this approach may offer substantial benefit to patients undergoing infertility treatment due to conditions such as oligozoospermia.

Embryo culture Exhaustive studies have been conducted aimed at optimizing preimplantation embryo culture in vitro, and, similar to IVM and IVF, a microfluidic platform may aid in this endeavor. The initial report on embryo culture using microfluidics by Raty and colleagues indicates that two-cell mouse embryos can be cultured to the blastocyst stage within static microchannels9,52

(Fig 63.6). These experiments demonstrate that, compared to 30 µl control microdrops, culture within the microchannel containing about 500 µl of media (10 µl within the actual channel itself) resulted in significantly greater 16-cell/morula formation at 24 hours, greater blastocyst formation at 48 hours and 72 hours, and a greater portion of hatched blastocysts at 72 and 96 hours. Subsequent experiments utilizing a similar device by Walters and coworkers from the same research group showed that in vivo derived four-cell porcine embryos could be cultured to blastocyst and transferred, resulting in live birth.53 However, in these experiments, no observable beneficial effects on embryo development were seen when compared to culture in control organ-well dishes. Building upon the initial chip-based static embryo culture studies, Hickman et al examined mouse embryo development in microchannels with media flow, controlled via a syringe infusion pump16 (Fig 63.7). Flow rates examined in this study (0.1 and 0.5 µl/h) did not enhance development compared to static culture. In fact, a flow rate of 0.5 µl/h resulted in significantly lower development of two-cell mouse embryos to morula and blastocyst stages, while producing higher

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Infusion pump

Inlet/loading port

Outlet port

Media flow Pyrex glass

Silicon

PDMS

numbers of abnormal embryos compared with controls. Thus, flow rate and manner of flow delivery may be important variables for embryo culture in microfluidic devices. Indeed, embryos sense sheer stress, which can induce apoptosis and be detrimental to embryo development.54 However, it is questionable if flow rates necessary for dynamic fluid flow in microfluidic channels would approach velocities that are high enough to cause concern. Additionally, it should be noted that these data on the impact of media flow and flow rate on embryo development should be reassessed, as culture conditions may have been suboptimal. In this particular study, control mouse embryos cultured in control static microchannels did not improve embryo development, as previously reported by Raty and colleagues from the same research group.9 One possible source of variation requiring future study was the increased number of embryos cultured in each device. Interestingly, utilizing an alternate chip design, Cabrera and co-workers presented preliminary data that one-cell mouse embryos could be cultured efficiently within microfluidic devices offering media flow.28 Embryos were loaded into a funnel reservoir, while medium was added and removed via a microfluidic channel connected to the bottom of the funnel via actions of a Braille actuator (Fig 63.8). It was demonstrated that regardless of media flow pattern (back and forth vs flow-through) or speed (fast vs slow), one-cell mouse embryos cultured in dynamic devices showed greater hatching of blastocysts and significantly higher cell number than static controls, yielding numbers similar to those obtained from in vivo derived blastocysts. Bormann and colleagues subsequently presented preliminary data validating beneficial effects of Brailledriven media flow in a microfluidic device on murine and bovine embryo development.27 A greater number of mouse embryos reached the morula stage at 48 hours, blastocyst at 72 hours, and hatched blastocyst at

Fig 63.7 A microfluidic perfusion system utilizing a syringe infusion pump to regulate media flow to culture preimplantation embryos.

96 hours compared with control static chips, whereas significantly more bovine embryos reached the blastocyst stage at 144 hours in microfluidic devices compared with control static devices. Follow-up experiments by Bormann and colleagues indicated that the beneficial effects of embryo culture in the dynamic culture device are additive and require a minimum 48 hours of culture at the beginning or end of 96 hours culture periods.26 Yet another approach to culturing embryos within microfluidic devices has employed not only dynamic media flow but also co-culture. Mizuno and coworkers have adopted a ‘womb-on-a-chip’ design, where endometrial cells are grown in a lower chamber, while embryos are cultured in an upper chamber, separated from the lower by a thin membrane,8,55 thus allowing embryos to interact with secreted factors from the endometrial cells (Fig 63.9). In this preliminary report, authors demonstrated that mouse ova fertilized on and resulting embryos cultured in these devices showed similar cleavage to two-cell embryos and similar blastocyst formation rates compared with 50 µl control microdrops.55 Furthermore, cell number was significantly higher in blastocysts fertilized/cultured in microfluidic devices. Subsequently, blastocysts obtained from microfluidic devices were transferred to recipient female mice and resulted in live offspring at rates similar to embryo cultured in static microdrops. Although these experiments are still preliminary, they add to the advancement of microfluidic technology in ART, showing that both IVF and embryo culture can be performed on the same device, and that resulting embryos yield live offspring. A similar co-culture approach was taken by the same group, culturing two-cell mouse embryos to blastocyst stage on the refined OptiCell microfluidic device. OptiCell microfluidic co-culture yielded chromosomally normal embryos that were capable of yielding live offspring.56

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(a) Loading funnel

Media reservoir

Braille pin actuators Braille display

(c)

Fig 63.8 Microfluidic device composed of PDMS utilizing Braille actuators to drive media flow two microchannels. Media flow is accomplished via Braille pin deflection of the thin bottom of the PDMS platform. Coordinated pin rise/fall allows peristaltic movement of a media bolus along the length of the microfluidic channels. (a) Top view. (b) Side view. (c) Image of Braille pin PDMS device and demonstration of Braille pin rise/fall used to control fluid flow through microfluidic channels. (a)

Inflow/outflow for upper chamber

Inflow for lower chamber

(b)

Embryo cage

Polyester membrane Polyester membrane

Embryo cage

Lower chamber for endometrial cell culture Outflow for lower chamber

Endometrial cell culture chamber

(d) Access guide to embryo cage

(c)

Fig 63.9 The ‘womb-on-a-chip’ microfluidic perfusion system. Embryos are grown in a co-culture system, separated from endometrial cells by a thin polyester membrane. (a) Top view. (b) Side view. (c) Indication of media flow. (d) Image of PDMS ‘womb-on-a-chip’ microfluidic device (photo courtesy of Dr Teruo Fujii).

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Finally, Mizuno and colleagues published an abstract reporting the first instance of human embryo culture within microfluidic devices. Donated 2–4 cell stage frozen human embryos were cultured to the blastocyst stage, resulting in significantly higher rates of blastocyst development from microfluidic devices compared with control microdrops.8 Additionally, visual scoring of microfluidic-derived blastocyst development revealed higher-quality blastocysts with significantly higher cell numbers compared with static controls. Unfortunately, co-culture confounds interpretation of results obtained, as it is impossible to discern if effects are attributed to co-culture or microfluidic design. As the goal of embryo culture has been the development of defined culture media, it will be interesting to see if similar studies can be performed without the use of co-culture.

How do oocytes/embryos benefit from microfluidics? How does microfluidic technology work to improve embryo development? It has been hypothesized that unknown autocrine/paracrine factors secreted by gametes or embryos are localized owing to the confining nature of microfluidic devices, and that this localization of factors may carry certain benefits. Indeed, oocytes and embryos do secrete various factors, and research is ongoing to identify components of this secretome as non-invasive markers of embryo developmental competence.57,58 If secretion of beneficial factors from cultured cells is responsible for improved development, then quantity of embryos, volume of media, and surface area occupied would be extremely important factors to consider. Discussion of these issues in traditional culture platforms has been eloquently reviewed.59 Indeed, several studies have examined the beneficial effects of group embryo culture and media volume on traditional culture platforms of several species.60–62 Although direct comparisons are difficult, owing to differences in experimental parameters, it appears that in certain instances group culture does provide beneficial effects. Furthermore, in dealing with group culture, another important variable to consider is the quality of companion embryos.63,64 However, experiments exploring effects of embryo density within microfluidic devices are lacking. Although the autocrine/paracrine debate offers an attractive means by which embryo development may benefit from microfluidic culture, secretion of trophic factors from embryos does not necessary explain improved embryo development within microfluidic devices with dynamic conditions. With media flow, tropic factors would presumably be diluted or removed, yet advanced development is still observed. Alternatively, benefits within dynamic devices could be the result of removal of harmful embryo metabolic by-products, such as ammonia.65 If this is indeed the

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case, then undoubtedly a delicate balance exists on the correct number of embryos to culture in static devices to not negate any potential benefit of group autocrine/paracrine factors. However, it should be noted that studies removing spent culture media and replacing with fresh media from microdrops at various intervals show no benefit to embryo development.66,67 Thus, removal of by-products may not be the sole reason for improved embryo development within microfluidic devices. A common denominator of static and flow devices is the constrictive nature of the micro devices, confining embryos to a very small area. Thus, benefits may be somehow related to cell proximity and spacing. Indeed, spacing of embryos during group culture affects development. Two innovative studies attached either pig or cattle embryos to culture dishes at measurable distances and demonstrated that if embryo spacing was too great, blastocyst development was not achieved.68,69 This spacing theory is supported by studies showing advanced embryo development in confining ultramicro drops,67 culture within glass capillary tubes,60,70 culture within small concave wells (GPS dish),1 as well as culture within extremely small volumes/area in the ‘well of a well’ or WOW technique.71 If spacing is important and can improve embryo development when cultured in groups, then another variable to consider in designing microfluidic devices for ART is shape of the culture area,59 which can dictate if embryos are in direct contact or maintain physical separation. It remains to be seen if microfluidic embryo culture improves development of individually cultured embryos. It has been suggested by some that benefits of culturing embryos within extremely small volumes, including microfluidic culture, may result from a reduction in localized oxygen (O2) tension due to culture in these confined spaces. In traditional culture approaches, reduced O2 levels from atmospheric levels of around 20% to around 5–7% is beneficial for embryo development and quality.72–74 Interestingly, the majority of reported microfluidic culture experiments were performed in 5% CO2 in air, and thus, reduction in localized O2 would appear to be a plausible theory. However, bovine embryos cultured in dynamic microfluidic devices in 5% O2 still showed improved development over static culture in the same device.27 Also, it should be noted that hypoxic conditions are detrimental to oocytes and embryos.73,75 Thus, if localized O2 depletion is occurring in microdevices, culturing in reduced O2 tension could in fact result in hypoxia, decreasing embryo development. Fortunately, as the bovine study by Bormann and colleagues indicated, this does not appear to be the case. In support of this, mathematical modeling suggests O2 depletion does not appear to be a factor in microdrops, regardless of the number of mouse embryos cultured, as diffusion and convection currents mix the environment to prevent anoxic regions.76 Thus, localized reduction in O2 levels is questionable, especially when one considers the microfluidic devices

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used in ART studies are composed of PDMS, which is extremely gas permeable. However, at least one mathematical modeling study by Byatt-Smith et al suggested that, due to slightly larger size, culture of human embryos within static microdrops may become marginally hypoxic, especially when cultured in 5% O2.77 As a preventive measure to hypoxia, these same authors hypothesized that ‘embryos may develop more successfully in stirred, as opposed to still medium.’ Therefore, in studies utilizing dynamic microfluidic devices, media renewal due to flow should prevent any localized build-up or depletion of factors, including O2, thereby alleviating the possibility that advanced development is due to O2 depletion. In fact, it can even be argued that media flow provides a more uniform and stable oxygen concentration, actually preventing formation of any localized oxygen depletion gradient that may form in static culture systems. Yet another possibility for benefits of microfluidic culture, at least in dynamic flow devices, is gentle agitation of embryos or media surrounding embryos. This mechanical stimulation could theoretically aid in clearing of receptors or stimulate signaling pathways, subsequently promoting morula compaction and blastocyst development. Although interesting, this hypothesis still awaits further exploration. Unfortunately, each microfluidic device utilized to date is different in its construction (culturing in channels, culturing in funnels, culturing with/without flow, using co-culture, utilizing different number of embryos/volume), thereby making it difficult to ascertain where the benefit to embryo development actually lies. Nevertheless, it is obvious that a culmination of factors is dictating embryo development and quality within any culture device, including microfluidic platforms. Further research examining divergence of gene expression patterns, molecular signaling pathways, or some other biochemical endpoint is required to begin to elucidate possible explanations for the observed effects in microfluidic culture and to optimize this promising technology for use in ART (Table 63.2).

Microfluidics and non-invasive viability assessment Perhaps the most important benefit of implementing a microfluidic platform for ART is the ability to integrate diagnostic tools to monitor gamete and embryo environmental conditions and physiologic processes. Although environmental monitoring has not yet received attention in ART, e.g. using a nanosized optical sensor, it is possible to monitor the pH of interstitial fluid flowing through a microfluidic device.78 Using nano- or microsensors, one can monitor temperature fluctuations,79,80 media flow rate,81 and volume of the cell to indicate shifts in media osmolarity.82 Additionally, electrical sensors have been developed to measure real-time changes in levels of ROS as indicators of oxidative stress on microfluidic

Table 63.2 Suggested variables that should receive future study to assist in widespread implementation of microfluidic platforms in ART • Effects of embryo/gamete density • Importance of embryo/gamete spacing • Effects on cell transcriptome, proteonome, metabolome, and secretome • Influence on embryo developmental competence, implantation, and pregnancy • Optimization of media flow rates/patterns • Development of specialized culture media

devices.83,84 Conceivably, any one or a combination of these approaches could be adapted for use with ART microfluidic devices, as measurement of any of these environmental parameters may provide insight into improvement of culture conditions and current IVF laboratory practices. Furthermore, it has long been the goal of reproductive investigators to discover a means by which to interrogate gametes and embryos to identify markers of physiologic processes as a non-invasive means of predicting developmental competence and implantation potential. Current research is examining secreted proteins from embryos in the hopes of relating this information to cell quality.57,58 Implementation of ELISA (enzyme-linked immunosorbent assay) ‘on-chip’ have met with some success in adherent cell systems,85,86 and may prove useful in analyzing embryo secretomic profiles. Additionally, examination of oocyte or embryo metabolomic activity and profiles has also showed promise in offering a non-invasive predictor of embryo quality.87–89 A silicon microfluidic device has been designed to measure O2 consumption rates as an indicator of mouse preimplantation embryo metabolic activity,90 and preliminary data measuring glucose, lactate, and pyruvate levels of spent embryo culture media in a pneumatic controlled microfluidic device exist.91 Although embryos were not grown in microfluidic devices in these studies, ideally these types of analysis systems will be constructed on the same device as cultured cells, allowing rapid, real-time analysis without additionally stressing embryos by additional manipulation or removing them from the incubator environment.

Future directions In a now increasingly used quote, Bavister stated: ‘Ideally, the culture medium should change progressively during development to keep pace with the embryos needs, but continuous flow or perfusion culture systems are difficult to maintain on a micro scale and may be impractical.’92 It is apparent that advances in microfluidic technology may offer a solution to this impasse, as designs are quickly approaching a stage

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where continuous flow of media will no longer be problematic. Microfluidic chip design and technology have progressed dramatically, ensuring compatibility with essential requirements of the IVF laboratory. In fact, at least one report detailing culture of human embryos on chip,8 and at least one commercial company has refined its system enough to begin testing microfluidic culture devices with donated human embryos. The attributes of microfluidics facilitates a more in vivo-like environment for culturing embryos in confined spaces, and in small volumes of media, as well as facilitating media flow and progressive substrate change to meet the evolving nutritional requirements of the developing embryo. Finally, microfluidic design allows the embryologist to explore spatial considerations not afforded by the two-dimensional surface of a dish. Thus, the scale of microfluidic devices offers the ability to shuttle in a new era in embryo culture. Emerging technological advancements offer the capability of not only regulating embryo culture fluid dynamics but also substrate compositional dynamics, essentially marking the end of ‘fixed’ chemistry in media. By combining a microfluidic platform with three-dimensional matrices of oriented macromolecules to create a ‘moist’ rather than a fluid environment, more like that of the female reproductive tract, embryos may benefit in ways previously unattainable in standard culture systems. Through chemical–physical interactions, oriented macromolecules within the female reproductive tract are hypothesized to support embryo cellular homeostatic mechanisms, imparting responsiveness or plasticity to the embryo.93,94 Thus, implementation of oriented three-dimensional matrices for in vitro embryo culture may render embryos more competent to compensate for encountered homeostatic imbalances. This approach has shown some promise on a macroscale for follicle and oocyte culture.95,96 However, as pointed out, the physical presentation of these macromolecules to gametes or embryos is problematic. Pool stated: ‘to mimic the physical-chemical conditions present in vivo, a potential culture system must be able to present an array of macromolecules, an assemblage that changes qualitatively, with time, from fixed sites in a minimum volume of water.’94 Microfluidics offers a platform that can alleviate these previous limitations and offer certain benefits over macro systems. One can imagine a microfluidic device housing multiple individual chambers in which a single embryo is encapsulated within a transparent three-dimensional oriented organic matrix, perfused with culture media that changes composition over time to meet the changing metabolic needs of the cell. Appropriate selection criterion would allow subsequent transfer of the biodegradable scaffold and enclosed embryo. Suitable biodegradable matrixes, such as poly (DL-lactideco-glycolide) (PLGA)97 and poly (glycerol sebacate) (PGS),98 have recently received attention in other cell systems. Furthermore, with continued advancement

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and use of 3-dimensional matrices, such as hydrogels, it is even imaginable to have an organic, self-regulating microfluidic device providing peristaltic media flow,29,99 truly embracing a physiologic phenomimetic approach to embryo culture. Perhaps, more importantly, not only can microfluidic devices be used to manipulate gametes and culture embryos more efficiently but also they lend themselves to integration with other systems. Microfluidic chips have been designed that can perform multiple aspects of in vitro embryo production13,32,55 and it is extremely feasible to imagine a microfluidic device integrated multiparametric, real-time, on-chip-diagnostic assay to assist in selection of the most viable gametes/embryos. Eventually, automation of such microfluidic devices may allow for reduced manipulation and prevent unneeded imbalances in embryo homeostasis, even alerting the embryologist and taking preventive actions if monitored variables fall out of a specified range. However, diligence in these pursuits is called for. Although the field has received tremendous initial interest, the lack of peer-reviewed publications demonstrates the infancy of the technology. Continued research and exacting experimentation are required to realize the full potential of microfluidic technology for ART (Table 63.2).

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77. Byatt-Smith JG, Leese HJ, Gosden RG. An investigation by mathematical modelling of whether mouse and human preimplantation embryos in static culture can satisfy their demands for oxygen by diffusion. Hum Reprod 1991; 6: 52–7. 78. Baldini F, Giannetti A, Mencaglia AA. Optical sensor for interstitial pH measurements. J Biomed Opt 2007; 12: 024024. 79. Chang YH, Lee GB, Huang FC, et al. Integrated polymerase chain reaction chips utilizing digital microfluidics. Biomed Microdevices 2006; 8: 215–25. 80. Lucchetta EM, Munson MS, Ismagilov RF. Characterization of the local temperature in space and time around a developing Drosophila embryo in a microfluidic device. Lab Chip 2006; 6: 185–90. 81. Lien V, Vollmer F. Microfluidic flow rate detection based on integrated optical fiber cantilever. Lab Chip 2007; 7: 1352–6. 82. Ateya DA, Sachs F, Gottlieb PA, et al. Volume cytometry: microfluidic sensor for high-throughput screening in real time. Anal Chem 2005; 77: 1290–4. 83. Amatore C, Arbault S, Bouton C, et al. Monitoring in real time with a microelectrode the release of reactive oxygen and nitrogen species by a single macrophage stimulated by its membrane mechanical depolarization. Chembiochem 2006; 7: 653–61. 84. Amatore C, Arbault S, Chen Y, et al. Electrochemical detection in a microfluidic device of oxidative stress generated by macrophage cells. Lab Chip 2007; 7: 233–8. 85. Eteshola E, Balberg M. Microfluidic ELISA: on-chip fluorescence imaging. Biomed Microdevices 2004; 6: 7–9. 86. Herrmann M, Roy E, Veres T, et al. Microfluidic ELISA on non-passivated PDMS chip using magnetic bead transfer inside dual networks of channels. Lab Chip 2007; 7: 1546–52. 87. Gardner DK, Lane M, Stevens J, et al. Noninvasive assessment of human embryo nutrient consumption as a measure of developmental potential. Fertil Steril 2001; 76: 1175–80. 88. Lane M, Gardner DK. Selection of viable mouse blastocysts prior to transfer using a metabolic criterion. Hum Reprod 1996; 11: 1975–8.

89. Seli E, Sakkas D, Scott R, et al. Noninvasive metabolomic profiling of embryo culture media using Raman and near-infrared spectroscopy correlates with reproductive potential of embryos in women undergoing in vitro fertilization. Fertil Steril 2007; 88: 1350–7. 90. O’Donovan C, Twomey E, Alderman J, Moore T, Papkovsky D. Development of a respirometric biochip for embryo assessment. Lab Chip 2006; 6: 1438–44. 91. Urbanski JP, Johnson MT, Craig DD, et al. Noninvasive metabolic profiling using microfluidics for analysis of single preimplantation embryos. Anal Chem 2008 (in press). 92. Bavister BD. Culture of preimplantation embryos: facts and artifacts. Hum Reprod Update 1995; 1: 91–148. 93. Pool TB, Martin JE. High continuing pregnancy rates after in vitro fertilization–embryo transfer using medium supplemented with a plasma protein fraction containing alpha- and beta-globulins. Fertil Steril 1994; 61: 714–19. 94. Pool TB. Recent advances in the production of viable human embryos in vitro. Reprod Biomed Online 2002; 4: 294–302. 95. Pangas SA, Saudye H, Shea LD, et al. Novel approach for the three-dimensional culture of granulosa cell–oocyte complexes. Tissue Eng 2003; 9: 1013–21. 96. Combelles CM, Fissore RA, Albertini DF, et al. In vitro maturation of human oocytes and cumulus cells using a co-culture three-dimensional collagen gel system. Hum Reprod 2005; 20: 1349–58. 97. Vozzi G, Flaim C, Ahluwalia A, et al. Fabrication of PLGA scaffolds using soft lithography and microsyringe deposition. Biomaterials 2003; 24: 2533–40. 98. Fidkowski C, Kaazempur-Mofrad MR, Borenstein J, et al. Endothelialized microvasculature based on a biodegradable elastomer. Tissue Eng 2005; 11: 302–9. 99. Eddington DT, Liu RH, Moore JS, et al. An organic self-regulating microfluidic system. Lab Chip 2001; 1: 96–9.

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64 The evolving role of the ART nurse: a contemporary review Joanne L Libraro

As the ‘ART’ of assisted reproductive technologies advances, so too does the role of each committed and determined staff member and/infertility treatment center. Today, to millions of infertile couples around the world, in vitro fertilization (IVF) has become synonymous with hope and the possibility of fulfilling the dream of parenthood. In the early days, treatment was considered something available only for the privileged or wealthy. Today, even more patients seek and successfully pursue treatment under one of many available IVF programs. The ‘ART’ was seen as a ‘laboratory science’ research oriented and experimental. As time passed, physicians, nurses, laboratory personnel, and researchers developed the prerequisite skills necessary to remain current in the field of reproductive endocrinology and infertility (REI). Additionally, the available technologies have become accepted as standard treatment modalities. Dating back nearly 50 years ago at Bourn Hall under the guidance of Robert Edwards and Patrick Steptoe, the future role of ART nurses was conceived and established.1 Jean Purdy (Fig 64.1), the first recognized ‘nurse’ of distinction in the field of REI, created and established the foundation for clinical growth, scientific development, holistic supportive therapies, and nursing research. The birth of Louise Brown – the first ‘test-tube’ baby – on July 25, 1978 not only thrilled her parents but also introduced the world to the future of assisted reproductive technologies. This triangle of success comprised Robert Edwards, Patrick Steptoe, and Jean Purdy. Today, REI nursing professionals are defining the future nursing standards in the field of reproductive health. These eager, focused nursing professionals grasp the opportunity to participate in the continued evolution of the contemporary REI nursing role in a similar way that Jean Purdy first made her own contributions. Nursing roles in infertility have been transformed since the introduction of IVF, and now, more than ever, nurses wishing to specialize in reproductive technologies have many opportunities to extend their

clinical and theoretical skills. The critical role of the ART nurse is reviewed in this chapter.

Assisted reproductive technologies: a rapidly progressing field ART procedures require time and energy, and many couples face a long and arduous journey in their goal to become pregnant. Having accepted the need for ART, couples generally face many layers of decisionmaking – emotional, cultural, ethical, financial, and religious – before initiating treatment. Because of the unique relationship established with the couple seeking treatment, the ART nurse plays a critical role in their care. Since the late 1980s and 1990s, technology in the field of reproductive medicine has evolved at a rate that no one could have predicted. In 2007, at any given ART center, patients could expect the staff to include board-certified REI physicians, nurses, psychologists, embryologists, andrologists, reproductive urologists, genetic counselors, financial counselors, reproductive endocrine laboratory staff, and administrative staff. Additionally, many IVF centers with academic affiliations provide a 3-year fellowship for obstetrician-gynecologists (OB/GYNs) interested in pursuing reproductive endocrinology and infertility. For patients undergoing treatment, the education and coordination of their treatment regimen can be overwhelming. A dedicated, skilled, and empathetic ART nurse can successfully coordinate and facilitate the entire treatment process, thereby providing patients with the support and guidance needed.

Infertility nursing The emergence of centers offering access to reproductive technologies has allowed nurses to extend their clinical role far beyond that of the traditional nursing model. In many ART centers, nurses are working with increased autonomy within the framework of a highly

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Counselor

Nurse researcher

Patient educator

Coordinator of schedules

Fig 64.2 Nurses working in assisted reproductive technologies (ART) today have many roles.

Fig 64.1 Jean Marion Purdy, 1945–85. Reprinted from Edwards et al,1 with permission from Elsevier.

specialized team of professionals. Therefore, the nurse becomes the ‘center’ of the patient’s treatment cycle, managing multidisciplinary responsibilities as the: • • • • • • •

ART clinical nurse patient educator IVF coordinator counselor nurse researcher egg donor coordinator PGD (preimplantation genetic diagnosis) coordinator • fertility preservation coordinator • male factor infertility coordinator. Today, nurses working in both small and large ART centers have diverse roles that require a wide variety of skills, encompassing, but not limited to, medical care, psychological support, quality assurance, and patient education. Experienced practitioners bring many of these core skills from other areas of nursing. Specific training in the field of infertility embellishes these transferable skills. Furthermore, the treatment of infertility, through the use of IVF and other related techniques, has an ethical and religious dimension that may provoke considerable challenge for nurses and patients: e.g. the use of donor sperm and/or embryos, PGD, fertility preservation, or issues related to multiple pregnancies. It is the diversity of the nursing role in ART,

combined with the fact that couples rely primarily on the nurse for education and, importantly, support, that ART nursing should not be the goal of a recent nursing school graduate. Those most likely to succeed as ART nurses are mature, experienced, flexible, and professional individuals who consistently seek to redefine their own role within a rapidly evolving field (Fig 64.2). To be successful in this highly demanding field, ART nurses require not only an appropriate level of education, training, and ongoing support but also must have a resilient personality to manage the considerable emotional and physical pressures they encounter. At the very least, nurses working in this specialty must be confident and assertive and able to perceive that they have the ability to change, or at least control, the events and systems around them. Nurses whose personality traits can be described as ‘shy,’ ‘apprehensive,’ and ‘reactive’ are more likely to suffer burnout. Indeed, there is evidence suggesting that ART nurses are at high risk of burnout. This is positively correlated to the length of time spent working in reproductive endocrinology and to a low perceived sense of emotional support.2 As the nursing shortage in the USA continues, ART nursing positions remain difficult to fill. Thus, the workload carried by many ART nurses can be compounded by this resource challenge. In addition, the complexity of contemporary ART adds to the challenges that many programs face when hiring and retaining nursing staff.

Assisted reproductive technologies clinical nurse Nurses have traditionally performed – or at least participated in – many of the technical steps involved in initiating a patient’s ART cycle. Following initial consultation with the physician, the ART nurse becomes intimately involved in each patient’s treatment cycle. In addition to their role in patient education, counseling, and treatment planning and coordination, nurses have commonly performed certain technical aspects of IVF treatment cycles, including: • medical history and record review • pretreatment evaluation and required testing

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• evaluation of daily laboratory results • medication administration and instruction • preoperative, intraoperative, and postoperative care • pregnancy evaluation and follow-up • ovulation induction monitoring and intrauterine insemination (IUI) partner and donor programs.3

Patient educator ART nurses are a key source of information for infertile couples. Available techniques and practices differ from clinic to clinic. Comprehensive patient education and support should be considered mandatory at every ART clinic. The goal of IVF patient orientation and education is to ensure that patients understand the ‘big picture’ of their treatment. Ultimately, successful patient education fosters confidence in couples undergoing treatment. As a result, patients are more likely to be confident about providing informed consent, to be satisfied with their treatment, and to be more inclined to accept its final outcome.4 Key aspects of patient education include discussions that focus on: 1. The couple’s specific infertility challenge 2. Overview of program practices (i.e. who’s who, how the clinic runs, etc.) 3. Available ART technologies such as intracytoplasmic sperm injection (ICSI), PGD, assisted hatching, testicular sperm extraction (TESE), third-party reproductive, fertility preservation, etc. 4. Timing of the patient’s cycle, including drug injections, overview of hospital experience from oocyte recovery to embryo transfer, etc. 5. Preparation of the cycle, including prestimulation protocols, stimulation protocols, and possible adverse events 6. Post-transfer management, such as pregnancy testing, follow-up appointments, etc. 7. Teaching reconstitution and administration of medications 8. Available support services that are center-specific, center-supported, or outside advocacy groups 9. Possible IVF concerns, such as premature ovulation, poor ovarian response, ovarian hyperstimulation syndrome, etc. ART remains a complex specialty with ever-changing medical terminology, a wide number of acronyms and abbreviations, and intricate drug regimens and protocols. For this reason, it is important for the ART nurse to prepare patients fully for what is to come during the ART process. These professionals focus on alleviating anticipated stress and fear associated with ART treatment, and foster a sense of confidence to succeed. In many ART centers, one-on-one training occurs with the couple, in addition to, or as a substitute for,

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group classes. The importance of involving the partner or spouse in the educational process cannot be overemphasized. Fortunately, the unique relationship developed with the ART nurse facilitates the involvement of the couple. During these sessions the nurse will gain insight into each patient’s specific needs, and provide necessary information specific to clinical procedures as these relate to the patient’s religious, cultural, psychological, and medical needs. Good patient compliance with drug regimens is imperative for a successful outcome, and this is more likely to be achieved if couples understand all aspects of their designated ART treatment. It is well documented that success or failure of treatment depends on a clear understanding of the current treatment regimen, regardless of the patient’s prior experience with the process. For example, human chorionic gonadotropin (hCG) is the single most important injection of the patient’s treatment cycle. Nonetheless, errors in hCG administration have been attributed to poor cycle outcome. Patients have made reconstitution errors, such as injection of one-tenth of the dose or administration of diluent only, or timed the injection incorrectly.5 A recent report found that 10 patients (15.2%) undergoing IVF or ovulation induction treatment received hCG incorrectly, and in these cycles only one pregnancy occurred.6 In the early days of IVF, a nurse could expect to spend approximately 1.5 hours on patient education, while today, patient education sessions may extend to more than 2.5 hours. Initially, the nursing time dedicated to patient education was related to the limited number of technologies available for infertility treatment. For example, medication administration was limited to urinary preparations of human menopausal gonadotropin (hMG) and hCG, and both were administered intramuscularly. In the mid-1990s, with improvements in laboratory procedures, the movement from subzonal injection to ICSI, the evolution from urinary to recombinant medications, improvements in PGD procedures, and the expansion of other available technologies, the time and complexity of patient education increased dramatically. In particular, the late 1990s saw the introduction of recombinant follicle-stimulating hormone (FSH), followed by other recombinant gonadotropins. These medications were suitable for subcutaneous self-injection by the patient or her partner. In recent years, the advancement of endometrial co-culture techniques, the broadening application of PGD testing, the addition of gonadotropin-releasing hormone antagonists as an alternative to the agonists for the prevention of a premature luteinizing hormone (LH) surge, and the development of new and more complicated protocols involving techniques such as testicular sperm extraction/aspiration have brought additional patient education challenges to the ART nurse. All these advances are associated with the need for nurses to provide appropriate and accurate education to patients, which of course requires the ART nurse to update her knowledge continually.

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Despite a continued willingness to extend therapeutic options for patients, there are ongoing efforts to simplify treatment regimens and, thus, minimize confusion. ART nurses frequently consult with colleagues on ways to streamline the educational process for patients, which ultimately improves quality of patient care.

Educational resources Most ART programs provide written, center-specific materials outlining the procedures for their clinic. As nurses on the forefront of patient education, they are constantly updating, revising these educational materials as new information becomes available. In addition, the use of demonstrations remains an essential part of patient education. In the past, medication preparations needed reconstitution. In recent years, the introduction of liquid formulations and medication injection devices has changed the required education due to the diversity of the preparations available. The most obvious example of this is the education provided to patients and partners to self-inject medications. Nurses use handson, tactile-type teaching with instructional materials, such as injection buttocks, videos, etc., to encourage patients’ understanding of the process. Utilizing this method also allows the nurse to assess the patient’s comprehension and ability to perform medication administration and/or reconstitution. Today, nurses utilize a number of available resources during their education sessions, including CD/DVD, web-based, and written instructions which are available for distribution to patients. Additionally, national patient organizations provide written materials (books, newsletters, fact sheets), CD/DVD, and reliable websites that are constantly being updated with current treatment options. One such national lay organization, RESOLVE, offers a wide range of services, including a helpline and call-in hour, publications, and fact sheets in addition to local chapters that organize regional meetings and support groups. Another well-established organization, the American Fertility Association (AFA), helps patients to access a variety of resources, including a 24-hour helpline, a range of educational seminars, physicianreferral lists, therapists, peer-support groups, and a monthly newsletter. Fertile Hope is a national nonprofit organization dedicated to providing reproductive information, support, and hope to cancer patients and survivors whose medical treatments may compromise their future fertility.

IVF nurse coordinator The nurse as a coordinator is part of the traditional nursing role that continues to this day. In ART programs, the overall goal of the nurse coordinator is to serve as a clinical liaison to provide close contact between scientists, clinicians, nurses, and patients to assist couples to move smoothly through a treatment cycle. This requires a great deal of flexibility, as well

Table 64.1 Coordinating the assisted reproductive technologies (ART) team: some aspects to consider Alerting the pharmacy regarding drug requirements Scheduling interventional procedures (e.g. ultrasound examinations, oocyte retrievals) Documenting treatment results and outcomes Updating patient records Communicating results to patients Amending existing protocols or developing new ones Updating the staff counselor on specific patient needs Obtaining legal consents for new or updated procedures

as extreme attention to detail and good delegation skills. Rather humorously, it has been described as ‘organizing the world based on another woman’s menstrual cycle … or organized chaos.’4

Coordinating the unit The success of an ART program is dependent upon the coordination of the entire ART team, including clinical and academic responsibilities and, prior commitments such as lectures, vacations, and conferences. Table 64.1 highlights some of the aspects of treatment that must be incorporated into the overall treatment plan for all patients. Many ART centers are growing and expanding to provide care at satellite-based centers. This enables couples who live at significant distances from the main center to remain at home and/or working for as long as possible and still have access to care. Additionally, the success of specific technologies achieved by some ART centers has attracted potential patients from both near and far. Coordinating care for patients who live abroad is much more complex than for those who live in closer proximity to the ART center. The requirements for long-distance coordination vary according to the couple’s situation and selected treatment options. All in all, well-organized management of care contributes to the smooth running of an efficient unit, ensuring patient satisfaction and overall success.

Patient coordination: focus on communication As ART programs continue to grow, both in numbers and in expertise, there are ongoing initiatives to assure optimal quality of care and patient satisfaction. Patients are particularly concerned about interacting with many different members of staff as they progress through a treatment cycle, particularly with respect to issues of communication. The introduction of ‘nursing teams’ in many ART centers, with patient-assigned nurse coordinators, helps to assure that patients experience individualized treatment and continuity of care. In one ART center, which has expanded via satellite offices, the establishment of a primary-care nursing team (an IVF coordinator and assistant coordinator assigned to each physician) has allowed either nurse to be present with the primary physician and fully available to the

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patient. The nursing team handles the medical aspects of the treatment cycle, while another coordinator (thirdparty liaison coordinator) deals with the non-medical, more administrative aspects of care (e.g. consents and contracts). Following a 1-year evaluation period, it was noted that accountability and communication improved, both within the teams and with the center, and there were fewer complaints from patients regarding communication and call-back issues.7 In other centers, a modified approach has also been successful. Specific nurse/doctor teams are established for the most interactive phases of treatment: e.g. during pretreatment down-regulation and the initial phases of ovarian stimulation. Once the patient is fully engaged in treatment, a more coordinated approach is utilized. Nurses also benefit significantly from this working practice, developing a closer rapport with patients and increased job satisfaction. More subtly, they tend to be able to empathize better with patients and to develop a greater insight into patients’ physical and emotional needs.8

Patient counselor Infertility, and its treatment, place considerable emotional demands on the patient and the couple. While infertility itself is recognized as a ‘life crisis,’ provoking a variety of emotional responses, the range of ART treatment options now available to couples also raises complex emotional issues. These options may present significant medical, legal, religious, and/or ethical implications. As patients proceed along the treatment journey, the psychological impact may become increasingly more important. Several studies have suggested that emotional and psychological factors may be a leading cause of patient drop out.9 In fact, there are several reports suggesting that up to 60–65% of patients who begin treatment may drop out before treatment completion.10–12 In addition, the percentage of patients dropping out of treatment appears to increase with each subsequent failed cycle.13 Although emotional/psychological factors are important, other factors may also contribute to patient drop out.13 In recognition of the importance of psychological factors in treatment success, many clinics offer supportive counseling on a routine basis. Since the ART nurse is often the first to recognize the couples’ need for counseling, she serves as an important advocate on their behalf.

Nurse vs counselor Although there is significant overlap in the emotional support provided by the ART nurse and a professional counselor, in general, counseling involves the use of psychological interventions based on theoretical frameworks for which specialized training is required. The nurse’s perspective includes a thorough understanding of the patient’s clinical scenario. The ART nurse is in a unique position to provide emotional support to the patient and her partner because of the close relationship

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that develops, based on a high level of trust, sensitivity, and discretion. This is very different from other clinical settings, because infertile patients rarely discuss the ‘private’ side of their infertility outside of the nurse/counselor relationship. Specific areas which the nurse can focus on to help promote psychological well-being include: 1. Talking through individuals’ emotional responses to their infertility. 2. Identifying couples’ sources of stress, such as the success of procedures like ICSI for severe male factor, PGD for diagnosis of chromosomal abnormalities, or donor gametes and the associated issues of disclosure, ethical, and religious ramifications. 3. Providing support to infertility patients’ concerns and emotions. 4. Discussion of therapeutic options, including: a. guidance on realistic expectations b. anticipatory emotional responses c. ethical and religious concerns. 5. Helping patients to maintain self-esteem and interpersonal relationships. 6. Encouraging patients to continue with ‘life’ outside of infertility. In many instances, there will be an overlap between the nurse as ‘educator’ and the nurse as ‘counselor.’ For example, as nurses discuss treatment options and verify patient understanding through appropriate questions and monitoring feedback, patients are able to provide informed consent, and ultimately feel more in control, therefore reducing potential anxiety. In addition, nurses collaborate with the psychological support team to offer patients additional services. ART nurses can take on a supportive role, drawing on clinical experiences to guide the couple in the decision-making process related to other treatment options or for those couples seeking closure after treatment failure.

Specialist counseling Some ART centers require patients to undergo psychological counseling prior to pursuing treatment. Certainly in cases where third-party parenting options are being considered, all ART programs mandate patient counseling. Additionally, some patients will require support that extends beyond the type required by most couples undergoing ‘routine’ ART procedures, and it is imperative that this should be recognized. Research indicates that three particular groups of patients are likely to benefit from specialist counseling: They are patients: • experiencing high levels of stress (e.g. after a failed treatment, during a multiple pregnancy, undergoing PGD, TESE, failed ICSI, etc.) • requiring donated gametes, surrogacy, or adoption (third-party reproduction) • seeking fertility services because of their special social or ethical circumstances.14

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Nursing research The driving force behind the acceptance of ART nursing as a separate specialty is nursing-directed research, ideally inspired, motivated, and supported through collaborations with physician colleagues. While the outcomes of nursing interventions are already used as sources for nursing-based research, generally the type of research undertaken by nurses has a more subjective approach, including investigation of the psychological, nurturing, and educational aspects of ART. This contrasts with the more objective research likely to be conducted by physicians. For example, a nurse-based study considering administration of progesterone for luteal-phase support would focus more on the tolerability of the drugs administered than would a physiciandriven study that would be more likely to concentrate on aspects such as in-phase endometrial development. Regardless of topic, nurses are already participating in clinical care research in partnership with other healthcare professionals, and are benefiting from the support given them by the National Institute of Nursing Research and Nursing Research Mentors. Increased acceptance of nursing-driven research will come through translation of their research into practice. Thus, it is clear that forums are needed to allow nurses to disseminate the knowledge they are acquiring. Every opportunity should be afforded nurses to present their studies, both at a local and at an international level, including the annual meetings of major societies such as the American Society for Reproductive Medicine (ASRM). In addition, there is a need for a specialized journal devoted to ART nursing. Such a journal could play an important role in stimulating ART nursing research, and in setting high standards for the publication of research studies.

The role of an effective nurse manager The nurse manager role is one of coordinating and directing daily clinical and administrative tasks to assure that the center functions effectively and efficiently on all levels. Frequently, the nurse manager comes out of the administrative role and interacts on a personal level with each member of staff as well as individual patients. The nurse manager is an integral member of the center’s quality assurance program. It is the responsibility of the ART nurse manager not only to assure that their staff receive the training needed but also to monitor and assess their activities continually so that the training standards are maintained. This ensures that the ongoing concerns of the patients and the ART center are addressed. ART nurses have the opportunity to contribute to all aspects of patient care, from the moment the patient enters the clinic to the time they leave, whereas in other areas of medicine the patients often receive ‘segmented’ and often disjointed and confusing information. The

quality of care, for which the nurse manager has an overall responsibility, contributes directly to the success of the ART program both in clinical terms and in terms of patient satisfaction. As mentioned earlier, burnout is a significant concern to the ART nurse manager because of the differences between ART nursing and the traditional nursing role. Contrary to most areas of medicine, where nurses can adopt a more detached attitude, ART nurses are more likely to become personally involved with the infertile couple, regardless of inconvenience to themselves. Teaching ART staff nurses how to allocate time to particular tasks, to organize their day, and to seek personal education and supportive care from physicians and staff psychologists is an ongoing role of the ART nurse manager. With the right support, nurses will not only remain healthy but also remain in a position to do their job effectively. Nurse managers’ interactions in centerspecific quality-assurance concerns assist with the evaluation of the ongoing needs of the department and its staff, and therefore assure that a healthy clinical balance is maintained. The ability to recognize staff educational needs and offer the appropriate materials and opportunities to address these is another way to assure quality of care provided, as well as addressing issues of burnout. Since ART is such a specialized field, the ART nurse manager not only becomes the nurse educator within the ART center (e.g. to other new nurses) but also is called upon to educate other specialties, such as the neonatal intensive care unit (NICU) or high-risk antepartum staff, on issues pertinent to ART.

Assisted reproductive technologies nurse training As technology advances, the need for ongoing educational support within ART centers has increased. As previously mentioned, ART nurses benefit tremendously from supportive physicians and colleagues within their center. Furthermore, many ART nurses are fortunate to gain educational support or insight for their research interests through participation in or attendance at educational symposia or being supported in their research endeavors through sponsored ART clinics. ART nurses are also supported in their evolving role by a number of organizations which offer professional development advice, research mentors, conferences, lecturing opportunities, information on policies, procedures, and position statements, stateof-the-art medical information, and networking opportunities (Table 64.2). For example, the Nurses Professional Group (NPG) of the ASRM provides a forum for networking and information exchange among nurses. It also offers continuing medical education (CME) opportunities (through roundtables, seminars, etc.) at its annual meeting. The NPG has developed Protocols and Procedures for Nurses, a

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Table 64.2 Professional organizations supporting the assisted reproductive technologies (ART) nurse American Infertility Association (AIA) (patient support group) Nurses Professional Group (NPG) of the American Society for Reproductive Medicine (ASRM) Regional nursing associations RESOLVE (patient support group) BioSymposia Society for Assisted Reproductive Technology (SART) Fertile Hope (patient support group)

publication that encompasses a variety of topics such as: • nursing management of patients undergoing ART • patient preparation for infertility-related treatments • protocols on procedures performed in reproductive medicine practice. While such resources are a valuable aid to the ART nurse, they are not a substitute for the one-on-one instruction that remains key to the successful training of a new ART nurse. Introductory training must include a review of basic gynecology, including the integration of the reproductive/endocrine cycle, the characteristics of normal and abnormal cycles, and physical anomalies which interfere with fertility, together with a less academic but equally important overview of the responsibilities of key ART team functions. It is crucial that each member of the team knows not only their own responsibilities but also those of their fellow team members and importantly how these roles interact. Ultimately, it is the practical experience of patient management and the experience gained through repeatedly performing assigned tasks that form the basic core of the ART nurse’s education and training. While expanding the skills of nurses will obviously increase the number of tasks they can perform competently within a treatment program, less obvious are the benefits such as increased job satisfaction and greater continuity of patient care. Well-defined responsibilities, standards, and protocols for clinical practice are required and should be continually refined. As an example, the recent inception of FDA (Food and Drug Administration) regulations established national guidelines for ART centers that required ongoing education for the experienced ART nurse and the development of new nurse training.

The future of assisted reproductive technologies The traditional role of the ART nurse has seen a noted expansion in clinical responsibility from the early days of ART nursing. This expanded role is due in

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part to the continued introduction of new techniques and treatment modalities. Despite the rapid advances made in ART and the constantly evolving role of nurses working in this field, there is currently no specific certification that officially recognizes IVF/ART nursing as a specialty. Unlike neonatal and high-risk antepartum nurses, who receive specialty training in an academic setting and who can qualify for certification, ART nurses have more limited educational resources. There is inadequate exposure to the field of infertility at any level of academic training, and when it is offered, often as a 1-day course, the level of its content is highly variable. As outlined earlier, ART nursing is emotionally demanding, and is not recommended as an entry-level position due to the level of autonomy that is required in this field. A certain level of professional maturity is mandatory; infertility treatment is elective and couples are usually well educated in terms of options in infertility treatment, and are often able to make informed decisions on their treatment regimen. The nurse needs to be confident enough to face the challenges presented by often very determined but very well-informed couples. As research and treatment options evolve for patients, so does the role of the nurse in reproductive medicine. In turn, the prospects for a variety of professional opportunities will develop, providing broader career options and greater job satisfaction.

Conclusions The ART nurse is in a unique position to extend a deep empathy for a patient’s infertility struggle, and plays a central role in the patient’s treatment. Appropriate continuing education and opportunities for clinical certification are essential to encourage and nurture the nurse’s professional growth and clinical expertise. Continued nursing research and collaborative development of state-of-the-art standards of care will help to assure that patients benefit from the ongoing emotional, educational, and practical support offered by the contemporary ART nurse. Although ART nursing may be perceived as ‘stressful’, it remains a profoundly rewarding career.

References 1. Edwards R, Purdy JM, Steptoe P. Implantation of the Human Embryo. London: Academic Press, 1985. 2. Rausch DT, Braverman AM. Burnout rates among reproductive endocrinology nurses: the role of personality and infertility attitudes. Presented at the 56th Annual Meeting of the American Society for Reproductive Medicine, San Diego, California, October 21–26, 2000: abstr. 3. D’Andrea KG. The role of the nurse practitioner in artificial insemination. J Obstet Gynecol Nurs 1984; 13: 75–8.

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4. James CA. The nursing role in assisted reproductive technologies. NAACOGS Clin Issu Perinat Women’s Health Nurs 1992; 3: 328–34. 5. Ludwig M, Doody KJ, Doody KM. Use of recombinant human chorionic gonadotropin in ovulation induction. Fertil Steril 2003; 79: 1051–9. 6. Markle RL, King PI, Martin DB, et al. Characteristics of successful human chorionic gonadotropin (hCG) administration in assisted reproduction. Presented at the 58th Annual Meeting of the American Society for Reproductive Medicine, Seattle, Washington, October 13–16, 2002: abstr. 7. Norbryhn G, Fontanilla T, Rogoff R, et al. Establish-ment of a primary care nursing team in a rapidly expanding multisite reproductive endocrine center. Presented at the 56th Annual Meeting of the American Society for Reproductive Medicine, San Diego, California, October 21–6, 2000: abstr. 8. Muirhead M, Lawton J. A team approach to assisted conception treatment. Hum Fertil 1998; 1: 40–3.

9. Domar AD. Impact of psychological factors on dropout rates in insured patients. Fertil Steril 2004; 81: 271–3. 10. Land JA, Courtar DA, Evers JLH. Patient drop out in an assisted reproductive technology program: implication for pregnancy rates. Fertil Steril 1997; 68: 278–81. 11. Olivius C, Friden B, Borg G, et al. Why do couples discontinue in vitro fertilization treatment? A cohort study. Fertil Steril 2004; 81: 258–61. 12. Malcolm CE, Cumming DC. Follow-up of infertile couples who dropped out of a fertility specialist clinic. Fertil Steril 2004; 81: 269–70. 13. Rajkhowa M, McConnell A, Thomas GE. Reasons for discontinuation of IVF treatment: a questionnaire study. Hum Reprod 2006; 21: 358–63. 14. Boivin J, Appleton TC, Baetens P, et al. Guidelines for counseling in infertility: outline version. Hum Reprod 2001; 16: 1301–4.

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65 Patient support in the ART program Sharon N Covington

Overview Reproduction is considered the most basic of human needs, propelled by powerful biological and psychological drives. When the ability to reproduce is thwarted, a crisis ensues – the life crisis of infertility. The psychological crisis of infertility has been well-documented in the literature. It is considered an emotionally difficult experience that impacts on all aspects of a couple’s or an individual’s life: relationships with others, life goals, social roles, self-image, self-confidence, and sexuality, to name a few.1 The losses associated with infertility are multifaceted, including the loss of hopes, dreams, future plans, marital satisfaction, self-esteem, sense of control, belief in the fairness of life, health, and wellbeing, and, most important, the ‘dream child.’ 2 Further, these losses evoke feelings of grief – shock, disbelief, sadness, anger, guilt, blame, and depression – which occur in a repetitive and predictable process as patients move through medical diagnosis and treatment. It is through the experience and expression of emotions involved in the grieving process that the infertile couple moves toward an acceptance of their infertile state, engages in the exploration of alternative plans, and begins to move forward with their lives.3 During the past 50 years, we have seen a shift from the psychogenic infertility model, in which demonstrable psychopathology was thought to play an etiologic role in infertility, to a psychological sequelae model, in which numerous psychological factors were considered the result of infertility.4 In this concept, infertility is viewed as an emotionally difficult experience affecting all aspects of an individual’s and a couple’s life. Thus, emotional distress is a consequence and not a cause of infertility, as conceptualized previously. The application of a broader spectrum of theoretical approaches has led to a less individualistic perspective and a more holistic approach to infertility. In this sense, the interactions among individuals/couples and social/medical components are considered and must be factored into medical treatment. These perspectives have also increased understanding of individual and couple

differences and resilience, the impact of reproductive medical treatments, and the efficacy of therapeutic psychological interventions.

Stress and assisted reproductive technologies Assisted reproductive technologies, while opening up expanded opportunities for the treatment of infertility, have generated their own psychological challenges for patients. For most couples, assisted reproductive technologies (ART) are the last, best option for having a child, and occur after long months, and sometimes years, of treatment failure, often at tremendous emotional, physical, and financial cost. Patients entering ART programs usually do so with the burden of grief and disappointment from infertility, acting depressed, angry, tired, dependent, and anxious. Although emotionally depleted, couples are attracted to a technology that offers hope where, a few years ago, none existed. They find themselves drawn into a new emotional turbulence of contrasting feelings of hope and despair, which seem to be generated in part by the experience of the technology itself. The intensity and high-tech nature of ART create a stressful atmosphere, where the stakes are high and the chance of success may be relatively low. ART is a gamble, and, like gamblers, patients may have unrealistically high expectations of success5–7 or feel compelled to try ‘just one more time,’ finding it difficult to end treatment without success. Of all infertility treatments, in vitro fertilization (IVF) is considered the most stressful,8,9 with 80% of IVF patients ranking it as ‘extremely’ to ‘moderately’ stressful.10 Furthermore, after a failed cycle, almost all couples report acute depression,11 with elevated anxiety and anger levels persisting weeks later.12 Despite the stressful consequences of infertility and ART, numerous studies report that the vast majority of patients are generally well adjusted.13–17 In one of the most extensive reviews of scientifically rigorous research on the psychological effects of infertility, Stanton and Danoff-Burg concluded that the majority of infertile men and women are psychologically resilient

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and maintain adequate psychosocial functioning.18 Boivin found little evidence that infertile patients, as a group, experience significant, long-term maladjustment on measures of anxiety, psychiatric disturbance, marital conflict, and sexual dysfunction, when compared with population norms.19 Overall, this group reports marital adjustment in the normal range, and that the crisis of infertility may actually improve marital communication and emotional intimacy.20–23

who used problem-focused coping had a higher level of well-being than those who used avoidance-coping or social support.27 Men, on the other hand, who are often action-oriented and solution-focused, frequently cope with infertility through greater involvement in work or sports-related activities. While men and women may have different coping strategies, the use and effectiveness of these techniques may be influenced by the point in the infertility process and the existence of a gender-specific infertility diagnosis.28

Gender differences and ART stress The majority of studies of stress during ART are in women, and, overall, women react more intensely to infertility and ART than do men.24 Prior to IVF, women report more anxiety and depression, less life satisfaction, lower self-esteem, and more anticipatory stress than their male partners.21 During IVF, the intensity of a demanding treatment protocol – daily ultrasound monitoring, blood draws for hormone levels, injections, invasive procedures for oocyte retrieval, and embryo transfer – is frequently given by women as a cause of psychological distress.9 If treatment fails, depression persists longer for women than for their partners, lasting up to 6 months.12 Years later, women will recall the stress of IVF as more stressful for them than for their partner, regardless of the success or failure of treatment.5 In one of the few studies that examined men’s distress during IVF, Boivin et al found that men who were undergoing intracytoplasmic sperm injection (ICSI) reported more distress on the days prior to retrieval than did other IVF men.25 However, in all other areas, ICSI and IVF men were similar in their adjustment to infertility and in their distress during the treatment cycle. These findings were in contrast to those of early studies of distress among men with male factor diagnosis, as these infertile men reported more negative feelings and psychiatric distress.11,26 The discrepancy between these studies may have been due to the fact that ICSI could circumvent the infertility, whereas at the time of the earlier studies the only medical option available was donor insemination. While the intensity of emotional reactions to particular aspects of ART may differ between men and women, the types of reactions are the same, with both experiencing a significant increase in anxiety and depressive symptoms from pre- to post-treatment.5,21 In addition, both men and women rank the relative stresses of each stage of IVF equally, and tend to overestimate the chances of success of IVF in general, showing a high level of hopefulness in their own cases.12 Men and women tend to cope differently with the stress of ART and infertility.20 As frequently noted, women are more expressive of feelings, and are more likely to seek emotional and social support during ART by informal activities such as talking to spouse, family, and friends. In terms of the effects of coping post-IVF treatment, Hynes et al found that women

Levels of stress during ART While general assumptions may be made about stress levels during ART, the experience for infertility patients will be personal and unique: each patient will experience the stress differently, based upon his or her own personality and life experiences. Newton et al note that stress has been conceptualized both as a stimulus or event (distressing circumstances outside the person) and as a response (internal disturbance).24 A contrasting approach describes stress as neither an event nor a response, but rather a combination of factors: the perceived meaning of the event and self-appraisal of the adequacy of coping resources.29 Thus, it is not the stress itself but the perception of the stress that determines how ART patients experience and handle it. The aspects of ART perceived to be stressful to patients are multifaceted and affect all parts of their life: marital, social, physical, emotional, financial, cultural, and religious. Time is stressful, both in the time commitment to an intense treatment which leads to disruption in family, work, and social activities, and, for some, in long waiting periods for IVF or third-party reproduction. ART stress impacts on the marital relationship with an emotionally laden experience, and, by removing the conjugal act for procreation, sexual intimacy is lost. Also, couples are stretched financially, paying for the high cost of ART treatment with a relatively low probability of success. Dealing with the medical staff and with the side effects or potential complications of medical treatment has its own stress: hot flushes, headaches, mood fluctuations, shots, sonograms, future health concerns, and decision-making about embryos and multiple pregnancies. Religious, social, cultural, and moral issues may also make ART stressful, especially for those dealing with third-party reproduction, when these values are in conflict with the choice of treatment. The first treatment cycle has been found to be the most stressful for patients, with high levels of confusion, bewilderment, and anxiety.6,9,12 This may be due to inexperience with the process, or possibly inadequate preparation of the patient by staff in terms of information and discussion of care. Slade et al found that for couples attempting three cycles of IVF, distress diminished during the middle cycle but rose after they discovered that the intervention had not been successful, with the last cycle being as stressful as the first.12

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Within a treatment cycle, patients view IVF/ART as a series of stages which must be successfully completed before moving on to the next phase of treatment: monitoring, oocyte retrieval, fertilization, embryo transfer, waiting period, and pregnancy test stages. The level of stress, anxiety, and anticipation rises with each stage, peaking during the waiting period. A number of studies have confirmed what clinicians have known anecdotally: in order of perceived stress for patients, waiting to hear the outcome of the embryo transfer is the most stressful, followed by waiting to hear whether fertilization has occurred, and then the egg retrieval stage.10,30 Patients are aware of the importance of these key phases in the IVF process, and the uncertainty of the outcome is highly distressing.

Methods Who provides patient support services in ART? Given the host of research on the emotional consequences of infertility and on the distressing nature of ART, it is clear that patients need psychological support as an integrated part of the medical treatment process. Technology has become more complex and so have the psychological, social, and ethical issues related to treatment, which challenges the resources of staff and patients. As a result of technologic advances in ART and recognition of the psychosocial issues and demands facing infertile patients, mental health professionals have become increasingly important members of the reproductive medical team.30,31 The specialization of ‘infertility counseling’ has emerged, combining the fields of reproductive health psychology and reproductive medicine, for mental health professionals including social workers, psychologists, psychiatrists, marriage and family therapists, and psychiatric nurses. Infertility counselors serve as a resource to patients and staff by providing specialized psychological services that support and enhance quality care. For example, the complex medical and psychological issues in third-party reproduction have social and legal implications that must be assessed carefully, and warrant involvement of a qualified mental health professional experienced in infertility counseling. In addition, the psychosocial impact on the offspring created by ART needs to be considered, and assistance given to families dealing with these issues preand post-treatment.31 Nonetheless, the responsibility for patient support in the ART program is the duty of all staff members, not just the domain of nurses or infertility counselors.32 Interactions with each staff member, from administrative staff to physician, influence a patient’s perception of care and, in turn, his or her stress level. Sensitivity, warmth, patience, and responsiveness create an environment of

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support. Also, general clinic routine and ambience reflect support and respect of patients when it is provided in an efficient, organized, clean, uncrowded, and esthetically pleasing atmosphere. All staff need to be sensitive to and knowledgeable about the psychological needs and stress of ART patients. While the primary focus of physicians, nurses, laboratory scientists, and other healthcare staff is the medical diagnosis and treatment of infertility, it must also entail ‘treating the patient, not the disease.’

Types of ART support services ART patient support services can be generalized from overall clinic administration and environment to specialized services which need to be provided by a mental health professional experienced in infertility counseling. For the purpose of this chapter, while specialized services provided by an infertility counselor are described, a detailed explanation of methodology is not addressed.4 Moving from specific to general, the method of providing patient support services can be categorized as: 1. 2. 3. 4. 5.

Psychological assessment and evaluation. Therapeutic counseling. Supportive counseling. Information and education. Clinic administration.

Psychological assessment and evaluation Psychological screening of participants using ART often varies from program to program, as currently only two countries (Australia and Canada) mandate counseling prior to ART treatment. While the Human Fertilisation and Embryology Authority (HFEA), which regulates assisted reproduction in the United Kingdom, has stipulated that psychosocial counseling must be offered to patients seeking IVF or donor gametes,33 one study found that less than 25% of patients took up the suggestion.34 In the United States, recommendations and guidelines for the provision of psychological services to ART participants are voluntary,35 and the decision concerning which patients should be screened or counseled, and for which procedures, is left to each individual fertility practice. Thus, available guidelines for assessment and evaluation are usually tailored to the specific requirement or preference of a particular program. Whether a clinic adopts formal or recommended guidelines or chooses to develop its own, the program’s policy regarding infertility counseling, screening, exclusion criteria, and so on should be clearly defined for the protection of the medical team, the infertility counselor, and patients.36 Notwithstanding the voluntary nature of screening ART participants, it has become the standard of care to require psychological evaluation of oocyte donors, surrogates, and gestational carriers by experienced mental

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health professionals. The evaluation usually involves both psychological testing of the donor/carrier, with the Minnesota Multiphasic Personality Inventory-2 (MMPI-2) being used most often,37 and clinical interviews with her and, when available, her partner. Assessment and counseling of recipients of donor gametes is also strongly recommended or required by many programs, especially when the donor/carrier is known or related. Other situations where programs may require screening and assessment involve patients undergoing IVF who are considered psychologically or physically vulnerable, previous IVF patients donating frozen embryos, single recipients of gamete donation, and older infertility patients.38 The established protocol for psychological evaluation and assessment within the author’s program includes: 1.

2.

3.

4.

Requiring all recipients of anonymous donor eggs, sperm, and embryos, and genetic parents using a gestational carrier, to see a staff infertility counselor. The psychoeducational counseling and assessment usually takes place in one or two counseling sessions, reading materials are provided, and issues related to raising children conceived through third-party reproduction are discussed. Requiring psychological evaluation of all anonymous oocyte donors. Psychological testing (MMPI-2) is administered, and then scored and interpreted. A minimum of two clinical interviews, one with the donor and one with her and her partner, are conducted with a staff infertility counselor to assess psychological functioning and discuss the process, motivations, and implications of gamete donation. Requiring all known donors or gestational carriers and recipients to undergo evaluation and counseling, which includes administering the MMPI-2 to the donor and gestational carrier. Clinical interviews are held with the donor or carrier and patient separately, including their partners, and a joint ‘group’ session is conducted to discuss how they will deal with issues in known donation. Legal consultation and contracts are also strongly recommended with gestational carriers. Requiring assessment and counseling of any infertility patient when the physician is concerned about psychological vulnerability or marital instability, or if a situation is presented to our internal ethics committee where additional psychosocial information is needed before a decision about treatment can be made.

Our mental health professional staff follow the criteria established for acceptance or rejection of participants in the recommended guidelines for ‘Psychological assessment of oocyte donors and recipients’ and the ‘Psychological guidelines for embryo donation’

developed by the Mental Health Professional Group of the American Society for Reproductive Medicine (ASRM).35 When a recommendation to withhold or postpone treatment is made by the infertility counselor, a team meeting takes place so that a decision is made by team consensus, rather than one member (usually the physician or the infertility counselor) being seen by the patient as the ‘rejector.’ It is useful to view and interpret these recommendations to the patient as protection of the parties involved rather than rejection, since it is the first responsibility of all healthcare providers ‘to do no harm.’

Therapeutic counseling Another aspect of patient support services involves intervention and treatment for the consequences of infertility, or for underlying mental disturbances that could affect medical treatment. Treatment modalities of individual, couple, and group counseling provide an opportunity to assist patients in understanding and handling the emotional sequelae of infertility; identifying and developing a coping mechanism to deal with treatment; managing the effects of infertility or psychosocial history on interpersonal functioning (anxiety, depression, etc.) and on marital, sexual, and social relationships; considering the implications of ART treatment; decision-making on treatment options and alternative family building; pregnancy and parenting following treatment; and ending treatment and building a life after infertility. ART programs may provide psychological assessment and therapeutic counseling services through an infertility counselor on the staff (an employee) or on site (an independent contractor), or may choose to refer to a qualified mental health professional who works independent of the clinic.39 Guidelines for when to refer patients for psychological assessment and intervention are displayed in Table 65.1.

Supportive counseling Supportive counseling involves reproductive healthcare providers giving both advice (counsel) and comfort (console) to their patients. Although nurses often assume primary responsibility for patient support, a team approach to advising and consoling is optimal. Services combine supportive and psychoeducational counseling, and may include: 1.

2.

Pre-IVF preparation session with an infertility counselor, which is offered as part of the treatment package. Monthly support groups for IVF participants, patients considering or using donor gametes, and those with general infertility (non-ART), secondary infertility, miscarriage, and pregnancy after infertility. These groups are open-ended, of no cost to patients, and run by a staff infertility counselor and, if needed, a nurse.

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Table 65.1

When to refer to an infertility counselor

Situations The following situations serve as guidelines for referral for psychological assessment, counseling, and/or intervention:40 the use or consideration of third-party reproduction psychiatric illness (past or present) history of pregnancy complications or loss significant physical illness (past or present) sexual or physical abuse (past or present) conflicted gender identity, homosexuality, or bisexuality chemical abuse or dependency marital instability or chaotic social functioning single patients older patients Symptoms Referral to a mental-health professional should also be considered when there is a change in current mental status and/or exacerbation of symptoms which are affecting normal functioning and relationships, including: depression or persistent sadness and tearfulness high levels of anxiety or agitation increased mood swings obsessive–compulsive behaviors strained interpersonal relationships social isolation loss of interest in usual activities diminished ability to accomplish tasks difficulty concentrating or remembering difficulty making decisions change in appetite, weight, or sleep patterns increased use of drugs or alcohol persistent feelings of pessimism, guilt, worthlessness persistent feelings of bitterness or anger thoughts of or reference to death or suicide

3.

4.

5.

6.

A monthly discussion series on infertility topics identified through a patient survey, such as adoption, donor issues, staff–patient communication, drug side effects, dealing with family and friends, decision-making, marriage enhancement, and when to end treatment. These informal groups are facilitated by an infertility counselor, physician, nurse, and/or an invited guest from the community who is knowledgeable on the subject. Stress management and relaxation classes taught by an infertility counselor and a nurse. Relaxation tapes and guided imagery tapes are also available to lend to patients for use before, during, and after retrieval and transfer. Referral resources within the community for patients who request alternative approaches to help with quality of life during infertility, such as mind–body programs, yoga classes, acupuncture, and homeopathy. Providing a network for patient-to-patient contact about aspects of treatment. Well-adjusted patients who have been through a procedure or have a specific diagnosis are asked by a staff member if they would be willing to speak one-on-one with other patients who request this contact. Common

7.

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requests for contact are situations where patients have undergone selective reduction or have carried multiple pregnancies. Giving each patient current information about local and national infertility support groups (e.g. RESOLVE, Inc.), such as monthly updates on meetings, support groups, living-room sessions, telephone counseling, newsletters, and articles.

Information and education Probably the most far-reaching opportunity for ART support is through patients’ easy access to written information and education about the medical and psychological aspects of infertility. Patients rely heavily on the educational materials that document the process and procedure of ART, and search out information at the clinic, through the media (TV, magazines, books, etc.), and on the internet. One study found that patients identified informational materials as their primary source of support, after talking with spouse, family, or friends.41 Information and treatment packets sent out to new patients should include material on the emotional aspects of infertility and on support resources available through the clinic and in the community. A clinic’s website is also an important source of support information, and could connect to other internet resources, such as RESOLVE, for easy patient access. Examples of information and education support services from the author’s program include: 1.

2.

3.

Monthly IVF and donor egg recipient preparation classes for new patients beginning a cycle. Presentations are made by a member of each treatment team – physician, embryology/laboratory, nurse, infertility counselor – and the administrative/finance office, who discuss protocol and process, describe treatment services, and answer questions. These classes are held in the evening, a light dinner is provided, written materials on the medical and emotional aspects of IVF or donor egg are distributed, and the informal atmosphere allows for easy exchange with patients. Ready access to pamphlets, articles, and written materials on the medical and emotional aspects of infertility, which are displayed in patient waiting areas. Ample supplies of these materials are available in the nursing, physician, and infertility counseling offices, as well as with administrative staff. For example, billing staff found that as patients were checking out from office visits they often talked about their stresses, and being able to give patients flyers on clinic support services or educational pamphlets was greatly appreciated. A ‘fact sheet’ of resources for patients with names, telephone numbers, and internet websites about clinic and community support services relating to infertility, endometriosis, premature ovarian failure, polycystic ovary syndrome, adoption, pregnancy,

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5.

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pregnancy loss or termination, multiple gestation and parenting, and single parenting. One-page ‘tip sheets’ on topics that offer suggestions about coping with the emotional aspects of infertility (IVF, marital relationships, etc.) and ‘summary sheets’ on medical treatments/procedures. Patient information ‘fact sheets’ are also available through the ASRM’s website (http: //www.asrm.org/fact/fact.html), and can easily be downloaded and given to patients. These summary sheets are especially helpful, as the volume of information given to patients may be overwhelming, and research has shown that patients retain only a small portion of information verbally given to them. A patient lending library of infertility-related books, videos, and audiotapes of instruction and information ranging from topics on sexual dysfunction and adoption to medical diagnosis and treatment of infertility. Resources that can be accessed or downloaded from the clinic’s website. These may include articles written by staff members on psychological and medical aspects of treatment, an ‘ask the expert’ column for patients to write in questions, and online webcasts to present information on treatment programs and psychosocial issues of infertility.

Clinic administration The manner in which an ART program is administered, along with the physical environment of the clinic, affects both patients’ stress levels and their perception of support. An esthetically pleasing, clean, well-maintained office staffed by friendly, professionally dressed, welltrained people goes a long way in communicating an impression of professional competence, caring, and confidence. Ways in which the author’s program provides support through clinic administration includes: 1. Patient waiting areas, with access to reading materials, water, telephones, and restrooms. During weekend monitoring, a continental breakfast is available for patients in this area while patients wait to see the physician. (If a clinic shares space with an obstetrics and gynecology department, sensitivity needs to be considered, and reasonable efforts made to separate pregnant patients and small children from infertility patients by adjusting appointments/schedules and/or seating arrangements.) 2. Private rooms where nurses or other clinical staff can instruct or consult with patients. 3. Private sections where billing and scheduling issues can be discussed by administrative staff with patients in a confidential manner. 4. A quiet, secure ‘donor room’ for men to give semen samples, with erotic magazines/materials, video player, and a comfortable chair or bed.

5. Private recovery areas after egg retrieval and embryo transfer with safe places to store belongings, television/video player or music, and a comfortable chair for husbands. 6. Soothing, calming background music piped throughout the office. 7. An annual or biannual ‘baby party’ for patients to come back with their children and celebrate with staff. 8. Miscarriage/pregnancy loss cards sent by the clinical staff when it is learned that, after a patient has been discharged from care, a pregnancy has been lost. 9. Primary-care nursing, where a patient is assigned to one nurse, facilitating better continuity and coordination of treatment. 10. A staff member ‘patient advocate/ombudsman,’ who patients may talk to when they perceive a problem with their care, or other conflict with the clinic that cannot be resolved. 11. Patient surveys, suggestion boxes, and written feedback, which encourage open communication regarding satisfaction, thoughts on improving care or services, and constructive criticism. 12. In-service training of all staff on the emotional needs of infertility patients, communication skills, stress management techniques, and on strategies to deal with difficult, demanding patients. 13. Staff support offering confidential assistance, direction, and referral for personal problems and professional burnout, by the staff mental health professional, or through an employee assistance program (EAP). Ultimately, happy staff members are productive workers, who give the best support and service to patients.

Results Although most patients undergoing ART are well adjusted and will cope adequately with the process, all will benefit from, and indeed need, emotional support during treatment. Numerous studies show that most patients believe psychosocial counseling is beneficial and that they would avail themselves of it, were it offered during treatment.19,42,43 While a minority of patients experience significant emotional distress and use formal counseling services, the vast majority of those who use formal counseling report having found it helpful.19 Several studies of patient satisfaction suggest that many patients are dissatisfied with support services (or the lack thereof) offered by their IVF centers.44–47 This information, coupled with the high dropout rates in ART programs, most likely due to psychological reasons,48–50 suggests that IVF programs need to provide better and more comprehensive psychosocial support services. Recent studies have indicated that even when cost is not a factor in pursuing treatment,

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over half of patients drop out of treatment before using up entitled insurance benefits.51–53 The most common reason given for treatment termination was psychological burden.51,52 Providing integrated psychological support services may be an important step in diminishing a patient’s depression and anxiety, lowering drop out rates, and possibly even increasing pregnancy rates – the goal of all fertility programs.49,54,55 Simple strategies for managing patients can help a great deal.56 Olivius and colleagues51 found that ease in contacting the clinic or clinician by telephone, seeing the same doctor during treatment, and receiving sufficient oral and written information about treatment and complications helped with patient distress. At the very least, written materials and educational resources on the medical and psychosocial aspects of infertility need to be readily available and given to patients by their programs. Further, the more holistically a patient is handled – supported medically and emotionally – the more likely she/he is to be treatment-compliant and satisfied with care, despite the outcome of treatment. In fact, the true mark of success of a program may be in the ability of the team to help patients feel that they, the patients, have done their best when treatment has failed (see Table 65.2 for a summary of strategies for ART patient support).

Future direction Reproductive medicine will continue to change as advancing technology presents increasingly complex options and choices for patients. As reproductive technology continues to advance and push the boundaries of social, psychological, religious, and ethical acceptance, the need for comprehensive support services for ART patients will continue to grow. Patients will request a more holistic approach to medical treatment, where their bodies and their emotions are treated with equal importance. As ‘educated consumers,’ ART patients will search for the most effective and comprehensive care program, often choosing a practice on the basis of whether psychological support services are integrated into treatment. There will continue to be a growing need for specialized clinical skills and services of mental health professionals trained in infertility counseling to provide this assistance to patients and staff. ART programs that have the foresight to integrate comprehensive support services with specialized mental health professionals as part of the treatment team will succeed.

Conclusion Infertility is an emotionally exhausting, psychologically demanding experience for patients and, at times, their caregivers. Since ART is considered the most stressful of all infertility treatments, patients

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Table 65.2 Strategies for assisted reproductive technologies (ART) patient support Before Educational classes presented by each member of the treatment team on IVF Pretreatment counseling session with a mental health professional/infertility counselor Psychosocial preparation and assessment of gamete donors, recipients, and surrogates with a mental health professional/infertility counselor Extensive written materials available and distributed on the medical, emotional, and financial aspects of ART Educational video tapes on the medical and emotional aspects of infertility and ART Support groups Stress management, relaxation, and guided imagery classes and audio tapes Resource lists of community support services including RESOLVE, Inc. During Access to the mental health professional/infertility counselor and other team members Telephone support with a primary-care nurse If a patient has met with an infertility counselor before starting the cycle, a brief visit in the OR on retrieval and/or transfer day Access to relaxation and stress management audio tapes Support groups After Psychosocial follow-up after a failed cycle or pregnancy loss Decision-making counseling regarding alternative therapies or ending treatment Counseling on alternative family building through adoption or third-party reproduction Counseling and support for the decision to remain child-free after infertility Counseling and preparation for multiple pregnancy, including selective reduction Counseling and follow-up for pregnancy after infertility, including support groups Counseling and follow-up for issues in parenting after infertility, including families created through donor gametes Support groups Patient feedback survey IVF, in vitro fertilization; OR, operating room.

who undergo it need as much support psychologically as they do medically from their clinical team. Specialized support services are needed for psychosocial preparation, assessment, and treatment of patients who are faced with the unique issues associated with and/or the consequences of assisted reproduction. These specialized psychological services must be provided by experienced mental health professionals trained in infertility counseling, who are, ideally, a part of the treatment team. Finally, patient support is the responsibility of all employees of an ART program, and staff must be knowledgeable about and sensitive to the emotional needs of their patients.

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References 1. Menning BE. The emotional needs of infertile couples. Fertil Steril 1980; 34: 313–19. 2. Mahlstedt PP. The psychological component of infertility. Fertil Steril 1985; 43: 335–46. 3. Stanton AL, Dunkel-Schetter C. Psychological adjustment to infertility. In: Stanton AL, DunkelSchetter C, eds. Infertility: Perspectives from Stress and Coping Research. New York: Plenum Press, 1991: 3–16. 4. Covington SN, Burns LH. Psychology of infertility. In: Covington SN, Burns LH, eds. Infertility Counseling: a Comprehensive Handbook for Clinicians, 2nd edn. New York: Cambridge University Press, 2006: 1–19. 5. Johnston M, Shaw R, Bird D. “Test-tube baby” procedures: stress and judgements under uncertainty. Psychol Health 1987; 1: 25–38. 6. Reading AE. Decision making and in vitro fertilization: the influence of emotional state. J Psychosom Obstet Gynecol 1989; 10: 107–12. 7. Visser A, Haan G, Zalmstra H, et al. Psychosocial aspects of in vitro fertilisation. J Psychosom Obstet Gynecol 1994; 15: 35–45. 8. Kopitzke EJ, Berg BJ, Wilson JF, Owen D. Physical and emotional stress associated with components of the infertility investigation: professional and patient perspectives. Fertil Steril 1991; 55: 1137–43. 9. Boivin J, Takefman J. The impact of the in vitro fertilization – embryo transfer (IVF–ET) process on emotional, physical, and relational variables. Hum Reprod 1996; 11: 903–7. 10. Connolly KJ, Edelmann RJ, Bartlett H, et al. An evaluation of counselling for couples undergoing treatment for in vitro fertilization. Hum Reprod 1993; 8: 1332–8. 11. Litt MD, Tennen H, Afflect G, Klock S. Coping and cognitive factors in adaptation in in vitro fertilization failure. J Behav Med 1992; 15: 171–87. 12. Slade P, Emery J, Lieberman BA. A prospective, longitudinal study of emotions and relationships in in vitro fertilization treatment. Hum Reprod 1997; 12: 183–90. 13. Connolly KJ, Edelmann RJ, Cooke ID, Robson J. The impact of infertility on psychological functioning. J Psychosom Res 1992; 36: 459–68. 14. Hazeltine FP, Mazure C, De L’Aune W, et al. Psychological interview in screening couples undergoing in vitro fertilization. Ann NY Acad Sci 1985; 442: 504–22. 15. Paulson JD, Haarmann BS, Salerno RL, Asmar P. An investigation of the relationship between emotional maladjustment and infertility. Fertil Steril 1988; 49: 258–62. 16. Downey J, Husami N, Yingling S, et al. Mood disorders, psychiatric symptoms and distress in

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31. Covington SN. The role of the mental health professional in reproductive medicine. Fertil Steril 1995; 64: 895–7. 32. Covington SN. Reproductive medicine and mental health professionals: the need for collaboration in a brave new world. Orgyn 1997; 3: 19–21. 33. Human Fertilisation and Embryology Authority. Code of Practice, 2nd edn. London: HFEA, 1995. 34. Hernon M, Harris CP, Elstein M, et al. Review of organized support network for infertility patients in licensed units in the UK. Hum Reprod 1995; 10: 960–4. 35. American Society for Reproductive Medicine. 2006 Guidelines for gamete and embryo donation. Fertil Steril 2006; 86 (Suppl 4): S38–50. 36. Klock SC, Maier D. Guidelines for the provision of psychological evaluations for infertile patients at the University of Connecticut Health Center. Fertil Steril 1991; 56: 680–5. 37. Klock SC, Stout EJ, Davidson M. Analysis of Minnesota Multiphasic Personality Inventory-2 profiles of prospective anonymous oocyte donors in relation to the outcome of the donor selection process. Fertil Steril 1999; 72: 1066–72. 38. Covington SN. Preparing the patient for in vitro fertilization: psychological considerations. Clin Consider Obstet Gynecol 1994; 6: 131–7. 39. Covington SN. Infertility counseling in practice: a collaborative reproductive healthcare model. In: Covington SN, Burns LH eds. Infertility counseling: a Comprehensive Handbook for Clinicians, 2nd edn. New York: Cambridge University Press, 2006: 493–507. 40. Burns LH. An overview of the psychology of infertility. Infertil Reprod Med Clin North Am 1993; 4: 433–54. 41. Boivin J, Scanlan LC, Walker SM. Why are infertile patients not using psychosocial counselling? Hum Reprod 1999; 14: 1384–91. 42. Baram D, Tourtelot E, Muechler E, et al. Psychosocial adjustment following unsuccessful in vitro fertilization. J Psychosom Obstet Gynecol 1988; 9: 181–90. 43. Mazure CM, Greenfeld DA. Psychological studies of in vitro fertilization/embryo transfer participants. J In Vitro Fert Embryo Transf 1989; 6: 242–56.

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44. Sabourin S, Wright J, Duchesne C, Belisle S. Are consumers of modern fertility treatments satisfied? Fertil Steril 1991; 56: 1084–90. 45. Laffont I, Edelmann RJ. Perceived support and counselling needs in relation to in vitro fertilization. J Psychosom Obstet Gynecol 1994; 15: 183–8. 46. Sundby J, Olsen A, Schei B. Quality of care for infertility patients. An evaluation of a plan for a hospital investigation. Scand J Soc Med 1994; 22: 139–44. 47. Souter VL, Penney G, Hopton JL, Templeton AA. Patient satisfaction with the management of infertility. Hum Reprod 1988; 13: 1831–6. 48. Land JA, Courtar DA, Evers JL. Patient dropout in an assisted reproductive technology program: implications for pregnancy. Fertil Steril 1997; 68: 278–81. 49. Domar AD. Impact of psychological factors on dropout rates in insured infertility patients. Fertil Steril 2004; 81: 271–3. 50. Penzias AS. When and why does the dream die? Or does it? Fertil Steril 2004; 81: 274–5. 51. Olivius C, Friden B, Borg G, Bergh C. Why do couples discontinue in vitro fertilization treatment? A cohort study. Fertil Steril 2004; 81: 258–61. 52. Smeenk JMJ, Verhaak CM, Stolwijk AM, Kremer JA, Braat DD. Reasons for dropout in an in vitro fertilization/intracytoplasmic sperm injection program. Fertil Steril 2004; 81: 262–8. 53. Malcolm CE, Cumming DC. Follow–up of infertile couples who dropped out of a specialist fertility clinic. Fertil Steril 2004; 81: 269–70. 54. Terzioglu F. Investigation into the effectiveness of counseling on assisted reproductive techniques in Turkey. J Psychosom Obstet Gynaecol 2001; 22: 133–41. 55. Smeenk JMJ, Verhaak CM, Stolwijk AM, Kremer JAM, Braat DDM. Psychological interference in in vitro fertilization treatment. Fertil Steril 2004; 81: 277. 56. Olivius C, Friden B, Borg G, Bergh C. Psychological aspects of discontinuation of in vitro fertilization treatment. Fertil Steril 2004; 81: 276.

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66 The relationship between stress and in vitro fertilization outcome Andrea Mechanick Braverman

Overview The impact of patient stress on the process of in vitro fertilization (IVF) is a complex and multifaceted interplay between the mind and the body. The old model of psychogenic causation, e.g. the Freudian approach that speaks of the woman’s fear of impregnation or motherhood, has long been supplanted by the careful exploration of the interplay of stress on the endocrine system. Considerations of dispositional characterological factors such as optimism1 or happiness2 have also led to the hypothesis that such factors may play a role in treatment outcome. Many studies have considered the relationship between stress (or other psychosocial variables) and its effect on pregnancy outcome per treatment cycle.1–5 The results have been mixed, and have often been confounding factors when the concepts of stress reduction and support as agents of cause or intervention in infertility and pregnancy outcomes are considered. In a comprehensive review of psychosocial interventions in infertility, Boivin noted that ‘analysis of these studies showed that psychosocial interventions were more effective in reducing negative affect than in changing interpersonal functioning,’ and that pregnancy rates were not likely to be affected by these interventions.6 Boivin also noted that counseling interventions which focused on affective expression regarding the emotional aspects of infertility were significantly less effective in producing a positive change than were education and skills training. Psychosocial intervention has looked at pregnancy and implantation rates,7,8 but not at treatment persistence and retention. Some of the major confounds that occur while considering psychological distress and pregnancy outcome include: the relationship between distress and anxiety/depression; influences of diagnosis or influence of information or attitudes of the medical team; habituation effects of chronic stress; other life stressors; coping styles; and baseline psychological issues. In Domar et al’s8 review of the association of psychological distress and pregnancy outcome, the author concludes: ‘Women undergoing ART procedures

report significant levels of negative psychological symptoms, both prior to beginning and especially after experiencing an unsuccessful cycle. Most of the research conducted with women undergoing assisted reproductive technologies (ART) treatment supports the theory that emotional distress is associated with treatment success.’ However, the authors note the limitations of many of these studies, including lack of control groups or small sample sizes. More recently, studies have turned their attention away from the tremendously complex relationship between stress or depression and pregnancy outcome, focusing instead on the causes behind the discontinuation of treatment and treatment perseverance. These studies clearly demonstrate that treatment dropout is associated with psychological factors.1–5 Given the high pregnancy rates using IVF over cycles, even involving patients with poorer prognoses, the ability for patients to remain in treatment, i.e. treatment persistence, gives a patient her best opportunity for achieving a pregnancy. Treatment persistence will allow a patient to optimize her biological potential.

Sources of stress The experience of infertility transcends borders and socioeconomic status. Nearly every society throughout history has produced art and literature that has spoken of the desire for offspring and the pain (or shame) that ensues when pregnancy does not occur. For many individuals, infertility is among the first life crises that they may face, having previously dealt with pregnancy’s counterpoint (the prevention of pregnancy) through vigilant contraception. Messages from friends, family, and society reinforce the notion that fertility is within an individual’s control. Public figures or movie stars who pursue infertility treatment with success at ages well into their 40s and 50s only contribute to some individual’s incredulity that there may be a fertility issue with either or both partners. The inevitable fact that age may play a substantive role in fertility may be lost within these competing social messages.

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The stress of infertility is felt in many ways by men and women and may change over time and as individuals experience failure in the cycles. In a groundbreaking 1985 study, Freeman and colleagues9 evaluated 200 couples experiencing infertility. Half the women and 15% of the men endorsed the notion that infertility was the worst event of their lives. In a more recent study, Verhaak and colleagues10 surveyed 148 IVF patients along with their 71 partners and measured numerous psychological factors (anxiety, depression, personality characteristics, the meaning of fertility problems, coping, marital relationship, and social support) at pretreatment, post-treatment, and final treatment stages. Six months later, participants were assessed for anxiety and depression. Among women, anxiety and depression increased after unsuccessful treatment and decreased after successful treatment. There was no change in anxiety and depression levels for men after either successful or unsuccessful treatment. Some of the major categories of stress that have been traditionally recognized are addressed below, but none of these factors unequivocally demonstrate a clear role in pregnancy outcome. Without question, entering into medical treatment for fertility places the individual and couple into a ‘patient’ mode. The stress of being in medical care alone can be a psychological burden for some patients. Contributing to this stress may be the language of infertility to which patients are regularly exposed, such as the use of pejorative terms like ‘failed cycle,’ ‘incompetent cervix,’ ‘shooting blanks,’ and ‘advanced maternal age,’ which may impact patients’ self-esteem and body image, and which may serve to reinforce that potentially stress-inducing notion that the individual is a patient with medical problems.

required tests, such as a postcoital.12 Sexuality may be linked with procreation during fertility treatment and is often divorced from recreation or intimacy. Timed intercourse can add to the burden of feeling measured, pressured, and stressed. Some women report feeling distanced from their bodies because of procedures or timed intercourse; men report feeling performance pressure with timed intercourse. Adding to these stressors are the feelings associated with unhappiness, anger, or disappointment in one’s own body. None of these feelings or stressors enhances the sense of being a sexual person with sexual desires. This further adds to the burden on the relationship.

Relationship with partner

For many individuals, being in ‘patient’ mode means that their bodies are not working correctly; this circumstance can take a toll on their self-esteem as well as on their body image. The disappointment in their own bodies felt by the patient may be exacerbated by their unconscious belief that fertility should ‘come naturally,’ that it should be in their exclusive control. The stated message by friends, family, and others to ‘just relax and you’ll get pregnant’ serves to reinforce the notion that individuals are failing if they cannot make their bodies work correctly. Doubts may arise in a patient’s mind regarding his or her sense of masculinity or femininity. For example, a man who has low motility may confuse emotionally that diagnosis with personal feelings of the loss of virility. The evaluation and scrutiny of the intimate and private areas of a relationship contribute to the pressure of being evaluated for adequacy.11

Research has demonstrated that men and women experience, and are affected by, infertility in different ways, and that their coping strategies may differ as well.13 Repeated treatment failure may take its toll on the relationship. Partners may disagree about the timing of when to seek evaluation or pursuit of treatment; couples may also need to navigate differing levels of optimism about treatment outcome. Congruence can depend upon the degree to which the partners perceive the severity of the stress of the infertility; lack of congruence may in itself add to stress within the relationship. Partners may adopt different coping strategies that may be intrapersonally effective but may add to the relationship burden. In a study of German men, researchers found that men tended to suppress their emotions and had more difficulty communicating and identifying their emotions.14 Take, for example, the situation in which one partner feels great relief by being able to process the thoughts and feelings which arise and where another finds discussion stressful and anxiety producing. The very coping strategies which bring personal relief only add to the relationship stress where one partner may feel ‘shut down’ and the other partner feels unfairly burdened, depending on which coping strategy is utilized. Couples may also experience lack of congruence with regard to disclosure or nondisclosure of their fertility issues. The partner diagnosed may feel stigmatized by the disclosure; the other partner may feel burdened by the demands of not sharing the information with important support persons. Overall, men with infertility tend to somaticize their reactions; this has been observed across cultures. Some cultures are protective of the stress infertility places on men, because only female factor infertility is recognized in those cultures. In Western cultures, men may repress their feelings related to the infertility; this repression may correlate with sexual dysfunction during treatment.15

Sexuality and intimacy

The burden of infertility and its treatment

Sexuality can also be affected by infertility. Sexual enjoyment has been found to be lessened during certain

Many different emotions have been identified as arising from infertility: anger, denial, grief, guilt, anxiety,

Self-esteem and body image

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and depression. The literature remains equivocal about the impact that the length of diagnosis has on psychological burden and adaptation. Issues from the past may weigh emotionally on the individual, e.g. previous pregnancy termination(s) or sexually transmitted diseases which may have contributed to the fertility problem. The constant cycle of hope and disappointment has led to the description of infertility as an ‘emotional roller coaster.’ Treatment for infertility places very concrete demands on the individual and couple, which adds to the stress and burden. For some, the time demands of physician consultation, monitoring, inseminations, or IVF may present real problems on a patient’s demanding job or upon an individual who is juggling childcare. Women must choose between being open or private about their treatment, not just with those in their personal lives but also within their careers; too many unexplained absences from work may result in a poor performance review. Many individuals and couples may also feel that they are in limbo, foregoing new jobs or promotions due to concerns about access to treatment or even financial coverage (depending on the country). For all, the decision about being open or private about their fertility situation can frequently arise from simple questions such as, ‘Do you have children?,’ often asked innocently in social situations. Social situations are sometimes avoided in order to escape painful stimuli such as encountering pregnant women or having contact with babies. This social isolation can also add to feelings of being different or of being cut off from the usual support structures that the individual or couple typically depends upon. Families may fail to understand why the individual does not participate in expected family events such as visits to the maternity ward after a delivery, attending baby showers, or even attending holiday events.

Overview of stress and pregnancy outcome data A primary consideration of the stress and pregnancy outcome inquiry must focus on whether stress is causative of factors which would prohibit pregnancy or whether it is the diagnosis and/or treatment of infertility which causes stress. This consideration is further complicated by the issue of how stress is defined by researchers and mediated by patients. Studies that have addressed stress as it is activated by the hypothalamic–pituitary–adrenal (HPA) axis have been unable to clearly delineate the exact pathways or mechanisms.16–19 Patients are bombarded with the adage to ‘just relax and you’ll get pregnant,’ which leads to the integration of the assumption that stress has an immediate and direct impact upon their fertility. Despite the conflicting literature, it is generally concluded that stress does have an impact on the body.6,7 Stress also has an

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impact on energy level, optimism, patience, and perseverance. Stress is also mediated by personal coping styles. For example, one person’s reaction to a very stressful event, e.g. public speaking, may elicit a large stress response. Yet, the intrapersonal experience of that stress may be energizing and allow the individual to feel that he is performing better. In a different study,20 the authors concluded that infertile women have a different personality profile and their stress levels (as measured by their prolactin and cortisol levels) were elevated compared to the controls. Coping with stress may ultimately provide assistance to conception through stress reduction. Developing more effective coping strategies helps people reduce treatment termination, thereby allowing patients to truly maximize their biological potential. Improved coping strategies may also reduce relationship stress and improve communication, leading to more congruence between partners in pursuing treatment. Stress reduction should also lead to an improved sense of well-being.6 The relationship between stress and outcome is multifactorial – both complicated and curvilinear. Take, for example, the individual who experiences anxiety during the waiting period between insemination and the pregnancy test. If the anxiety increases over time, the patient’s desire to avoid the distress may grow greater than her desire to achieve pregnancy. Or the negative experience of anxiety may lead to avoidance behaviors with treatment tasks and reduce the efficacy of treatment: e.g. missed monitoring appointments, or poor timing with coitus or medication. Another conflict that may prevent good coping strategies is when an individual’s belief conflicts with his or her needs. Dysfunctional beliefs lead to poor choices in coping strategies. For example, consider the patient who believes that medical intervention is ‘unnatural.’ This belief may lead that person to pursue treatment slowly and, for many medical reasons, this slowness may be costly. A 39-year-old woman who operates on the belief that medical intervention is unnatural may delay more aggressive treatment for a critical year or longer, thereby missing more optimal fertility opportunities. In another example, if a male partner believes that conception can take place without a doctor’s intervention and his partner is ready to pursue treatment, conflict can quickly arise and tax the couple’s communication skills.

Cognitive therapy Cognitive therapy is an effective tool for understanding how stress can arise when a person’s beliefs are dysfunctional. The formulation of the patient’s dysfunction is based on the patient’s internal experiences and how those experiences are distorted through negative beliefs, assumptions, inferences, and conclusions. Change is mediated by the task of examining the accuracy of these beliefs.21 In cognitive therapy,

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the therapeutic relationship takes a ‘back seat’ as compared to a psychodynamic approach. It is the empirical investigation of these internal experiences and the opportunity to test the automatic thoughts, assumptions, and negative beliefs that yield the opportunity to correct the faulty dysfunctional constructs.22 It is the ability to change the underlying cognitive structures that will in turn change the patient’s affective state and pattern of behavior. Behavioral techniques and homework help to elicit cognitions that contribute to or cause problematic behavior and also help the patients test their maladaptive cognitions and assumptions, thus mobilizing the patients into constructive activities and enabling them to develop better coping strategies.9 In contrast to the psychoanalytic approach (which works by making the patients conscious of their unconscious past), cognitive therapy identifies current thinking and behavior.23 For example, the combination of helping patients understand the narcissistic injury of their infertility, as well as its impact on their interpersonal relationships and life planning, is critical. A woman may feel stress or inadequacy when her belief that ‘I cannot be a real woman unless I get pregnant’ leads her to avoid interpersonal relationships in order to escape the painful feelings that arise from feeling isolated from the fertile world. In addition, the recognition, as well as differentiation, of infertility as a crisis leads to stress. For some clients, infertility awakens or aggravates long-term issues in their lives, such as anxiety about intimacy, poor communication skills, etc. The cyclic nature of infertility treatment creates the feeling in the patient of an immediate infertility crisis, rather than the patient identifying it as a chronic condition. In many situations, a woman may present as if she has persistent depressive symptoms, but in reality these symptoms remit during the 2 weeks of the follicular stage of her cycle. The stress may be chronic in that it exists all the time, but the woman experiences it intensely during the waiting period post ovulation. Understanding that feelings of worthlessness, purposelessness, poor self-esteem, poor body image, isolation, and withdrawal (because of painful stimuli relating to fertility), among others, are inherent in the infertility experience is important for a thorough understanding of how change in the individual’s reactions can be made by identifying these dysfunctional cognitions and exchanging them for functional ones. For example, a woman who has always struggled with body image issues may find that infertility exacerbates these feelings; educating and disentangling the issues with the woman by identifying the dysfunctional thoughts gives her the opportunity to diminish the emotional impact and substitute other thoughts and behaviors. The stresses of infertility arise from many sources. Below are some examples of how dysfunctional

thoughts are identified and responded to within a cognitive therapy model. •

•

•

•

•

•

•

All or nothing thinking: I can never feel like a real woman if I can’t be pregnant; or My ability to gestate a pregnancy is a small part of my femininity. Overgeneralization: Everyone will think differently of my child because of the gestational carrier; or Some people may be curious about the gestational carrier pregnancy; or My child will still have the same genetics as it would had I carried the pregnancy. Selective negative focus: I cannot care for my own child in utero; or I can care for my child by selecting the best gestational carrier and building a positive working relationship. Disqualifying the positive: I will never carry my own baby; or I will miss the pregnancy experience but I have the rest of my child’s life to experience; or I will not carry a pregnancy but I can still have my own genetic child. Arbitrary inference: People think I’m selfish because I don’t have any children; or People simply think we have not chosen to start our family yet. Emotional reasoning: Because I feel sad about not being pregnant, my child will feel sad that I couldn’t carry him or her. ‘Should’ statements: I should not feel sad, therefore I am not handling this well; or It is alright to feel sad and handling this event means working through the sadness.

Behavioral medicine Behavioral medicine offers many strategies for coping with and managing stress. Employing a variety of strategies, the intervention will focus on introducing techniques such as cognitive behavioral strategies, relaxation techniques, and guided imagery. Patients also consider how nutrition, acupuncture, massage therapy, and yoga may effectively manage their stress. Another potential aspect of infertility stress management involves communication training so that patients communicate more effectively with the medical professionals involved in their care. Better communication increases the ability to receive and understand information as well as allowing patients to feel that they can effectively negotiate meeting their needs.

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Complementary alternative medicine: acupuncture, acupressure, and other alternatives Infertility treatment has come a long way since the days when psychogenic reasoning placed the blame for the woman’s infertility on the infertile woman herself. Modern medicine has begun to consider the mind–body connection in the treatment of infertility. Hotly and passionately debated within the mental health and medical professional communities, the mind–body connection is sometimes considered an entity or process with a commonly understood definition when, in fact, the mind–body connection is still more conjecture than fact. Yet, in a recent study, when asked to what extent religion and spirituality influenced patients’ health, (56%) responded ‘much or very much’; yet only 6% believed that it changed ‘hard’ medical outcomes. With regard to ways of coping with health issues, 75% felt that religion and spirituality helped patients to cope and 75% felt it gave patients a positive state of mind.24 It is estimated that, in the USA alone, complementary and alternative medicine (CAM) is utilized by at least one-third of all adults:25 that number increases to 62% of adults when prayer that is used specifically for health reasons is included. Other research clearly shows that, globally, CAM is either a standard part of patient care or an emerging option.26 Treatment for infertility has evolved to include an understanding that the most effective treatment involves treating both the mind and the body. Although evidence-based studies are still emerging for treatments such as acupuncture and relaxation approaches, more programs are opening their doors and their referrals to CAM. Accompanying the concept of treating the whole person is the paradigm of collaborative care in the treatment of the patient. A team approach – composed of a physician, nurse, mental health professional, acupuncturist, yoga instructor, or other professional – represents the new model for providing patient care. Patient associations are emerging as leaders in providing these models for collaborative care. For example, in the USA, the American Fertility Association and RESOLVE regularly bring these diverse providers together for the benefit of patients, through the use of forums, inperson seminars, or internet seminars. CAM has different meanings in different cultures and different countries. What is complementary in one country may be an accepted staple of regular treatment in another. Research internationally is expected to begin to integrate all these approaches and lead us to a more global understanding of how harmony between the mind and body can facilitate good health and, conversely, how disharmony or dysfunction between the mind and body can contribute to poorer results for the patient.

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Acupuncture The literature about the efficacy of acupuncture has been equivocal. The basic tenets of acupuncture posit that its efficacy is achieved by balancing the flow of Qi (energy) through the patient’s body.27 Fertility mechanisms for women undergoing acupuncture involve the stimulation of β-endorphin secretion, which has its impact on the gonadotropin-releasing hormone (GnRH) pulse generator and then upon gonadotropin and steroid secretion. This process creates a favorable environment within the uterus for implantation of the embryos by virtue of the increased blood flow.28 Although some studies have demonstrated a higher clinical pregnancy rate for acupuncture,28–30 other studies have shown no positive effects.27,31 Acupuncture’s efficacy has been explored by comparing traditional acupuncture (needle insertions along the meridians and points), to electroacupuncture, to laser acupuncture, and to sham acupuncture. Criticism of the comparability of all these approaches has been made in the Western medical community and in traditional Chinese medical communities alike.32 The first published study of a prospective randomized trial of acupuncture was published in 200230 and demonstrated a significantly higher pregnancy rate in the acupuncture group (n = 80) vs the controls (n = 80), with pregnancy rates of 42.5% and 26.3%, respectively. In a later study that was not published but presented at the European Society for Human Reproduction and Embryology,33 Paulus et al used sham acupuncture in a group, and compared the results to a group where traditional acupuncture was used; no significant difference was observed. The authors noted that the pressure from the sham needles could indeed have created an effect. As Domar32 observed, concerns were raised because the study was not ultimately published; Domar also goes on to suggest that studies should account for the effect that the belief by the patient that acupuncture is effective must be controlled for; he argues that it may be the belief which is the agent of change, leading to increased pregnancy rates, rather than the acupuncture itself. In a recent study,28 patients were randomized into three groups: a control group; patients who received acupuncture on the day of embryo transfer; and patients who received acupuncture on the transfer day and then 2 days later. Clinical ongoing pregnancy rates were higher in both treatment groups than in the control group, but did not reach statistical significance. The authors concluded that acupuncture significantly improves the outcome for IVF but that adding treatment 2 days after offered no other improvement. In a different study, 225 patients were randomized to receive either luteal-phase acupuncture or placebo acupuncture.34 Both clinical and ongoing pregnancy rates were higher in the acupuncture group. The authors concluded that acupuncture was safe and effective for women undergoing IVF.

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In another recent study of 228 patients, patients were randomized to receive acupuncture treatment or noninvasive sham acupuncture.27 The acupuncture group received three treatments. Traditional Chinese medicine was used to diagnose the infertility, and treatment was rendered accordingly. In the sham acupuncture group, points near but not on the actual acupuncture points were used. No significant differences were found between the groups, but the authors suggested that a small treatment effect could not be excluded because the odds were 1.5 higher for the treatment group. The authors concluded that acupuncture was safe for women undergoing embryo transfer. Finally, in a review article by Stener-Victorin and Humaidan,35 the authors reviewed four studies and noted that three of these found a higher efficacy rate for the acupuncture groups. They cautioned that the different study protocols created challenges in drawing conclusions, but could state that acupuncture has a positive effect and no adverse effects on pregnancy outcome.

Conclusion Many factors contribute to the stresses placed on, and experienced by, women and men who have fertility problems. Research has yet to disentangle and adequately address the relationship between stress and infertility. Counseling with cognitive or behavioral approaches offers tools for individuals and couples in coping with their infertility. There is emerging literature suggesting that patients are utilizing complementary alternative medical approaches and that acupuncture may be a safe and effective tool for assisting with infertility treatment.

References 1. Smeenk JM, Verhaak CM, Stolwijk AM, Kremer JA, Braat DD. Reasons for dropout in an in vitro fertilization/intracytoplasmic sperm injection program. Fertil Steril 2004; 81: 262–8. 2. Olivius C, Friden B, Borg G, Bergh C. Why do couples discontinue in vitro fertilization treatment? A cohort study. Fertil Steril 2004; 81: 258–61. 3. VanderLaan B, Karande V, Krohm C, et al. Cost considerations with infertility therapy: outcome and cost comparison and preferred provider organization care based on physician and facility cost. Hum Reprod 1998; 13: 1200–5. 4. Sharma V, Allgar V, Rajikhowa M. Factors influencing the cumulative conception rate and discontinuation of an in vitro fertilization treatment for infertility. Fertil Steril 2002; 78: 40–6. 5. Roest J, van Heusden AM, Zeilmaker GH, Verhoef A. Cumulative pregnancy rates and selective drop-out patients in in-vitro fertilization treatment. Hum Reprod 1998; 13: 339–41.

6. Boivin J. A review of psychosocial interventions in infertility. Soc Sci Med 2003; 57: 2325–41. 7. Domar AD, Clapp D, Slawsby EA, et al. The impact of group psychological interventions on distress in infertile women. Health Psychol 2000; 19: 568–75. 8. Domar AD, Clapp D, Slawsby EA, et al. Impact of group psychological interventions on pregnancy rates in infertile women. Fert Steril 2000; 73: 805–11. 9. Freeman EW, Boxer AS, Rickels K, Tureck R, Mastrionni L Jr. Psychological evaluation and support in a program of in vitro fertilization and embryo transfer. Fertil Steril 1985; 43(1): 48–53. 10. Verhaak CM, Smeenk JM, van Minnen A, Kremer JA, Kraaimaat FW. A longitudinal, prospective study on emotional adjustment before, during and after consecutive fertility treatment cycles. Hum Reprod 2005; 20(8): 2253–60. 11. Keye WR. The impact of infertility on psychosexual function. Fertil Steril 1980; 34: 308–9. 12. Boivin J, Takefman JE, Brender W, Tulandi T. The effects of female sexual response in coitus on early reproductive process. J Behav Med 1992; 15: 509–18. 13. Newton CR. Counseling the infertile couple. In: Covington SN, Burns LH, eds. Infertility Counseling: a Comprehensive Handbook for Clinicians, 2nd edn. New York: Cambridge University Press, 2006. 14. Conrad R, Schilling G, Langenbuch M, et al. Alexithymia in male infertility. Hum Reprod 2001; 16: 587–92. 15. Petok WD. The psychology of gender-specific infertility diagnoses. In: Covington SN, Burns LH, eds. Infertility Counseling: a Comprehensive Handbook for Clinicians, 2nd edn. New York: Cambridge University Press, 2006. 16. Berga SL. Functional hypothalamic chronic anovulation. In: Adashi EY, Rock JA, Rosenwaks Z, eds. Reproductive Endocrinology, Surgery, and Technology, Vol 1. Philadelphia: LippincottRaven, 1996. 17. Chrousos GP, Torpy DJ, Gold PW. Interactions between the hypothalamic–pituitary–adrenal axis and the female reproductive system: clinical implications. Ann Intern Med 1998; 129: 229–40. 18. Ferrin M. Clinical review 105: stress and the reproductive cycle. Clin Endocrinol Metab 1999; 2: 309–14. 19. Haimovice F, Hill JA. The role of psycho-neuroendocrine-immunology in reproduction. In: Hill JA ed. Cytokines in Reproduction. Austin, TX: Landes Bioscience, 1998. 20. Cxemickzy G, Landgren BM, Collins A. The influence of stress and state anxiety on the outcome of IVF-treatment: psychological and endocrinological assessment of Swedish women

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entering IVF-treatment. Acta Obstet Gynecol Scand 2000; 79(2): 113–18. Beck AT, Rush AJ, Shaw BF, Emery G. Cognitive Therapy of Depression. New York: Guilford, 1979. Clark DM, Fairburn CG, eds. Science and Practice of Cognitive Behavior Therapy. New York: Oxford University Press, 1997. Bergin AE, Garfield SL, eds. Handbook of Psychotherapy and Behavior Change, 4th edn. New York: John Wiley & Sons, 1994. Farr AC, Sellergren SA, Lantos JD, Chin MH. Physician’s observations and interpretations of the influence of religion and spirituality on health. Arch Intern Med 2007; 167: 649–54. Barnes PM, Powell-Griner E, McFann K, Nahin RL. Complementary and alternative medicine use among adults: United States. Adv Data 2004; 27: 1–19 Astin, JA. Why patients use alternative medicine: results of a national study. JAMA 1998; 279(19): 1548–53. Smith C, Meaghan C, Norman RJ. Influence of acupuncture stimulation on pregnancy rates for women undergoing embryo transfer. Fertil Steril 2006; 85: 1352–8. Westergaard LG, Mao QM, Krogslund M, et al. Acupuncture on the day of embryo transfer significantly improves the reproductive outcome

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in infertile women: a prospective, randomized trial. Fertil Steril 2006; 85: 1341–5. Margarelli PC, Cridenda DK. Acupuncture and IVF poor responders: a cure? Fertil Steril 2004; 81: S20. Paulus WE, Zhang M, Strehler E, El-Danasouri I, Sterzik K. Influence of acupuncture on the pregnancy rate in patients who undergo assisted reproduction therapy. Fertil Steril 2002; 77: 721–4. Quintero R. A randomized, controlled, doubleblind cross-over study evaluating acupuncture as an adjunct to IVF. Fertil Steril 2002; 81: S11–12. Domar AD. Acupuncture and infertility: we need to stick to good science. Fertil Steril 2006; 85: 1359–61. Paulus WE, Zhang M, Strehler E, Seybold B, Sterzik K. Placebo-controlled trial of acupuncture effects in assisted reproductive therapy. Hum Reprod 2003; 18(Suppl 1): 18. Dieterle S, Ying G, Hartzmann W, Neuer A. Effect of acupuncture on the outcome of in vitro fertilization and intracytoplasmic sperm injection: a randomized, prospective, controlled clinical study. Fertil Steril 2006; 85: 1347–51. Stener-Victorin E, Humaidan P. Use of acupuncture in female infertility and a summary of recent acupuncture studies related to embryo transfer. Acupunct Med 2006; 24(4): 157–63.

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67 The impact of legislation and socioeconomic factors in the access to and global practice of ART Fernando Zegers-Hochschild, Karl G Nygren

Introduction Throughout the world, the availability of infertility services is the result of public health policies associated with a variety of socioeconomic, political, and, on many occasions, religious influences. Wide disparities exist in the availability, quality, and delivery of infertility services within developed countries, but most of all between developed and developing countries. Relatively few of the world’s infertile population have complete equitable access to the full range of infertility treatment at affordable levels. Even in wealthy countries, such as Japan and the United States, access to assisted reproductive technologies (ART) is marked by high disparity and inequality in the access to treatment, partly due to high costs and legislative decisions. Access of men and women to health care and specifically to the treatment of infertility requires not only awareness of being infertile and the knowledge that there are treatment alternatives but also large amounts of funds are required, irrespective of whether they are provided by national health authorities, by individuals themselves, or by a combination of both. In countries where access to infertility treatment is granted by law, fertility is understood as a right to which all women and men have equal access. Centralized policies are then established in order to have access to these goods. An example of policies regulating who and under what conditions access is granted is reflected in the establishment of an age limit for women where treatment will be provided. Another example is a restriction in the number of embryos to be transferred in ART. When access to infertility treatment is not part of a governmental policy, individuals must rely on their personal wealth and/or private insurances covering medical care. Under this scenario, what regulates access to diagnosis and treatment is left to a free market policy, leaving out of reach all those individuals who cannot afford the costs involved. Furthermore, in

most countries, companies that provide private health insurances do not cover the costs involved in the treatment of infertility. Coverage of infertility treatments offers some additional difficulties. While nobody would argue against the use of all available tools in order to save the lives of people with cancer, the use of modern reproductive technology is controversial, and many legislators wonder whether specific treatments should be available or funded in order to generate a new life. Interestingly, there is much more social acceptance and legislative agreement in saving lives than in generating new ones. Irrespective of whether a country is over- or underpopulated, there seems to be less public concern in prolonging the lives of the elderly than in generating new young lives. It is a rule of life that those individuals promoting laws and regulations have already passed by the burden of existing: all they need to worry about is the quality of their aging and death. On the other hand, for those who have not yet come to existence, there are no chances of influencing policymakers unless the latter themselves have experienced infertility or been moved by someone with this condition. Countries around the world either do not regulate or regulate ART in many different ways. It is the purpose of this chapter to review how different legislation as well as socioeconomic, demographic, and religious factors influence the access to and the practice of ART.

Factors influencing worldwide contribution to ART cycles Information on the number of ART procedures performed is now available, thanks to worldwide data collected by the International Committee for Monitoring Assisted Reproductive Technology (ICMART).1,2 While in 1993, 40 countries reported 350 000 cycles, in the year 2003 (last year reported),

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n = 667711

Middle East 5.0% Latin America 2.9% Asia 12.5% Australia & New Zealand 5.0%

Europe 55.5%

Fig 67.1 Regional contribution of ART cycles to the World Report (2003).

North America 19.1%

67.0

45

42.8

Percent of women

40 35 30 25

20.1

20

12.8

15 10

4.5

5

2.5

2.8

2.1

1.3

54 countries reported almost 700 000 cycles, representing an increment of 20% in a decade. The major contributor to ART cycles is Europe, which performs approximately 370 000 cycles, followed by North America, reporting almost 130 000 cycles in 2003. Between 1993 and 2003, the number of initiated cycles increased seven times in Europe and four times in the USA. However, neither the relative contribution nor the percent increment in cycles follows a homogeneous pattern. In 2003, Europe represented 55.5% of initiated cycles, while Latin America, the Middle East and Australia & New Zealand contributed with less than 7% each (Fig 67.1). In many ways, the proportion of ART cycles is a reflection of availability of this expensive form of treatment to infertile couples.

Inequality in the access to ART When availability is expressed as number of ART cycles per million inhabitants, the proportion of treatment cycles fluctuates between 1000 and 2000 cycles

il az Br

le hi

py

t C

nt ge Ar

Eg

a in

n pa Ja

SA U

K U

ed Sw

D

en

m

ar

k

en

0

Fig 67.2

Access to ART.

per million inhabitants in Germany, Sweden, Denmark, and Belgium, and from 500 to 700 cycles in the UK, Switzerland, and Austria.3 The disproportion is even greater between European countries and other regions of the world. Using the same calculations, the availability in the USA is only about 400 per million, 185 in Japan, and 60–100 cycles per million in countries in Latin America (Argentina, Brazil, and Chile) and Egypt in the Middle East. Because of the variability in the age distribution of different populations, expressing number of ART cycles per million inhabitants does not consider the number of women that would really require infertility treatment. For example, the need for infertility treatments should be higher if the mean age of the female population is under 30 years old than if the mean age of the female population is older. Fig 67.2 describes access to ART by dividing the number of initiated cycles per country by the number of women aged 25– 40 years old, assuming 10% infertility and 30% of those requiring ART (3% of all women aged 25–40 years old). Using this calculation, differences between

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Table 67.1

887

Number of ART cycles (2002) according to public/private health expenditure (2003)

GDP per capita (dollars)

Country (% general government expenditure)

30–35 000

Sweden Denmark UK Australia Japan

(13.6%) (13.5%) (15.8%) (17.7%) (16.8%)

86.2 83.0 85.7 67.5 81.0

14.8 17.0 14.3 32.5 19.0

1241 2106 625 1291 185

>40 000

USA

(18.5%)

44.6

56.4

413

8–12 000

Brazil Argentina Chile

(10.3%) (14.7%) (12.7%)

45.3 48.6 48.2

54.7 51.2 51.4

66 119 65

<5000

Egypt

(8.2%)

42.6

57.3

89

Public health (%)

Private health (%)

ART cycles/million

GDP, gross domestic product.

Sweden and Denmark on the one hand and Latin American countries and Egypt are vast. Interestingly, the major source of difference in access to modern reproductive technology does not only depend on the wealth of the country. It is also a reflection of the distribution of wealth. An example is the USA, the richest country, with a disproportionate poor access to ART treatments of its population.

Factors affecting access to ART Access to ART can be the result of multiple variables. Table 67.1 describes the relationship between availability of ART and the way funds allocated to health are distributed among the population. Countries from northern Europe allocate 13–14% of their gross domestic product (GDP) to health expenditure, and more than 80% of it is allocated into public as opposed to private health expenditure. On the other hand, the USA, with the highest GDP per capita of US$40 000, also allocates the highest percentage of funds to health (18.5%) but, due to a different economic policy, only 44.6% goes on public health expenditure. Countries in Latin America and Egypt are not only much poorer, they also follow trends similar to the USA, allocating the majority of their restricted funds into private rather than public health. Consequently, coverage of infertility treatments is reduced in these countries. Japan, is an exception; while it allocates a high proportion of its GDP to health, and most of it as public health expenditure, its low coverage is the result of a political decision to refrain from funding infertility treatments altogether. The first conclusion would be that access to ART treatment is strongly influenced by socioeconomic policies. Countries where infertility treatment is considered a right, to which all individuals are entitled as equals, distribute their wealth through public facilities and have a much higher coverage of treatments. On the contrary, countries where access to infertility

treatments is partly regulated by the market, requiring out-of-pocket funding, have a much lower coverage of fertility treatments, which, in turn, decreases the number of treatment cycles.

Access to ART treatment and insurance coverage Access to health and insurance coverage are intimately related. Only a few countries – Australia, Belgium, France, Greece, Israel, Slovenia, and Sweden – have national health plans that cover a full range of treatment. Differences between them reside in the number of ART cycles that are covered by the national health plan and in the regulation imposed to have access to this facility (the age limit of the female partner, the maximum number of embryos to be transferred, etc.). An interesting observation results from the fact that countries with full coverage also deal with the costs involved in pregnancy, delivery, and neonatal care. Today, single embryo transfer is the rule in Sweden and for young women in Belgium. Therefore, the costs they have absorbed by covering ART have been compensated for by decreasing the number of multiple births and high costs involved in the care of preterm babies. Some other countries in Europe and the Middle East, such as the UK, Denmark, Finland, and Tunisia, have only partial coverage from public sources. What is more striking is that in Latin America, a region strongly influenced by Catholic tradition (which opposes ART), access to infertility treatments is left out of coverage both by public and private insurances. Since ART treatment is only covered by out-of-pocket funding, this results in a source of inequality and disparity in the availability of health resources. The absence of insurance coverage determines that only wealthy couples have access to treatment and, as will be seen later in this chapter, is at least in part responsible for a high rate of multiple births.

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Table 67.2

Access to ART (2002) according to age of female population and fertility rate

Country/region

Female median age (est, 2006)

Fertility rate

Population growth

Access % (25–40)

Sweden Denmark UK

42.2 40.4 40.7

1.66 1.66 1.74

0.2 0.3 0.3

42.8 67.0 20.1

Japan

44.7

1.4

0.1

4.5

USA

37.8

2.09

0.9

12.8

Brazil Argentina Chile

29.0 30.7 31.3

1.91 2.16 2.00

1.1 1.0 1.0

1.3 2.8 2.1

Egypt

24.3

2.83

1.8

2.5

Access to ART and demographic factors It is interesting to note that coverage of infertility treatments in many countries or regions is strongly associated with the mean age of women, the fertility rate (ratio between the number of births and the number of women exposed to the risk of pregnancy), and the population growth rate (the rate at which the number of individuals in a population increases). Table 67.2 describes the association between access to ART in different countries and the mean age of their female population, their fertility rate, and population growth rate. Countries having the highest access to ART treatment are those with the lowest population growth rate and highest age of women. In contrast, younger populations with higher fertility rate, as in most countries in Latin America and the Middle East, have less coverage or no coverage at all. Again, as discussed before, Japan is an exception, because the median age of the female population is almost 45 years old, with a low population growth rate, and despite this, infertility treatment is not covered. Perhaps the underlying factor responsible for these disparities is that in countries with an older female population and a negative growth rate, the nation as a whole needs to deal with population renewal. On the contrary, in countries with a young female population and a high growth rate, it is not the country but the individual who has to deal with his/her reproductive needs. This might perhaps explain why no Latin American country considers infertility as a disease and, therefore, infertility treatments do not fall into the public health agenda. The desire to have more children is not a national priority in regions with a young female population and a high fertility rate. Furthermore, in the absence of legislation regulating the practice of ART, access to fertility treatments is not on the agenda of national health policies, and companies responsible for health insurance do not cover expenses related to fertility treatments.

Furthermore, for many legislators in Latin America, procedures such as in vitro fertilization (IVF) and embryo transfer are considered morally unacceptable and a luxury that should not be sustained with state funds. An extreme example of this moral dictatorship is Costa Rica where the high courts decided that IVF is morally unacceptable and illegal, forbidding the practice of ART in the country. The result of this policy is that a small proportion of wealthy citizens can afford traveling abroad, while the vast majority of couples requiring ART remain childless.

Influence of tradition and religion in the practice of ART It is often difficult to ponder the influence of religion, tradition, and other cultural factors in the application of laws regulating reproductive health. The difficulty is not necessarily the result of an imposition of a certain religious morality; many times, economic and political forces are strongly bound to religious organizations, which, in the end, influence legislative processes.

Christian tradition Although religion and public laws have been separated for centuries in countries in Western Europe and the Americas, Christianity and, most of all, the Roman Catholic Church is by far the most outspoken religious body when it comes to moral behavior concerning sex and reproduction. Catholic tradition has a strong influence in Latin America, less in the USA, and even though most European countries have a more rational, evidence-based approach to ethics in reproductive health issues, the Catholic tradition can still exert strong influence. A recent example is Italy’s new law regulating the practice of ART. The fundamental basis for the Catholic opposition to any form of ART started in the late 1960s. In his encyclical

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Humanae Vitae, Pope Paul VI established that the uniting and procreative meanings of the conjugal act should not be voluntarily dissociated. Consequently, both contraception and assisted reproduction are considered immoral, as they voluntarily dissociate these two meanings: contraception, by allowing sexual intercourse devoid of its procreative meaning; assisted reproduction, by allowing procreation not mediated by sexual intercourse. Later, in 1987, the Vatican published a document ‘Donum Vitae,’ which contained an ‘Instruction on respect for human life,’ issued by the Congregation for the Doctrine of the Faith and signed by Cardinal Joseph Ratzinger (today, Pope Benedict XVI). This document stated that a person, as we understand it, exists from conception onwards and, therefore, condemned all forms of assisted reproduction, irrespective of its intention, the source of gametes, and marital status. This principle carried such power that later, the vast majority of countries in the Americas signed the ‘American convention on human rights, pact of Costa Rica,’ which states that ‘laws should protect the lives of those to be born – in general – from conception onwards’. Based on this principle, the Supreme Court of Costa Rica stopped ART in that country. In the rest of Latin America, ART is performed but no laws are available, in spite of the fact that most countries have law projects sitting in their parliaments. In the majority of cases, this is because no agreements have been reached between legislators as to whether preimplantation embryos are entitled to rights of their own. Needless to say that any form of embryo manipulation, genetic diagnosis, or research is performed in few countries without the possibility of discarding abnormal embryos. The influence of Catholicism on ART is less evident in the USA, where more value is placed on the right to autonomy, both from the perspective of couples and providers. It must be said, however, that recently, the opposition of the US government to therapeutic cloning and embryonic stem cell research is mainly the result of lobbying by the Catholic Church under the argument that a preimplantation embryo is entitled to the same rights of an existing person. For various reasons, the Council of Bishops in Europe have been more liberal in the application of directives arising from the Vatican. An example is the Catholic University of Leuven, Belgium, where ART, including embryo cryopreservation, is offered openly. Conversely, however, is the recent law passed in Italy which forbids fertilization of more than 3 oocytes, embryo cryopreservation, use of donor gametes, genetic diagnosis, etc. The reason behind this restrictive law is the result of pressure from the Catholic Church on the bases of human rights attributable to embryos from conception onwards. In a different attitude towards reproduction, all Protestant denominations (Baptist, Methodist, Lutheran, Mormon, Presbyterian, Episcopalian and others) are very liberal concerning infertility treatments and the promotion of reproductive science.

889

ART is accepted as long as gametes belong to spouses, and embryos are not intended to be destroyed.

Islamic tradition Differently to religious laws regulating the Western world, sharia, the code of law based on the Koran, also regulates political, public, and private lives. Its teachings and directions are open for interpretation as science and technology discovers new routes and they serve humankind and society.4 Concerning reproduction, almost all scholars agree that it is legitimate for infertile couples to pursue any form of therapy as long as both male and female gametes belong to the couple and pregnancy takes place in the woman’s uterus.5 Consistent with this concept of genetic heritage, Islam does not approve adoption. Thus, it is the duty of physicians to help infertile couples achieve conception with the freedom to use technology as long as this takes place inside the married couple.6 The embryo is entitled to due respect, and genetic diagnosis can be practiced as long as it does not harm the embryo.5 Although law allows for preimplantation genetic diagnosis (PGD) in Islamic countries, couples cannot practice their autonomy to decide upon the fate of their embryos. In Islam, embryos cannot be discarded.

Jewish tradition The application of the Jewish tradition is circumscribed to the teachings found in the Torah, subsequently followed by a compilation of traditions and interpretations, such as the Talmud and other ancient religious documents. Israeli laws are secular and rule public affairs, while private matters are the domain of Judaic law, enforced by special rabbinical courts. When it comes to procreation, both secular and religious laws are pragmatic and favor the stability and strength of the family, and in agreement with the first commandment, ‘Be fruitful and multiply,’ laws allow almost any form of assisted reproduction. Although marriage and/or a stable relationship are required to have access to ART, single mothers can also receive fertility treatments. Different religious branches of Judaism have marked differences in the interpretation of the law; nonetheless, in the final analysis, the decision to use modern reproductive technology is dealt with as a freedom for infertile couples and it is provided by the government. Israeli law allows gamete donation (with strict regulations on the source of male gamete), any form of ART, PGD, even for sex selection, oocyte donation, etc. Israel holds the highest number of IVF clinics per capita and the National Health Insurance Fund provides IVF treatment for up to two live births for childless couples and for single mothers. The coexistence of Jewish religion and law represents a remarkable example of equilibrium and tolerance

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and allies the strength found in tradition and the need to use science and technology to bear children and strengthen the family.

How legislation influences the practice of ART Number of embryos transferred and multiple births The issue of multiple births is one of the most serious complications generated by ART. The risks related to multiple births not only involve maternal and perinatal complications but also generate financial and social problems that have to be dealt with by the family alone. The rate of multiple births varies in different countries and regions. For 2002, the proportion of twins and ‘triplets and more’ was 31% in Latin America, 35.2% in the USA, and 24.9% in Europe (Fig 67.3). Perhaps, the most remarkable difference is in the number of triplets and more, which rises from 1.3% in Europe to 3.6% in the USA, and to 6.4% in Latin America. The differences in high-order multiple births in Latin America and the USA is in part the result of embryo reduction in the latter. In Latin America, between 1998 and 2004, a total of 29 115 babies were registered as born, half of them had cohabitated with at least one other fetus with a direct impact on perinatal mortality, which increased 2.4 times in twin births and 5.3 times with the birth of triplets and quadruplets.7 There is no doubt that the number of embryos transferred has a direct effect on the chances of becoming pregnant, and is the one single factor which by itself increases the risk of multiple gestation and birth. Indeed,

Proportion (%)

The purpose of reviewing religious morality is that, especially in the developing world, religion can have a strong influence on political decisions. In countries dominated by a Catholic tradition, which today are concentrated mainly in Latin America, much of the discussion is not centered on the rights of infertile women and men. On the contrary, most of the discussion is centered on the moral rights of an embryo. As a consequence of the above, ART is accepted because it is there, but no country has been able to reach a consensus on minimal standards to regulate the practice of ART. This lack of pragmatism in confronting biomedical and social realities is at least in part responsible for the low access to treatment and generates inequality, lack of autonomy, and therefore absence of certain diagnostic and therapeutic procedures, such as PGD and other forms of preventing the inheritance of genetic diseases. In countries where the influence of religion on public policies has been restricted, it is the right of persons that prevails, inasmuch as it does not affect society as a whole. In general, the more separation there is on how and who is entitled to impose religious and public laws, the more respect there is for the needs of women, men, and for the children to be born.

100

1.3

3.6

6.4

23.6

31.6

24.6

75.1

64.8

69.0

Europe

USA

Latin America

75 50 25 0 Triplets or more

Twins

Singletons

Fig 67.3 Regional variability in multiple birth in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) (2002).

this risk also increases as the age of woman decreases. In 2003, 67% of transfer cycles in Latin America included 3 or 4 embryos, compared with 29% in Europe and 59% in the USA. Many factors may be responsible for these regional differences, but the pressure for success placed by the couple and their family plays an important role. This pressure increases due to economic constraints. Thus, if for economic reasons, the couple can afford only one treatment cycle, the risk/benefit evaluation of multiple births as opposed to no birth is considered differently than if couples have 6 cycles for free. Table 67.3 compares the number of embryos transferred and the proportion of high-order multiple births in different countries during 2002. Data are presented according to whether the source of funding was public or out of pocket. There is no doubt that the most efficient way to decrease the number of multiple births is by reducing the number of embryos transferred and this is easier to do when the high costs are totally or partially covered by public or private sources.

Mode of fertilization – ICSI vs IVF Since its introduction, the use of intracytoplasmic sperm injection (ICSI) has increased yearly. Today, in certain regions of the world, ICSI is used in more than 70% of ART cycles. Worldwide, the proportion of ICSI over IVF increased from 24% in 1995 to 56.2% in 2003. However, this proportion has regional variations, which, similar to what happens with the number of embryos transferred, is influenced by different legislations, especially by socioeconomic variables such as who is responsible for the costs of treatment. In countries where ART is subsidized by public funds, the proportion of ICSI is relatively low: 53.2% in Australia and 56.3% in Europe. In regions where ART is paid for directly by consumers, the proportion of ICSI rises to over 75% in Latin America and 94.8% in the Middle East3 (Fig 67.4). Irrespective as to whether it is right or wrong, there is a tendency to avoid unexpected failed fertilization or low fertilization rate with regular IVF, and centers tend to use more ICSI to ‘ensure’ fertilization.

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Table 67.3

Source of funding influences the number of transferred embryos and high-order multiple births (2002)

Mean no. of embryos transferred

Source of funding

Country

Public

Denmark Sweden UK France

1.9 1.7 2.1 2.2

0.3 0.2 0.6 0.7

Out of pocket Out of pocket + private insurance Out of pocket Out of pocket

Brazil USA Egypt Chile

3.3 3.0 3.0 2.7

7.8 3.8 3.6 7.3

Out of pocket

Japan

2.3

0.6

0.0

Procedure distribution (%)

100 80

0.1

1.1

24.5

36.8

45.7

5.2

54.4

43.7

60 40

891

63.1 56.3

75.2

94.8

45.5

53.2

20 0 Europe

North Australia & New Zealand America

ICSI

IVF

Asia

Latin America

Middle East

GIFT

Fig 67.4 Procedure distribution according to region (2003). ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; GIFT, gamete intrafallopian transfer.

Examples of the effect of laws on the outcome of ART treatments Perhaps the best examples of the impact of legislation on the outcomes of ART can be found with the implementation of the new laws in Belgium and Italy.

The Belgium example In July 2003, the Belgium health authorities introduced legislation to improve financial access to ART treatments and to reduce multiple births. The main decision included that laboratory costs would be refunded for six cycles in a lifetime for women under the age of 43 years old. This benefit is conditioned by the number of embryos that can be transferred, which varies from 1 to 3, depending on the age of the woman and the cycle number. Furthermore, each center is obliged to report all its data to a centralized registry, who can evaluate trends. This policy not only eliminates inequality in access but also decreases the risks of multiple births. In this way, the reduced neonatal

High-order multiple births (%)

costs should be enough to cover the costs of treatment. This is perhaps one of the best examples on how a legislative body examines the available data and implements a solution that brings equality and benefit to all members of society. Belgium is a multireligious community cohabiting with a proportion of nonreligious community. It is important to note that this policy does not enter into a philosophical discussion of when does personhood begin. On the contrary, it looks at the economic, social, and biologic evidence with pragmatism and implements a legislation that can deal with them in the best possible way, allowing individuals to decide by themselves. The consequences of this legislation are still being evaluated, but so far, at least two conclusions can be extracted. First, there is no reduction in the chances of a couple having a baby; it can take more cycles, but the cumulative birth rate is not affected. Secondly, there is a marked reduction in the rate of multiple births. A good review of this data can be found in a recent publication by Van Landuyt.8

The Italian example In March 2004 a new Italian law imposed a number on limitations on the medical profession and on infertile couples. In fact, no more than 3 oocytes can be inseminated, because no more than 3 embryos can be generated. Furthermore, all embryos need to be transferred, since embryo cryopreservation, PGD, or any form of embryo manipulation is not allowed. In a different dimension, only married couples or heterosexual couples living a stable relationship can have access to ART treatment. No treatment is available for gay couples or single women. This law establishes the right of an embryo over the rights of her and/or his progenitor. This is especially confusing in a country where the termination of a clinical pregnancy is legal. Furthermore, by restricting ART to only married and cohabiting heterosexual couples, it specifically discriminates against infertile

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women, since if they were fertile, they could become pregnant without requiring a stable heterosexual relationship. This discrimination is only restricted to those infertile women requiring ART, since infertile women are not required to have a heterosexual stable partner to have access to ovarian stimulation or pelvic surgery or to any other form of infertility treatment. The ethical and clinical implications of this law have been discussed by Benagiano and Gianaroli.9 In the absence of an Italian registry capable of analyzing reproductive data before and after the law, few articles have dealt with its consequences. Recently Ciriminna et al10 reviewed the effect of the law in cases with severe male factors and, as expected, showed a significant negative impact on pregnancy and implantation rates. These examples represent two different ways to legislate in areas related to sex and reproduction: •

•

In the Italian experience, the main intention of legislators is to defend a moral principle: ‘the respect of a person from conception (fertilization) onwards,’ irrespective of the effects this might cause actual persons (men and women) in the family and in society. In the Belgian experience, the main intention of legislators is to defend the right of actual persons to receive medical treatments. It is also an objective to protect the quality of life of those to be born by facilitating the birth of singletons.

There is a world tendency to procure (or satisfactorily balance) safety over efficacy, particularly in countries where access to ART is guaranteed or at least facilitated by public resources. In the search for safety, a single embryo transfer (SET) policy has been established in Sweden, Finland, and Belgium. There is more than one strategy to reach this goal, and these three countries have arrived at a SET policy by different roads. In Sweden, currently in the lead of the SET transition, 70% of all embryo transfers are now SET, with the eradication of triplets, and a reduction of twinning from over 25% to only 5%. This transition is a result of a combination of several factors: a professional decision, soon supported by the national patient organization, and later followed by governmental regulation. The process took 4 years, from 2002 to 2005. The main factor behind this move was a cooperative effort between the professional societies of gynecologists and pediatricians and governmental authorities, where the medical risks for IVF children were thoroughly investigated. A national IVF register of all women giving birth after IVF was formed and, using the personal identification number given to each Swedish citizen, cross-links were made to five different population-based health registers already in operation. Very convincing evidence emerged that the much higher risk profile of IVF children was caused not by the IVF technique per se but by elevated multiple

delivery rates. A large randomized clinical study followed by national data demonstrated that pregnancy rates did not drop, after a substantial increase in the proportion of SET. This, finally made the case for SET as the norm. A very important driver for the transitions was the back-up by the lay press. In the midst of this process, the law was changed to say that SET must be the norm. The change of law merely confirmed what was already happening, and was therefore welcomed in the country. Discussion on economy was never involved. In Finland the transition to SET as the norm was the result of professional decision only, with no governmental interference, and again the economy was not an issue. In Belgium, on the other hand, economic arguments on high national costs for postnatal healthcare of prematurely born multiple-birth IVF children convinced the government to change reimbursement policies to strongly favor SET. There is thus not one single road to procuring an equilibrium between safety and efficacy: in this case through the SET policy. In Sweden national data on safety and efficacy, followed by governmental regulation of the practice of IVF achieved this effect; in Finland the same process but with no governmental intervention; and in Belgium a change of governmental reimbursement policies. But the effect is the same: a much reduced risk for the children. It is interesting to note that in countries where the influence of religious morality is well balanced by strong and independent lay organizations, laws tend to follow realistic and sensible evaluation of reality and public decisions are adopted after incorporating the lay public and society as equals in the discussion of public policies. On the contrary, countries with strong religious influence tend to moralize in such a way that the value of embryos become the dominating issue overlooking the rights of actual persons – in this case, infertile couples in the pursuit of effective and safe treatment of infertility. The examples of the Nordic countries in contrast with countries in Latin America and now Italy are a true reflection of this reality.

References 1. Adamson GD, de Mouzon J, Lancaster P, et al. World collaborative report on in vitro fertilization, 2000. International Committee for Monitoring Assisted Reproductive Technology (ICMART). Fertil Steril 2006; 85(6): 1586–622. 2. De Mouzon J, Adamson GD, Ishihara O, et al. IVF Monitoring Worldwide (ICMART). Proceedings of the 23rd Annual Meeting of the European Society for Human Reproduction and Embryology, July 1–4, 2007, Lyon, France. 3. Andersen AN, Goossens V, Gianaroli L, et al. Assisted reproductive technology in Europe, 2003.

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4. 5.

6.

7.

Results generated from European registers by ESHRE. Hum Reprod 2007; 22: 1513–25. Serour GI. Traditional sexual practices in Islamic world. Global Bioethics 1995; 1: 35–47. Yepren S. Current assisted reproduction treatment practices from an Islamic perspective. Reprod Biomed Online 2007; 14 (Suppl 1): 44–7. Serour GI. Medically assisted conception dilemma of practice and research. Islamic views. In: Serour GI, ed. Proceedings of the First International Conference on Bioethics in Human Reproduction Research in the Muslim World, 1992, Cairo, Egypt. 11 CPSR 2: 235–42. Zegers-Hochschild F, Galdames V, Schwarze JE, eds. 15th Anniversary of the Latin American Registry

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(RLA) of Assisted Reproduction. 2003–2004 Report. Latin American Registry of Assisted Reproduction 2007. 8. Van Landuyt L, Verheyen G, Tournaye H, et al. New Belgian embryo transfer policy leads to sharp decrease in multiple pregnancy rate. Reprod Biomed Online 2006; 13: 765–71. 9. Benagiano G, Gianaroli L. The new Italian IVF legislation. Reprod Biomed Online 2004; 9: 117–25. 10. Ciriminna R, Papale ML, Artini PG, et al. Italian Society of Embryology, Reproduction and Research (SIERR). Impact of Italian legislation regulating assisted reproduction techniques on ICSI outcomes in severe male factor infertility: a multicentric survey. Hum Reprod 2007; 22: 2481–7.

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68 Recent ethical dilemmas in ART Françoise Shenfield

Almost 30 years after the first birth from in vitro fertilization (IVF), and over 20 years since the first ovum donation (OD) birth, we still face numerous dilemmas in assisted reproduction, some newer than others. Assisted reproductive technologies (ART) have been integrated into the framework of our family structuring and our societies, and many initial issues raised by these new technologies cannot even be thoroughly discussed in books solely dedicated to the subject.1–3 The choices made below must perforce be eclectic and reflect current concerns. However, one must always start at the beginning, and before explaining the choices made in this chapter, the introduction must mention the embryo. The necessity of embryo research and its implications for the status of the embryo entity are still under challenge at a time when the therapeutic potential of embryonic stem cells gives further arguments to its necessity in a consequentialist fashion. Symbolically, in France, where the 2004 legislation4 allowed some embryo research, within very limited boundaries, the hopes induced by possible cell therapy have meant that some embryo stem cell research is now possible. In Belgium, a new law5 also allows research on stem cells. Some dilemmas are only alluded to, as they have been the subject of many previous discussions, although specific aspects are now more in focus. They relate in general to the use of gamete donation, to the source of these gametes, and the way donors are recruited. But currently, the theme of identity, reflecting in a sense the social place of our children in a constantly changing (mostly) Western European framework, seems to be acutely relevant. We have by now seen an almost universal ban on reproductive cloning, but gamete donation still evokes contradictory attitudes worldwide. It is of note, for instance, that this is forbidden to strict Muslims who behold the genetic link as paramount in family relations. But also, in the context of gamete donation where it is socially accepted as practice of compensation for total sterility, recent concern about the anonymity6 of the process must be reflected upon. Other topical concerns are linked to the more recent achievements of preimplantation genetic diagnosis (PGD), particularly the endeavor to obtain a matched sibling for an

already sick child by this technique, and screening for late-onset genetic disease; cryopreservation of reproductive tissues and oocytes and its implication for the reproductive future of the well and the sick; and, last but not least, prevention of the most common complication of ART, multiple pregnancy. Thus, these topics have been chosen to represent the modern ethical dilemmas. The first issue raised, however, is one of justice, a personal as well as meta-ethical and societal concern, with many serious consequences. There is no doubt that this issue should take precedence, as it colors many other problems in the real world and in ART. Can our patients be offered totally autonomous choice when/if there is no state support and subsidy in their plight to become a family, and they may feel pressured to risk multiple pregnancy in order to compensate for the paucity of cycles of treatment they can afford, especially with IVF? Fortunately, more national and international bodies are pressing for single embryo transfer (SET) in optimal cases, so that hopefully the price some children of ART still pay in terms of morbidity, and their family in terms of costs (psychologic as well as financial) and added stress, will eventually lessen.7

Justice for our patients and concerns for the future child(ren) There is a wide discrepancy in access to ART among different health systems worldwide. Two main factors limit access: the selection criteria used by national legislation or local codes of practice (age, marriage, etc.), and economic factors, whether or not treatment is funded by the state, in a direct or indirect fashion. It is impossible to compare access to all infertility treatments internationally (ovulation induction, surgery, gamete donation, IVF), but one may have an idea of the disparity of access as a function of the number of IVF cycles performed in proportion to different countries’ populations. Thus, striking differences between countries of similar populations and economic wealth are worth mentioning. For instance, the latest European IVF monitoring (EIM) figures8 show

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that the best provisions are in Northern Europe and France. It is unlikely that this discrepancy should be linked to different needs of respective patient populations. However, in the UK, access is either totally free through the National Health Service (NHS), or totally paid for privately by patients, in the respective proportions of approximately 25/75%. This is compounded by unequal access in different areas, with wide variations among regions or Primary Care Trusts responsible for their budgets, which themselves use heterogeneous criteria to select patients. Furthermore, infertility treatments are not reimbursed by private insurance in the UK. There is still a lack of political will to improve systematically both national and rational access to fertility treatments, and in spite of repeated discourses of intent, there is not even a guarantee that the necessary monies would follow the agreed theoretical provisions. However, we can look up and learn from some examples of collaboration between policy makers and clinicians, as recent change in Belgium has taught us that one may have a good success rate with SET, and to motivate caregivers by a fair but strict insurance.9 Patients are thus not tempted to ‘maximize’ the only attempt at IVF they can afford, and to prefer multiple embryo transfer (within the limits of respective national legislations), without due regard to the risks imposed on the potential children. Embryo reduction may be offered, but is never a simple decision for aspiring parents, and our patients should be spared this further dilemma. At the time of writing, the Human Fertilisation and Embryology Authority (HFEA) has issued a consultation document on this very subject, presenting alternatives and possible solutions: one might select the embryo most likely to implant, the way in which results are reported, and, especially, educate patients, politicians, and the public at large. For instance, the report of ‘success’ rate could be put in terms of likelihood of clinical pregnancy per cycle started (or per couple), including the replacement of frozen–thawed embryo transfers. The prevention of multiple pregnancies (which could be reported as complications rather than successes of ART), with their untoward consequences, both for the parents and for the potential children, has become a matter of concern to fertility specialists and patients alike.9

Recent advances and their ethical implications Preimplantation genetic diagnosis The availability of multiple embryos created in vitro is essential in order to perform PGD for couples in need. PGD is the ultimate step in antenatal screening, retrogressing from diagnosis on the fetus in utero to the embryo in vitro. As far as PGD itself is concerned, it may be appropriate to introduce the term ‘pregravid diagnosis,’ as indeed the mother to be is not pregnant

until a fertilized embryo (after PGD or not) has been replaced and implanted in utero following the necessary IVF. There are specific new dilemmas engendered by PGD, such as human leukocyte antigen (HLA) typing for curing a sick sibling, but in general the ethical dilemmas of PGD are not dissimilar to those encountered in antenatal screening. Indeed, the United Nations Educational, Scientific, and Cultural Organization (UNESCO) International Bioethics Committee (IBC) addressed similar concerns in its report on genetic screening and testing:10 underlined were the problems of limits of the technique (such as accuracy and quality control) and those concerning information, privacy of patients, and public policy, as well as universal values and civic freedom. Of relevance is the fact that the IBC came to a consensus as to when therapeutic terminations of pregnancies were, in its opinion, ‘out of the question’: for requests of enhancement of human characteristics, for avoidance of traits within the range of ‘normality,’ and avoidance of predisposition to treatable diseases. Such constraints could similarly apply to PGD, although one in particular is put to the test by the screening of late-onset disease, such as familial polyposis coli. The gene is undoubtedly one which indeed predisposes its bearer to this familial cancer, but whether it falls foul of the spirit of the UNESCO declaration is arguable in the sense that cancer may or may not be ‘treatable.’ The IBC also stated that there must be careful monitoring of screening programs, and it must be said that in many instances the transparency with which screening is performed must be commended. For example, all cases of PGD worldwide are recorded centrally.11 The ‘promotion of informed reproductive decisions’12 is indeed a caring and sensitive terminology to qualify this pregravid diagnosis, and European Society for Human Reproduction & Embryology (ESHRE) taskforce reflections underline the importance of a multidisciplinary approach. Let us not forget, however, that the danger at societal level is that those opting out will be portrayed as irresponsible and stigmatized. Needless to say, counseling is of great importance in all these decisions. What is the most awe-inspiring denominator of this complex equation? The answer must be: the potential harm to the fetus and future child born after the use of the technique, and the need for surveillance of this particularly ‘precious’ offspring, in turn entailing recording the births and follow-up of the children. This means a specific dilemma between the fundamental principle of confidentiality, on behalf of the carers to the patients, the respect of their autonomy and right to privacy, and the danger of the psychologic consequences of this intrusion for the children.13 The problem of confidentiality with regard to the child sometimes seems insoluble, as it entails a parental, if not a state, decision, as is the case in the cessation of anonymity in gamete donation in Sweden.

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The eugenics debate must also be mentioned, as it has been argued that some couples would demand, after preimplantation diagnosis, the assurance of a ‘perfect’ baby. Actually, in practice, couples say that they want a ‘normal’ rather than a ‘perfect’ baby.14 But is the basic philosophy of preconception and preimplantation diagnosis akin to eugenics, in that it selects gametes or embryos? Although concerns that ‘a more and more restrictive definition of normality and humanity would ensue from a wide application of PGD’15 must be at least considered, it must be said that if eugenics is defined by its focus on population, and not individual couples’ choice to reproduce, this term of eugenic practice does not apply. Finally, one may actually wish to use a new terminology rather than eugenics in view of the tainted historic background of eugenics, and in particular its radical movement at the beginning of the 20th century.16 Also, one may diagnose X-linked disorders with PGD, and this has naturally rekindled the debate about sex selection for social reasons, when, for example, several ‘healthy’ embryos of different gender potential would be available for embryo transfer. Although some accept a compromise with the possibility of family balancing (never for the first child and always for a child of another sex),17 it implies that families with same-sex children may be somewhat unbalanced. Even more important, the argument that sex selection is likely to reinforce sexist attitudes already too prevalent in most societies must be the most powerful against sex selection on a whim.18 It thus seems clear that a unique advantage of PGD lies in it being a pregravid test, one step beyond the prenatal diagnostic tests already in use for several decades. It has been seen as an additional prenatal diagnosis rather than as an alternative.19 A newer dilemma is that of choosing by PGD an embryo free of disease which may also become a child who would be an HLA match to an already sick sibling.20 The term ‘parity for donation’ qualifies this practice, as well as spelling out the particularly difficult dimension of this endeavor. Two different cases may arise: the child conceived by PGD and embryo transfer is also at risk of the disease affecting the older sibling, or is not, and PGD is performed solely for HLA typing. The main argument against this kind of request by the parents is the instrumentalization of the future child, infringing the Kantian categorical imperative, in its second formulation, ‘Act so that you treat humanity, whether in your own person or in that of another, always as an end and never as a means only.’ In HLA-matching cases, the endangered wellbeing of the existing sibling serves as the compelling reason to accept the technique. Even from the point of view of the future child, it may be seen as beneficial to be able to save its sibling as a matter of solidarity. Sensitive counseling may help the parents to foresee difficult events, such as failure of the initial aim. What if the planned child does not save the life of the

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elder sibling? How can the guilty feelings be assuaged in a situation where goodwill was assumed on behalf of a future person. Another problem is that of the acceptability of a motive for the selection of embryos. There the ‘postnatal’ test is useful, as it states that it is ethically acceptable to create a child that can be used for a certain goal if it is acceptable to use an existing child for the same goal. However, the motive of parents’ selfinterest is not acceptable. Finally, a few words about PGD for late-onset genetic cancers, as a license has recently been given to a British center by the HFEA for this very purpose. The ethical arguments are sharpened by the fact that the child is not affected by the disorder, but the adult may be. Uncertainty, which varies with the genetic penetrance, and the possibility of therapeutic improvement during the lifetime of the possibly affected embryo which will become the patient, have meant that this dilemma is perhaps more complicated than others raised by PGD.21 At the time of writing, the French equivalent of the HFEA, l’Agence de la Biomedecine, is holding a series of meetings to thrash out these very issues and advise the French Health Ministry.

Preserving one’s future fertility Cryopreservation of reproductive tissues for possible future use illustrates the difficulties at the interface between research and therapy and of consenting on behalf of others, while freezing oocytes,22 especially for one’s own use in the future, raises complex questions. Sperm cryopreservation has long been routine and helpful to preserve the fertility potential of (often) young men threatened by cancer and iatrogenic sterility. It is widely used worldwide, but posthumous treatment illustrates the different societal interpretations concerning the welfare of the potential child born fatherless. Although it is allowed, with prior counseling and consent of the man preserving his sperm, in the UK,23 to this day in French law, assisted reproduction treatments are solely to be used for a couple’s parenting project, defined as the man and woman which constitute this couple, of a reproductive age, who are alive and legally consenting. At a time when legislation was not yet in place, a refusal by the French Tribunal de Grande Instance to transfer two cryopreserved zygotes to a widow24 was made even more poignant by the fact that a month later, a tribunal in western France allowed the same procedure in a similar case. By contrast, in the UK, posthumous treatment is allowed with explicit prior consent in writing and after the opportunity of counseling has been given to the gamete donor(s). After the Blood case,25 where no consent had been available at the time of sperm retrieval owing to the terminal illness of Mr Blood, a report26 published on the basis of a

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wide consultation document concluded that ‘the importance of the rule requiring consent cannot be underestimated and that no exceptions should be made beyond the current rules which take into account the situations of necessity or which authorise medical intervention where this is in the best interest of the person unable to consent at the time.’ Thus, prior consent is the key to the use of cryopreserved sperm. But in this very particular case, Mrs Blood has been allowed officially by the court to give their father’s name to her two sons conceived with her deceased husband’s sperm; this has paved the way for the recent change which allows the father’s name to be on the birth certificate, with written consent and counseling.27 One important element governing the freezing of oocytes is the degree to which women are able to combine their desire to have children with their other aspirations for education, career, and (partner) relationships. As a result, a growing group of women require medical assistance for reproduction, often resulting in the unsuccessful fulfillment of their desire to have children. When they finally decide to start their families, they have already reached a low point in their ovarian supplies. On the basis of these facts, at least some women may want to make use of the possibility of retaining their fertility by freezing oocytes or ovarian tissue. Recent progress in oocyte freezing, particularly with vitrification, may now change the outlook for women, as one may hope for a much better freeze–thaw success rate. It would also alleviate the dilemmas raised by embryo cryopreservation for those who consider the embryo to have the moral status of a person. However, we face even more complex issues in the case of childhood cancers. About 15% of treatments of childhood and adolescent cancer carry a substantial risk to future fertility. This risk varies according to the presenting pathology and required treatment. For example, testicular cancer and total body irradiation (TBI) have different specific outcomes for the fertility of the patients. Other risks include the possibility of mutagenicity or congenital malformations in the survivors’ offspring, or miscarriage due to pelvic irradiation in the female sufferer. Up until the advent of IVF and related techniques, the only alternative to foregoing one’s future reproductive ability was, for the male, the cryopreservation of sperm prior to treatment. Egg donation, intracytoplasmic sperm injection (ICSI) with ejaculated or testicular gametes, research into oocyte and gonadal tissue freezing, and even the theoretical possibility of human reproductive cloning have since offered new hopes to cancer patients and complicated the ethical and legal issues even further, especially during the crucially vulnerable adolescent period. The first concern is of a psychologic nature. Although the burden of the disease process is often reflected in the compliance problems that many children and adolescents experience, children often

assume that offers are prescriptive, and may view an offer to consider the possibility of storing gametes or gamete tissue as a ‘must’ rather than a ‘may.’ This problem should be elicited during the process of obtaining informed consent. In the legal sense, of course, in the UK we are accustomed to differentiating between the must of legislation, and the ‘should’ of codes of practice from Royal Colleges or other bodies, which sometimes may be cited in court as references or treated as normative. In the ethical sense, it is obvious to many that in spite of all professionals having their duty of care at heart (both ethically with a beneficent and nonmaleficent intent, and legally), this particular field is even more fraught than others, with possible conflict between these duties and the need to respect the autonomy of the child, while also considering the wishes of the child’s parents. Furthermore, the more recent possibility of preserving the reproductive outlook for women was followed with intense media exposure after presentation of the first successful follicular development in autografted, previously cryopreserved, ovarian tissue.28 Other concerns over the years have included the possibility of damage to the offspring linked directly to cryopreservation itself. No extra risk has been observed in the children of adults treated for cancer when they resulted from the use of such cryopreserved gametes, as well as post-radiation/chemotherapy treatment.29 Although there are practical differences for young boys and girls, the child/adolescent is contemplating death and fertility at the same time, as well as potency and reproduction. Testicular tissue freezing may be offered to young boys who do not produce sperm in their ejaculate. In order to obtain consent from a patient, the prerequisite of capacity and understanding are necessary. The legal age of consent for medical treatment is 16 years old, whereas for therapeutic research the proxy must be satisfied that, on reasonable assessment of the risk/benefit ratio of the procedure, it is in the best interest of the child to participate.30 Furthermore, there is in the field of cryopreservation an inherent inequality owing to the unsatisfactory results and technical difficulties in freezing female gametes and ovarian tissue. Thus, while the treatment of males, or the repair in the psychoanalytic sense of the couple’s infertility by artificial insemination of frozen–thawed sperm, is current practice, it may be argued that cryopreservation of prepubertal testicular and ovarian tissue is still research, whether gametes are matured later in vitro or in vivo, or tissues autografted. The consent one needs to obtain from the patient is thus of a different kind, further complicated by the distinction between therapeutic or nontherapeutic research. Meanwhile, while we may be in a transition phase before easier freezing of oocytes and when there is no certainty that ovarian tissue may be used to obtain gametes or as an autograft, adult women or parents have inquired whether oocytes could be stimulated

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and fertilized in vitro as embryos. Specific ethical dilemmas are linked to the freezing of embryos themselves, with regard especially to the duration of cryopreservation and their ultimate fate.31 This is complicated by the use of donor sperm, yet another complex decision, usually made within the context of a couple where the male sterility is absolute and incurable, although by no means exclusively. Here again, different legislative approaches to the treatment of single women reflect different ethical appraisals of the reproductive rights of women and the welfare or interest of the child to be, as set out in the HFEA Code of practice.32 Finally, if a child eventually dies of the initial disease (as do 40% of TBI patients), or suffers a recurrence or a second cancer before being in a couple, provisions must be made to deal with the outcome of frozen gametes or tissue. Might the parents have any access to the gametes which represent the only life potential of a dead child, at a time when grief may be so overwhelming as to distort the natural disquiet about posthumous conception as seen in the recent controversy raised by the Blood case (R. v Blood, 1997)? English legislation allows posthumous treatment after counseling and written consent, but the concept of procreation totally outside a couple and decided by future grandparents rather than parental figures does not seem to be compatible with the account to be taken of the welfare of the child as required by both the Human Fertilisation and Embryology (HFE) Act 199023 and the spirit of the Children Act 1989,33 which places emphasis on parental responsibility rather than parental rights. However, before addressing the eventual use of cryopreserved tissue, it seems appropriate to hope that the same storage regulations for immature as well as mature gametes be applied in the UK, thus closing the present legal loophole in order to protect both patient and offspring. Indeed changes in UK law are subject presently to debates and parliamentary commissions appraisal, and it is likely that the clause requiring to consider the ‘need for a father’ will go: this would put firmly the spirit of the act within women’s reproductive rights context, and also fit in with antidiscriminatory legislation, confirming the practice of many UK centers to treat single women or same sex couples (another practice also forbidden in France, but not in Belgium or Spain).

Stem cells and cloning At this stage, stem cell nuclear transfer (SCNT) represents only a theoretic variation of embryonic stem cell technology in order to avoid recipient rejection occurring with the use of an allogenous source. In the case of ethical analysis of reproductive cloning, the debate has practically died down as regards international disapproval.34 The debate entailed possible threats to the notions of identity, dignity, and unicity of the individual represented by a clone, or threats to

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reproductive autonomy coming from the sense that society would treat the individual as somewhat predetermined. This would entail a lack of liberty, even if relative, but too awesome to contemplate for the future cloned person induced by this increase in genetic determinism. It seems, however, that the most important argument is in the realm of the psychologic arguments: the narcissistic venture of the parent(s) threatens the building of the identity of the child, mostly by decreasing the possibility of separation from the initial model and the child’s autonomy. Some countries, such as the UK and Belgium, have opted to ban cloning in their national laws.5 In the case of the stem cell debate, the embryo is still at the core, because of its symbolic representation of our future. But the repulsion caused almost universally by reproductive cloning has not been universally matched by the same feelings or arguments on the use of stem cells from embryos. At this moment, the number of countries allowing therapeutic cloning is growing.35 In the UK, embryo research has been licensed under strict conditions since the HFE Act 1990, permitting only research linked to reproduction. After a democratic process involving a report by the Chief Medical Officer and a vote in both chambers, new categories were added to the statute on January 31, 2001, allowing this time ‘research for serious disease.’ Interestingly, in a bid to slow the licensing of this new application, a ‘pro-life’ lobby has asked a judge to assess whether an embryo created by SCNT would actually qualify to be such an entity in terms of the HFEA Act 1990. Surprisingly, the High Court actually ruled (November 15, 2001) that this entity is not an embryo under the Act (it would be another kind of embryo, and if so which kind?), but the Law Lords thankfully reversed this decision on appeal last year. Thus, the HFEA may now issue licenses for SCNT and stem cell research from spare blastocysts. An interesting paradigm is the use of affected PGD embryos for such research, as this may satisfy those who do not approve of the destruction of such embryos. Of course, research embryos are eventually destroyed, but they will have hopefully contributed to better health care of actual persons (in the legal sense, where the person must be born alive to deserve this title). Last but not least, the possibilities of embryonic stem cell research raise the question of the origin of human oocytes for such work. We know that such research may help in preclinical safety studies to assess possible risks of new technologies for medically assisted reproduction (e.g. oocyte cryopreservation, in vitro maturation), develop cell therapy, models to study specific genetic diseases, e.g. amyotrophic lateral sclerosis (ALS), or fundamental research in order to study basic biological mechanisms of, for instance, early embryo development. But the possible risks to the egg donor (ovarian hyperstimulation syndrome [OHSS], infection) have already made the titles of several debates.36

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The ethical considerations of our ESHRE Task Force on law and ethics37 have only just been published and stress that the risks for women must be minimized, and that ‘respect for autonomy should be a core principle, provision of counseling by an independent counselor, and that…an additional safeguard is the exclusion of women as donors who are involved in the research project or work in the same department, … or of illiterate women.’ A major concern is of course the compensation offered to the donor. It is advised that ‘the compensation should be given irrespective of the number and the quality of the oocytes retrieved and should depend solely on the efforts made by the subject, and that particular attention should be given to the problem of egg sharing,’ which more specifically applies to the UK where, for pragmatic reasons, a compensation in kind is offered for oocytes. In the case of egg sharing, this compensation consists mostly of a partial or full IVF cycle. Based on the principle that the money is compensation for discomfort and inconvenience suffered during the process, the compensation should be very modest in the case of egg sharing, as these disadvantages were primarily accepted by the patient for her own treatment. Finally, on the problem of internationalization of scientific research in general and stem cell research in particular, ESHRE advises that ‘at all times, the researchers should be able to show that the oocytes used in the protocol were obtained according to the ethical standards. However, strong scepticism about the effectiveness of the control exercised on the practice in clinics in some countries makes this a highly unrealistic rule. Unless appropriate control by independent authorities can be proven, the most effective way to avoid malpractices and trade in oocytes is not to import human oocytes. Simultaneously, no oocytes for research should be collected from women coming from abroad.’ It is also an ethical imperative to consider alternatives to using human oocytes and burdening women, and new developments, highlighted the UK in March 2007, were greeted by many headlines,38 which referred to the creation of an animal–human hybrid and chimera embryos for research purposes. The Government advisory body made the announcement at their most recent plenary meeting, in response to the current debate about whether research that involves the mixing of human and animal material should be permitted in the UK. We know that the HFA Act 1990 is currently under review, and the Government’s White Paper published last December proposes that the creation of hybrid and chimera embryos should be banned in legislation. Furthermore, the HFEA has already received applications from two research teams who want to use cow or rabbit eggs to generate human embryonic stem cells. However, it was decided to defer making a policy decision until a public consultation is carried out. It is also known that the Human Genetics Commission (HGC) will be responding favorably to the HFEA’s

consultation, although several of its members felt that the regulatory framework was already in place to deal with this type of research but were conscious that public reassurance was necessary. Finally, the UK’s House of Commons Science and Technology Committee has conducted an inquiry into the Government’s proposals for banning the creation of animal–human chimera embryos for research purposes, and has backed the possibility of this technique. This resulted in the inclusion of ‘cybrid’ research in the new Bill, and the two licence applications were approved by the HFEA.

Conclusion Arguably, the only appropriate way to conclude this attempt at pulling the strands of ethical concerns, since ART has radically changed the outlook for our infertile patients, is to emphasize our responsibility to the vulnerable and place the interest of the future offspring before that of the patients hoping for reproduction. The profession has to decide, with society, whether our duty to the vulnerable next generation, the planned offspring, is not even stronger than that we have to our patients. Furthermore, this has to be undertaken now in a world which knows no boundaries, and where ‘trans border reproductive care’ (a term much preferable to ‘reproductive tourism’) is ever increasing.39 Whether any other matter related to human reproduction will lead to similar public debate and political activities remains to be seen, but it is reassuring to see the words ‘responsibilities’ and ‘duties’ stressed at international level. This may be seen optimistically as a landmark in the opportunity to communicate with the public at large about science in general, and reproductive science in particular, befitting the context of the Council of Europe Bioethics Convention, which expressed the need for international cooperation: ‘so that all humanity may enjoy the benefits of biology and medicine.’ Whatever the new ethical challenges we are about to face with the ever-changing new technologies of ART, we should certainly strive for this communication to be achieved within all layers of society in order to share the ethical appraisal and assume our joint responsibility, especially towards the vulnerable future child.40

References 1. Shenfield F, Sureau C, eds. Ethical Dilemmas in Assisted Reproduction. New York: Parthenon, 1997. 2. Shenfield F, Sureau C, eds. Ethical Dilemmas in Human Reproduction. New York: Parthenon, 2002. 3. Shenfield F, Sureau C, eds. Current Ethical Dilemmas in ART. London: Informa Healthcare, 2006. 4. lois 2004, Loi no 94-654 du 29 Juillet 1994, Relative au Don, Assistance Médicale à la Procréation et Diagnostic Prenatal. Paris: Journal Officiel du 30 Juillet 1994; Loi no 2004–800 du 6 aout 2004 relative a la bioethique.

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Recent ethical dilemmas in ART 5. Belgium: art.6 de la loi du 11 mai 2003 (recherche sur les embryons in vitro). Moniteur belge, 28 mai 2003: 29288. 6. Daniels K. Donor gametes: anonymous or identified? Best Pract Res Clin Obstet Gynaecol 2007; 21(1): 113–28. 7. Dickey RP. The relative contribution of assisted reproductive technologies and ovulation induction to multiple births in the United States 5 years after the Society for Assisted Reproductive Technology/ American Society for Reproductive Medicine recommendation to limit the number of embryos transferred. Fertil Steril 2007; 88(6): 1554–61. 8. The European IVF-monitoring programme (EIM) for ESHRE. Assisted reproductive technology in Europe, 2002. Results generated from European registers by ESHRE. Hum Reprod 2006; 21(7): 1680–97. 9. Pennings G, Devroey P. Subsidized in-vitro fertilization treatment and the effect on the number of egg sharers. Reprod Biomed Online 2006; 13(1): 8–10. 10. UNESCO International Bioethics Committee. Report of the subcommittee on genetic screening and testing, 1994. Paris: UNESCO, 1994. 11. Sermon KD, Michiels A, Horton G, et al. ESHRE PGD Consortium data collection VI: cycles from January to December 2003 with pregnancy follow-up to October 2004. Hum Reprod 2007; 22(2): 323–36. 12. Modell B, Kuliev AM. Services for thalassaemia as a model for cost-benefit analysis of genetic services. J Inherit Metab Dis 1991; 14: 640–51. 13. Milliez J, Sureau C. Pre-implantation diagnosis and the eugenic debate: our responsibility to future generations. In: Shenfield F, Sureau C, eds. Ethical Dilemmas in Assisted Reproduction. New York: Parthenon, 1997: 51–9. 14. Marteau TM, Croyle RT. The new genetics: psychological responses to genetic testing. BMJ 1998; 316: 693–7. 15. Testard J, Sele B. Towards an efficient medical eugenics: is the desirable always the feasible? Hum Reprod 1995; 11(12): 3086–90. 16. Missa JN. Eugenics. In: Sureau C, Shenfield F, eds. Clinical Obstetrics and Gynaecology. New York: Baillière Tindall, 1999. 17. Pennings G. Family balancing as a morally acceptable application of sex selection. Hum Reprod 1996; 11: 2339–43. 18. Shenfield F. Sex selection: why not! Human Reprod 1994; 9: 569. 19. Pembrey M. Preimplantation diagnosis as an alternative to prenatal diagnosis. Presented at the International Conference on Genetic Diagnosis, from Prenatal to Preimplantation, June 1998, Rennes, France. 20. Pennings G, Liebaers I. Creating a child to save another: HLA matching of siblings by means of preimplantation diagnosis. In: Shenfield F, Sureau C, eds. Ethical Dilemmas in Human Reproduction. New York: Parthenon, 2002: 51–66.

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21. Braude P. Preimplantation diagnosis for genetic susceptibility. N Engl J Med 2006; 10: 541–3. 22. Dondorp W. Freezing the hands of time: fertility insurance for healthy women? In: Shenfield F, Sureau C, eds. Ethical Dilemmas in Human Reproduction. New York: Parthenon, 2002: 1–21. 23. Human Fertilisation and Embryology Act 1990. London: HMSO 1990. 24. Toulouse Tribunal de Grande Instance, Mme Veuve Pires v. CECOS 1993. 25. R. v. Human Fertilisation and Embryology Authority ex parte Diane Blood [1997] 2 All ER 687. 26. McLean S. Review of the common law provisions relating to the removal of gametes and of the provision of consent provisions in the HFEA Act (1998) School of Law, University of Glasgow, 1998. 27. Human Fertilisation and Embryology Deceased Fathers Act 2003. 28. Oktay K, et al. Press release, American Society for Reproductive Medicine, 27 September 1999. 29. Dodds L, Marrett LD, Tomkins DJ, Green B, Sherman G. Case-control study of congenital anomalies in children of cancer patients. BMJ 1993; 307: 164–8. 30. Kennedy I, Grubb A. Research: the incompetent patient. In: Medical Law, Text with Materials, 2nd edn. London: Butterworth, 1994: 1052. 31. ESHRE Taskforce for Law and Ethics. The cryopreservation of frozen embryos. Human Rep 2001; 16: 1049–50. 32. Human Fertilisation and Embryology Authority. 7th Code of Practice. London: HFEA, 2007: www. gov.hfea.uk. 33. Children Act 1989. London: HMSO, 1989. 34. Shenfield F, Babinet C, and Teboul G. Cloning: reproductive crime or therapeutic panacea, where are we now? In: Shenfield F, Sureau C, eds. Current Ethical Dilemmas in ART. London: Informa Healthcare, 2006. 35. Greece: art.1 of law i 3089/2002 (assistance médicale à la reproduction humaine) relatif, notamment, à l’article 1455 du Code civil grec. Website: cie-csg/ Legislationpdf/Grèce 36. Editorial, Safeguards for donors. Nature 2006; 442: 601. 37. ESHRE Task Force on Ethics and Law 12: oocyte donation for non-reproductive purposes. Hum Reprod 2007; 22(5): 1210–13. 38. UK Medical body backs inter-species embryo research. Bionews 20-6-2007: http://www.BioNews. org.uk. 39. Pennings G. International parenthood via procreative tourism. In: Shenfield F, Sureau C, eds. Current Ethical Dilemmas in ART. London: Informa Healthcare, 2006: 43–57. 40. ESHRE Task Force on Ethics and Law 13: the welfare of the child in medically assisted reproduction. Hum Reprod 2007; 22(10): 2585–8.

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Index Page numbers in bold indicate Figures, page number in italic indicate Tables accreditation European Cooperation for Accreditation and, 25–26 European standards for, 25–26 ISO standards and (see International Standardization Organization) management requirements and, 28–30 process, 27, 32–36 technical requirements for, 30–35 (see also International Standardization Organization) activins, 139 age. See maternal age airborne toxicants, 3, 6, 20 air quality, 31 amenorrhea, 460, 470, 542, 721, 762, 808 American Society for Reproductive Medicine, ASRM, 10–11, 812, 823, 864 amniotic sac microenvironment, 779, 781 androgen(s) conversion, 336, 471, 472, 595 follicle levels of, 85, 584, 723 function, 595–98 insentivity, 460 Kallmann’s syndrome and, 346, 347, 348, 349 low responder patient and, 595–98, 595, 599 oocyte quality and, 520 plasma levels, 517, 726 receptors, 346, 347, 491, 726 regulation of, 520 secretion, 323, 490, 490, 491, 493, 503, 511, 513, 533, 595, 595–96, 724 See also hyperandrogenism; testosterone andrology laboratories, 10–12, 18, 21, 46 anejaculation, 171, 657–59, 661, 665 anesthesia acupuncture, 703–4, 704 epidural, 678 equipment, 17–18 general, 465, 659, 663, 678–79, 682, 701–3 ketamine, 703 laparoscopy and, 674, 682 locoregional, 661–62, 666 midazolam, 703 narcotics, 703 neuroleptanesthesia, 703 nitrous oxide, 703 oocyte retrieval and, 674 propofol, 703 regional, 702–3 sedatives, 701–2 testicular biopsy and, 667–68 transvaginal ultrasound-guided oocyte retrieval and, 559, 701, 787 ZIFT and, 682

anovulation, 449–51, 461, 462, 494, 498, 530, 593, 711, 722 anti-Müllerian hormone (AMH) cycles in, 521, 579 follicular levels of, 87–88, 723, 740 regulation and, 111, 521 serum levels of, 87–88 source of, 520–21, 739 apoptosis embryos and, 222, 851 follicles and, 123, 328, 596 oocytes and, 94, 122, 123, 601 sperm and, 68–69 stem cells and, 833 aromatase inhibitors, 336–37, 452, 469, 471–72, 471, 471, 503, 509, 597–98, 611, 613, 717, 723, 731 assay acridine orange, 74 acrosome, 47, 48 chromomycin A3, 72–73 comet, 75–76 hemizona, 48–49, 49 in situ nick translation, 73–74 mannose binding, 49 penetration, 48 sperm chromatin structure, 76–77 terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling (TUNEL), 76, 77 assisted hatching acid Tyrode’s, 183, 193, 194 implantation failure and, 617, 621, 626 indications for, 182 laser-assisted, 168, 183–84, 184, 193, 194, 359 low responder patient and, 168, 182–83, 602–3 repeated implantation failure and, 618, 621–22 results, 186, 187, 602–3 assisted reproductive technologies (ART) access to, 885–88, 888 child development and, 177 embryo culture and (see embryo culture) embryo transfer and (see embryo transfer) endometriosis and (see endometriosis) ethics and (see ethics) funding for, 891 growth in, 241, 275 history of, 859 insurance coverage for, 887 international frequency of, 885, 886, 887, 890, 891 in vitro fertilization and (see in vitro fertilization) laboratory (see assisted reproductive technologies (ART) laboratory) legislation and, 890–92 microfluidics and, 844

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Index

multiple pregnancy and (see iatrogenic multiple pregnancy; multiple pregnancy) nurse (see assisted reproductive technologies nurse) patients, 797–99 patient support for, 867–72, 871, 873 physicians and, 799–802 polycystic ovaries and (see polycystic ovaries) religion and, 888–90 success rates, 578 ultrasound in (see ultrasound) assisted reproductive technologies (ART) laboratory accreditation of (see accreditation) air quality of, 31 building materials for, 5–6 ‘burning in’, 6–7 cleanliness of, 31–32 computers in, 10, 19, 664, 774 design, 2–5 layout, 2, 31 maintenance, 7 personnel, 2, 10–14, 11, 12, 13, 14 quality control and, 19–20 renovation of, 5 testing in, 20 assisted reproductive technologies nurse coordinating role of, 443, 862, 862 counseling role of, 863 history of, 859 management and, 2, 864 patient education and, 861–62 professional organizations for, 865 research, 864 responsibility of, 443, 559, 645, 860–61, 860 training for, 864–65 See also assisted reproductive technologies (ART) laboratory: personnel atresia, 85, 112, 122, 493, 495, 499, 521, 524, 577, 584, 593, 608, 723, 761, 779 autoimmune disease, 335, 808 autologous endometrial coculture, 620, 625, 628–29 azoospermia, 9, 171, 312, 323, 343, 345, 345–48, 346, 451, 460, 465–66, 657–58, 489–90, 660, 662, 664, 666–68 bicornuate uterus, 463, 464, 638–39 blastocyst development, 184, 193, 214, 226, 228, 244, 245, 419, 422, 423, 624, 849, 853 freezing, 277, 281, 282 (see also cryopreservation; slow freezing) hatching (see assisted hatching) metabolism, 248, 249, 250 (see also metabolomics) thawing, 277, 281, 282 transfer, 174–77, 207, 228–29, 230, 231, 623, 686–87, 765, 800 (see also embryo transfer) bleeding intraperitoneal, 787–78 intratesticular, 667 menstrual, 540, 542, 554 retroperitoneal, 787–78 vaginal, 562, 589, 787 bone marrow transplantation, 333, 335, 338, 403 British Human Fertilisation and Embryology Authority, 1 cancer breast, 334, 334–38, 338, 472, 774 cervical, 209, 335, 338, 338 childhood, 78, 312, 335, 898 fertility preservation and, 337 ovarian, 335, 471 cerebral palsy, 241, 707, 795–96, 796, 802 chemokines and chemokine receptors, 427–30, 430

chromosomal abnormalities damaged DNA and, 67 (see also DNA: damage) embryo selection and, 381 incidence, 128, 210, 366, 381–82, 623 maternal age and, 371, 382 polar biopsy and, 357 Clinical Laboratory Improvement Amendments of 1988 (CLIA ’88), 10–11, 15 clomiphene citrate (CC), 85, 336, 449–50, 469–71, 470, 540, 548–49, 554, 584, 673, 712–13, 761, 763, 797, 808 College of American Pathologists, 1, 10, 15 computers, 10, 19, 664, 774 condoms, 40, 54, 78, 673–74, congenital bilateral absence of the vas deferens (CBAVD), 171, 343, 345, 345, 347, 349, 466, 658, 666–67, 669 controlled ovarian hyperstimulation (COH) effectiveness of, 106, 118, 156, 712 endometriosis and, 712 follicle stimulating hormone and, 502 (see also follicle-stimulating hormone) gonadotropin releasing hormone agonists and, 514, 531 oocyte retrieval and, 713–14, 753 ovarian hyperstimulation syndrome and, 759 ovarian reserve and, 87 protocols for, 85, 122, 553, 572, 674 See also ovarian hyperstimulation syndrome controlled rate cooling, 255–58, 289 corpus luteum, 85, 122, 160, 330, 333, 461, 489–90, 519, 565, 592, 711 cryopreservation blastocyst, 229, 231, 279 cancer patients and, 337 DNA damage following, 76 effectiveness, 231, 255, 275, 282–83, 296, 298 embryo (see embryo cryopreservation) epididymal sperm and, 172, 176 equipment for, 4 injury, 260–61, 268, 290, 292, 295 legislation and, 305 oocyte (see oocyte cryopreservation) ovarian tissue and (see ovarian tissue cryopreservation) slow freezing in (see slow freezing) testicular sperm and, 172, 323–25 vitrification in (see embryo vitrification) cryoprotectants cell damage and, 268, 276, 290 characteristics of, 276, 277, 285, 291, 298, mechanism of, 291, 324 types of, 276, 312–13, 334 vitrification and, 268, 269, 291, 294, 314 See also cryopreservation culture medium composition of, 17, 53, 159, 164, 194, 220, 222–24, 223, 224, 225, 248, 838 influence of, 224, 225 monoculture, 224–25 sequential, 224–25 storage of, 3, 227–28 See also culture systems culture systems chemical characteristics of, 226–27 co-, 279, 619, 624–25, 627–28 design of, 29, 142–43, 219, 220, 232, 233 embryo:volume ratio in, 227 incubation chamber in, 225–26 medium for (see culture medium) quality control in, 228 cumulus cell morphology, 88 removal, 89, 105 See also granulosa cell

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Page 905

Index cumulus-oocyte complexes (COCs) apoptosis of, 122, 123 collection of, 122, 123 culture, 142–43 evaluation of, 104, 124–25, 125, 126, 127, 127 morphology, 112 transfer of, 106, 108 cyclic adenosine monophosphate, 111, 113, 116–17, 142, 491 DNA age and, 69 (see also maternal age) assays, 49–50, 73–77, 78, 129, 314, 366, 376, 395, 431, 405, 407, 409 cancer and, 69 contamination and, 314 damage, 57, 67–71, 78, 658, 848 (see also DNA: fragmentation) fragmentation, 67, 78, 316 HIV, 58 microarrays, 393, 395, 408, 431 polar body, 374 polymerase, 73, 406, 407, 412 recombinant, 489, 493 repair, 67–68, 594, 598, 599, 773 sperm, 67–71, 78, 658, 848 staining, 45–46, 71, 71–72, 73, 75, 129, 130 donation. See egg donation; embryo donation Doppler ultrasonography. See ultrasound egg donation clinical outcomes of, 812–13 donor recruitment for, 810 donor screening for, 810–12, 811 endometrial stimulation for, 812 endometrial synchronization and, 812, 813 history of, 807 indications for, 807–9, 808 recipient screening for, 809, 809, 810 elective single embryo transfer (eSET), 207, 708, 800 electroejaculation, 658–61, 665 embryo bank (see embryo bank) biopsy, 193, 195–200, 202, 196 classification, 395 controlled rate cooling of, 255–58, 289 cryopreservation (see embryo cryopreservation) culture (see embryo culture) development, 225, 227, 233–35, 234, 244, 836 donation (see embryo donation) genetic analysis of (see preimplantation genetic diagnosis) metabolomics, 250–51, 251 mouse, 20, 20–21, 23, 29, 182, 188, 191, 196, 200, 220, 223 232, 249, 276, 417–18, 422, 702–3, 830, 832, 849–51 nutrient consumption, 248, 248 physiology, 94, 221, 251, 278, 283–84, 420 proteome (see proteome) quality, 30, 67, 87–88, 95, 106, 134, 228, 242–50, 382, 479, 693, 854 secretome, 421–23, 422, 422, 423 selection, 241–42, 242, 244, 246, 246, 247, 251 stem cells, 827–29, 828, 831, 832–35, 837–39 thawing, 277, 279, 306 transfer (see embryo transfer) transport, 307–8, 308 vitrification (see embryo vitrification) embryo bank consents, 306 embryo infection and, 308–9 embryo management, 307–8, 314

905

laboratory accreditation and, 305 legislation regarding, 305 timing of thawing and, 306–7 embryo cryopreservation cryoprotectants in, 291–92 injury from, 290–91 outcome, 278, 278, 282, 282–83, 717 procedure for, 200, 233, 257, 289–90 quality control in, 283–84 replacement cycles and, 277–78 trouble-shooting, 284–85 See also cryopreservation embryo culture extended, 9, 178, 229 factors in, 220 medium (see culture medium) metabolomics and, 250–51, 251 microfluidics and, 850–53, 851 systems (see culture systems) techniques for, 9 temperature for, 34 embryo donation acceptability of, 813–14 history of, 807 indications for, 807–9, 808 recipient screening for, 809 success of, 814 See also egg donation embryologists, 2, 13 embryonic-endometrial cross-communication, 427–30, 428, 430 embryo transfer (ET) complications from, 787 gentle atraumatic transfer and, 694–95 outcome, 207, 220, 229, 230 procedure, 159–60, 228–29, 231, 693–94, 696–97 pronucleate oocyte and, 242 single (see single embryo transfer) ultrasound-guided, 696 embryo vitrification developmental stage and, 295–96 embryo choice and, 296 outcomes, 298 procedures, 268–71, 268, 269, 269, 270, 271, 292–95, 293, 294, 298–99 species and, 295–96, 296, 297 See also slow freezing endocrine-disrupting chemicals (EDCs), 773, 775, 776 endometrial cavity polyp, 463 endometriosis controlled ovarian hyperstimulation and, 712–13, 714 (see also controlled ovarian hyperstimulation) early embryo development and, 714–15 gamete intrafallopian transfer and, 716 (see also gamete intrafallopian transfer) incidence, 335 infertility and, 711–12 intrauterine insemination and, 712 pregnancy rates and, 713, 713, 715–16, 716 surgery for, 716–17 symptoms, 711 endometriotic cysts, 562, 641–42, 788 environmental toxicants, 3, 6, 20, 773, 774, 775 epidermal growth factor, 114, 117, 119, 135, 138–39, 759 equipment computerized semen analyzers, 18 computers, 10, 19, 664, 774 CO2 control, 17–18 microscopes (see microscopes) microtools, 9, 20, 165, 167–69, 182, 193, 194 pH control, 17–18 temperature control, 16–17

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estradiol actions of, 85, 429, 469, 517–19, 740, 813 cycles, 518, 530, 565–66, 565, 566, 572 levels, 329, 331–32, 336, 475, 494, 642, 682, 722, 725, 761 production, 329, 332, 336, 472, 490, 503, 514, 516, ethics cloning and, 899–900 committees, 307, 366, 808, 815, 817–20, 824–26, 870, 896, future fertility and, 897–99 justice issues and, 895–96 medical laboratory, 26, patient material and, 22 preimplantation genetic diagnosis, 896–97 religion and, 895 stem cells and, 899–900 (see also embryo: stem cells) European Cooperation for Accreditation (EA), 25, 30 European Society for Human Reproduction and Embryology (ESHRE), 27, 821, 881, 892, 896 European Union (EU) Tissues and Cells Directive, 26–27, 33, 37, 313–15 extrauterine pregnancy, 686, 790–91 fallopian tubes assessement of, 464–65, 637, 643 gamete intrafallopian transfer (GIFT) and (see gamete intrafallopian transfer) hydrosalpinx and (see hydrosalpinx) morphology, 642–43 removal of, 626 zygote intrafallopian transfer (ZIFT) and (see zygote intrafallopian transfer) fertility. See infertility; male infertility; subfertility Fertility Clinic Success Rate and Certification Act of 1992, The, 10 fertilization egg morphology and, 106 intracytoplasmic sperm injection and, 176 in vitro (see in vitro fertilization) oocyte competency and, 134 phases of, 103, 209 rate, 47, 49, 54, 57–59, 60, 69–70, 89, 107, 133, 171, 176, 176, 260, 501, 541, 588, 702, 714–15, 722, 723, 727–28, 849 fine-needle aspiration (FNA), 661, 661, 666–67 fluorescent in situ hybridization (FISH), 71–73, 195, 343, 350, 357–58, 371, 393, 618, 622, 829 fluorescent DNA staining, 45 follicles atresia of (see atresia) cryopreservation of, 328 (see also cryopreservation) development of, 489–92, 490, 500–4 hormonal regulation of, 123, 123, 489–92, 490, 491, 511 (see also follicle-stimulating hormone; luteinizing hormone) luteinizing hormone effect on, 513–16 (see also luteinizing hormone) microenvironment of, 778–79 follicle-stimulating hormone action of, 111, 123, 489–90, 511, 591 low responder patient and, 502, 591–92, 591, 592, 593 luteinizing hormone and, 515 obesity and, 723 ovarian reserve and, 741, 808 ovulation induction and, 172, 477, 493, 497, 498, 500, 511, 725 priming, 123–24, 123, 156 purified (pFSH), 477, 477, 493, 500 recombinant, 431, 478–80, 725, 761, 861 serum levels, 85, 327, 490, 500, 530 sperm and, 69, 657 structure of, 591, 591 synthesis and secretion of, 489–90, 529, 591 ‘threshold’, 496, 512, 513 ‘window’, 511, 512 See also gonadotropin(s)

gamete intrafallopian transfer (GIFT) controlled ovarian hyperstimulation and, 674 (see also controlled ovarian hyperstimulation) endometriosis and, 716 frequency of, 891 history of, 673 laproscopic oocyte retrieval in, 675, 676, 677 patient selection for, 673–74 results of, 677–79, 678 transfer catheters for, 675–77 gene therapy, 627, 828, 835 genetic counseling, 2, 343, 347–48, genetic disorders, 177, 358–59, 374, 403, 657, 812, 836–37 genetic screening, 86, 199, 349–50, 398, 410, 454, 577, 601, 810–11, 829, 896 genetic testing. See genetic screening gestational surrogacy acceptability of, 817 complications of, 821–22 counseling for, 819 indications for, 818, 818 legal issues in, 822–23 patient management and, 819–20 patient selection for, 818–19 religious issues in, 824 success of, 820–21 gonadotropin(s) designer, 479–80, 480 high-dose, 579–80, 603 history of, 476, 477 human menopausal, 331–32, 476–77, 559, in vivo oocyte maturation and, 137–38 low responder patients and, 549, 579–80, 603, 723, 761, 808 ovarian stimulation and, 87, 118, 137–38, 156, 491, 494, 511–12, 519, 540, 553, preparations, 492–94 recombinant, 9, 478–80, 493–94, 498, 504 safety of, 480 secretion of, 9, 470 standardizing, 478–79 See also follicle-stimulating hormone; human chorionic gonadotropin; luteinizing hormone gonadotropin-releasing hormone agonists (see gonadotropin-releasing hormone agonist(s)) antagonists (see gonadotropin-releasing hormone antagonist(s)) function of, 529 receptors, 482–83 secretion of, 85, 347, 470, 480, 529 structure of, 480, 481, 483, gonadotropin-releasing hormone agonist(s) amino acid sequence of, 531 clinical applications of, 530–34 dose, 532 low responder patients and, 549, 580–89, 582, 585, 587, 604 luteal suppression and, 278, 500, 502, 516, 517, 593 mechanism of action, 480, 482, 483, 512 outcomes, 550 ovarian hyperstimulation syndrome and, 762, 763 pituitary desensitization and, 172, 475, 554, 724–25, 813 protocol, 86, 532–34, 534, 540–42, 547, 549, 580, 582, 585, 587, 674, 713, 727, 743, 761 synthesis and structure of, 480, 481, 530, 539 gonadotropin-releasing hormone antagonist(s) Cetrotide and, 542, 543, 546 in clinical studies, 539–42 clomiphene citrate and, 549–50 development, 539 endometrial quality, 544 estradiol and, 546 human chorionic gonadotropin and, 546–47, 548 leutinizing hormone and, 545–46, 547, 547

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Index low responder patient and, 549 luteal phase support and, 547–48, 548 mechanism, 483, 483 oral contraceptives and, 542–43, 543, 545 ovarian hyperstimulation and, 331 pregnancy outcome and, 549, 550 protocol for, 540, 540–43, 541, 543–44, 547–48, 548 Good Manufacturing Practice/Good Laboratory Practice guides (GMP/GLP), 26 granulosa cell (GC), 113 coculture, 624 follicle stimulating hormone and, 490–91, 493, 495, 511, 595, 723 function of, 87, 111, 114, 117, 119, 135, 138, 490, 513, 520, 595 luteinizing hormone and, 117, 125, 139, 474, 491, 584, 591, 595 morphology, 112, 113 VEGF and, 760–61 See also cumulus cell growth hormone, 139, 143–44, 151, 454, 494, 585, 594, 598 hatching. See assisted hatching heterotropic subcutaneous grafting, 330–31, 330 HIV. See human immunodeficiency virus Human Cellular and Tissue-Based Products, 10 human chorionic gonadotropin administration of, 118, 142, 172, 260, 330, 463, 645, 722, azoospermia and, 657 ovarian hyperstimulation syndrome and, 759 ovulation induction and, 306, 474, 497, 498, 539, 542, 547, 598, 721, 762 pharmacokinetics, 475, 476 priming, 156 recombinant, 478, 489 structure of, 475 human embryo biopsy procedures, 193, 195–200, 202, 196 human immunodeficiency virus (HIV) ART and, 58, 61, 429 combination antiretroviral (cARV) therapy for, 57 discordant couples and, 58 semen preparation and, 57, 59 human menopausal gonadotropin (hMG), 85, 107, 156, 172, 331–32, 353, 474–76, 475, 493, 495, 540, 559, 579, 606, 678, 821, 861 human proteome (see proteome) hydrosalpinx antibiotic treatment for, 755 definition of, 747 endometrial receptivity and, 750 fluid characteristics, 749–50 interventions for, 750–55, 751, 809 (see also hydrosalpinx: salpingectomy for) imaging, 462, 643, 748, 751 oxidative stress and, 750 pregnancy and, 449, 747–49, 749 salpingectomy for, 751, 752, 752, 753–54, 754 salpingostomy, 754 transvaginal aspiration for, 754, 754 tubal ligation for, 753 hyperandrogenism, 462, 520, 721–23, 727–28, hypogonadotropic hypogonadism, 347, 466, 470, 491, 494, 498–500, 500, 511, 513, 531, 592, 657–58, 724, 725 hysterosalpingo-contrast sonography (HyCoSy), 465 hysterosalpingography (HSG), 464, 620, 643, 747, 788 iatrogenic multiple pregnancy, 795, 796, 797, 800, 803 immunosurgery, 830, 831, 837 implantation assisted hatching and, 617, 621, 626 chemokines and, 428–30, 430 failure (see implantation failure)

907

pregnancy rates following, 177, 243, 280, 617 receptivity to, 431–32 implantation failure aneuploidy screening for, 618, 622–23, 626 blastocyst culture and transfer for, 618–19, 623–24, 625, 626 coculture methods for, 619, 620, 624–25, 625, 626 (see also culture systems: co-) prophylactic salpingectomy for, 617, 619–21, 626 repeated, 391, 617–22, 624, 627, 683 infertility burden of, 878–79 definition of, 451 endometriosis and, 711–12 environmental factors and, 453 history of, 477 incidence of, 459 investigation of, 459–64 male. See male infertility models of, 451–52 nursing, 859–60 treatment (see assisted reproductive technologies) unexplained, 450–51, 467 inhibins, 139, 491, 495, 520 insulin, 138, 472–73, 494, 721, 723–24, 626–27, 765, 776, 832, 835 insulin-like growth factor, 94, 119, 128, 138, 491, 597, 613, 723, 759 insulin resistance and, 460, 472–73, 721–23, 726–27, 765 insurance, 7, 387, 673, 740, 819, 873, 885, 887–89, 891, 896 International Standardization Organization (ISO) definition, 25 9001:2000, 26, 435–37, 436, 441, 15189:2007, 26–34 17025:2005, 26–28, 28, 29–36 intracytoplasmic sperm injection (ICSI) cumulus cell removal for, 105 factors affecting, 176 fertilization rates for, 176 history of, 9, 104 indications for, 171, 450 outcomes, 175–78, 178 procedure for, 104, 172–74, 174, 175 sperm cryopreservation and, 172 sperm handling for, 172–73, 174 intrauterine insemination (IUI), 712 endometriosis and, 712 failure of, 156, 465 indications for, 58, 448, 450, 712 outcome of, 46, 70, 649 in vitro fertilization (IVF) accreditation for, 26 age and, 371, 452–53, 577, 712 anesthesia and (see anesthesia) anovulation and, 449–51, 461, 462, 494, 498, 530, 593, 711, 722 assisted hatching and, 182 criteria for, 709 cryopreservation and, 255, 335 (see also cryopreservation) embryo stem cells and, 828–29 (see also embryo: stem cells) embryo transfer and, 693 (see also embryo transfer) endometriosis and, 447–48 (see also endometriosis) history of, 9, 275, 447, 531, 559, 701, 807 human immunodeficiency virus and (see human immunodeficiency virus) indications for, 447–51, 448, 448 international differences in, 890, 891 legislation and, 305, 888 (see also legal issues) metformin in (see metformin) microfluidics and, 849 (see also microfluidics) multiple pregnancies and, 707–9 (see also multiple pregnancy) organization of, 438, 439, 442

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outcomes, 9, 220, 231, 241, 448, 449, 453–54, 454, 617, 701, 707 polycystic ovaries and (see polycystic ovaries) repeated, 350 sperm preparation for (see sperm preparation) stem cells and, 93 (see also stem cells) stress and, 877 (see stress) surrogacy and (see gestational surrogacy) tubal dysfunction and, 449 ultrasound and, 461 (see also ultrasound) vitrification and, 289 (see also vitrification) See also assisted reproductive technologies in vivo maturation (IVM) activins and, 139 basal medium for, 134–35 clinical outcomes for, 119, 158, 160 clinical protocols for, 122 collection for (see oocyte: collection) description of, 155, 159 energy substrates and, 135–36 glutathione metabolism and, 136, 137 hormonal priming for, 156 hormone requirements for, 137–39 inhibins and, 139 meiosis-activating sterols and, 137 microfluidics and, 848–49 (see also microfluidics) oxygen tension and, 139 patient selection for, 155–56 polycystic ovaries and, 157, 728–29 protein source in, 136 ISO standards. See International Standardization Organization Kallmann syndrome, 346, 347, 348, 349 Kennedy disease, 346, 347, 349 Kruger’s strict criteria, 9 laparoscopy, 463, 465, 559, 626, 673–74, 676, 681, 747, 751, 789, 819 legal issues, 7, 10, 305, 822–23, 890–92 low responder patient androgens and, 595–98, 595, 599 aneuploidy screening and, 601–2, 602 aspirin and, 600–601 assisted hatching and, 168, 182–83, 602–3 follicle-stimulating hormone and, 502, 591–92, 591, 592, 593 gonadotropin-releasing hormone agonists and, 549, 580–89, 582, 585, 587, 604 high-dose gonadotropins and, 579–80, 603 human growth hormone and, 598 luteal phase manipulations and, 599–600 luteinizing hormone and, 592–95, 593, 595 modified natural cycle and, 590–91 natural cycle and, 589–90 oral contraceptive pill pretreatment and, 598–99 ovarian stimulation in, 578–79, 452 luteal support protocols estradiol supplementation and, 567 gonadotropin-releasing hormone antagonists and, 547–48, 548 progesterone supplementation and, 567, 567, 569, 571, 571, 572 recipient cycles and, 571 route of support for, 567–70, 568, 568, 569, 570, 570 stimulated ART cycles and, 571 thaw-transfer cycles and, 571, 572 timing of, 567 vaginal progesterone therapy and, 570–71 luteinizing hormone action of, 490–92, 513–16 ceiling hypothesis and, 491–92, 492, 493, 517 dose, 491–92, 492, 493

folliculogenesis and, 513 gonadotropin-releasing hormone agonists and, 516 (see also gonadotropin-releasing hormone agonist(s)) granulose cells and, 117, 125, 139, 474, 491, 584, 591, 595 low responder patient and, 592–95, 593, 595 (see also low responder patient) ovulation induction and, 492, 492, 504, 592 pharmacokinetics, 475, 515 serum levels of, 474, 530 threshold hypothesis and, 516, 517, 593 male factor infertility, 155, 160, 171, 316, 323, 450, 455, 674, 678–79, 685, 717, 725, 770, 790, 678 male infertility chromosomal aberrations and, 343, 344, 345, 346, 347, 348 congential bilateral absence of the vas deferens (CBAVD) and (see congenital bilateral absence of the vas deferens) cystic fibrosis and, 347 genetic testing for, 349–50 Kallmann syndrome and, 346, 347, 348, 349 Kennedy disease and, 346, 347, 349 myotonic dystrophy and, 346, 347, 349, 374, 405, 408, 836, 408, 836 Noonan syndrome and, 348 primary ciliary dyskinesia and, 346, 347 Yq11 and, 343, 345, 349, 466 maternal age aneuploid oocytes and, 377 birth rates and, 577–78, 738 chromosomal abnormalities and, 371, 382 ovarian reserve and (see also ovarian reserve) polar body biopsy and, 366–67, 367 (see also polar body) pregnancy rates and, 577–78, 738 reproductive age (see reproductive aging) meiosis arrest of, 103, 111, 112, 113–18, 135, 141, 141–42 M-phase promoting factor (MPF) and, 114, 114–15, 116 oocyte, 85, 103, 107, 113, 116, 142 progression stages of, 132 resumption of, 113–18, 114, 115, 116, 134, 137–38, 209, 474, 778 meiotic spindle (MS) cryopreservation and, 260–61 morphology, 89–91, 90, 91, 107, 107, 127 oocytes quality and, 90, 90–91, 92 retardance, 92, 92 menstrual cycle anovulation and, 449 (see also anovulation) bleeding, 540, 542, 554 irregular, 449, 459, 721 monitoring, 156–57 oocyte collection and, 156–57 phases, 85, 471, 490, 491, 520, 577, 599, 696 regulation of, 489, 704 mercury contamination, 779, 780 metabolomics, 250–51, 251, 395, 423 metformin diabetes and, 9, 472 effectiveness of, 472–73, 474, 727–28, 728, 765 mechanism of, 727–28 ovulation stimulation by, 472 pharmacokinetics, 473–74 microinjected oocytes fallopian transfer (MIFT) and, 683 microfluidics andrology and, 847–48 characteristics of, 843–44, 844 devices for, 844, 845, 847, 848, 850, 851, 852 embryo culture and, 844–45, 850–53, 851, 853–54 embryo manipulation and, 846–48 in vitro fertilization and, 844, 849–50 non-invasive viability assessment and, 854

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Index micromanipulation blastocysts and (see blastocyst: transfer) cumulus-oocyte complexes, 106, 108 embryo (see embryo transfer) environmental conditions for, 163 equipment for, 164–67, 164, 165, 166, 167, 168 history of, 163 laser-assisted, 168 media and, 164 procedure for, 169 system components, 163 vibration and, 167–68, 168, 169 See also gamete intrafallopian transfer; zygote intrafallopian transfer microscopes dissecting, 5, 105, 325 inverted, 5, 78, 105, 108, 125, 127–28, 163–65, 168, 172–73, 182–83 laboratory design for, 5 Polscope, 107, 127, 133, 257, 260 quality control of, 18 stereo-, 125–26, 165, 165, 269–70, 284, 291, 361, 410, 837–38 microsurgical epididymal sperm aspiration (MESA), 57, 63, 171, 345, 661, 668–69 microtools, 9, 20, 165, 167–69, 182, 193, 194 multifetal pregnancy reduction (MFPR), 795, 796, 797, 797, 803 multiple pregnancy avoidance of, 9, 286, 367, 682 causes of, 450, 676 high-order (HOMP), 796–97, 799, 801–2 iatrogenic (see iatrogenic multiple pregnancy) incidence of, 451, 471, 495, 495–96, 497, 682, 685–86, 796 mortality rates in, 802 neonatal phase of, 801 perinatal practice and, 803 pregnancy phase of, 800–801 See also multifetal pregnancy reduction myotonic dystrophy, 346, 347, 349, 374, 405, 408, 836, 408, 836 National Committee for Clinical Laboratory Standards (NCCLS), 14 nonobstructive azoospermia (NOA), 323, 343, 349–50, 466, 657, 662, 668 Noonan syndrome, 348 obesity, 455, 459, 635, 721, 723–24, 726–28, 742 oligoasthenoteratozoospermia, 59, 343 oligomenorrhea, 459, 470, 640, 721 oocyte aging, 95, 133–34, 375, 384 collection, 157, 159, 159, 559–61, 560, 560, 561, 562–64, 562 competence, 87, 94–95, 134, 138, 491, 594 cryopreservation (see oocyte cryopreservation) culture, 119, 120–21, 134, 855 cumulus cell removal, 89, 105 (see also cumulus cell) cytoplasmic inclusions, 93, 93 cytoplasmic maturity, 130–32, 131, 132, 140–42 denuded, 89, 105, 108, 118, 133, 135, 139, 149, 223 development, 86, 88–89, 93–94, 112, 131, 134, 142, 208, 257, 263, 491–92, 493, 592–98, 797 dimensions, 132 fertilized, 53, 134, 176, 207–9, 210, 214, 214–15, 255, 262, 625, 828 giant, 93, 94 in vivo maturation of (see in vivo maturation) maturation, 103, 113, 118–19, 133, 208 (see also oocyte: nuclear maturation) meiosis of, 85, 103, 107, 113, 116, 142 morphology, 89, 92, 100–101, 107, 125, 208, 209–10, 214 nuclear maturation, 127–28, 128, 128–30, 130, 141, 142 nucleolar precursor body (npb) of, 210–14, 211, 213, 214

909

polar bodies of (see polar body) pronucleate, 221, 223, 226, 233–34, 242, 306–7 quality evaluation, 85, 87, 88, 89–95, 92, 95 retrieval, 106, 119–20, 124, 713–14, 753 selection, 86, 93, 93, 257–58 vitrification (see oocyte vitrification) volume, 113, 256, 256, 259 oocyte-corona-cumulus complex, 88–89, 88 oocyte cryopreservation blastocyst, 229, 231, 279 controlled rate cooling in, 255–58, 289 disease transmission in, 271 effectiveness of, 255, 262–63, 263, 337 embryo, 336–37 embryo bank (see embryo bank) history of, 255, 267 indications for, 267 injury from, 260–61, 268, 290, 292, 295 insemination following, 260–61 laboratory design and, 4–5 oocyte preparation for, 261 oocyte selection for, 257–58 (see also oocyte selection) oocyte size and, 267 oocyte survival following, 259–62, 263 outcome, 258, 263, 271–72, 337 protocol for, 259 reproducibility, 258–59 slow freezing and (see slow freezing) timing of, 260–61 vitrification, 268–71, 268, 271, 394 See also cryopreservation oocyte vitrification contamination and, 271 outcomes, 271–72 See also oocyte cryopreservation oogenesis, 111, 130, 140 oophorectomy, 332, 334–35, 338 ovarian hyperstimulation syndrome (OHSS) classification, 759, 760 etiology, 759–61 gonadotropin releasing hormone and, 763–64 gonadotropin releasing hormone antagonists and, 764 human chorionic gonadotropin and, 762 paracentesis and, 767, 769 prevention of, 761–65 recombinant luteinizing hormone and, 762–63 risk factors for, 155, 462, 473, 491, 541, 567, 724, 761, 810 symptoms, 759, 760 treatment, 119, 766–69, 767, 768 ovarian reserve in vivo maturation and, 155 increasing, 604 indicators of, 521, 554, 579, 588, 594, 738, 743 maternal age and, 502, 678, 737, 740 testing, 617, 738–43, 741, 741, 742, 808 ovarian stimulation aromatase inhibitors for, 336–37, 471–72, 471, 471 clomiphene citrate for, 85, 469–71, 470 follicle stimulating hormone and, 554 (see also follicle-stimulating hormone) gonadotropins and, 476 (see also gonadotropin(s)) history of, 539 hyperstimulation and (see controlled ovarian hyperstimulation; ovarian hyperstimulation syndrome) human chorionic gonadotropin and, 474–76, 475, 475, 476, 478–80, 480, 540 (see also human chorionic gonadotropin) human menopausal gonadotropin and, 476–77, 477 insulin resistance and, 727–28 low responder patient and, 578–79, 452

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metformin and (see metformin) obesity and, 727–28 (see also obesity) protocols for, 86–87, 107 purified follicle-stimulating hormone (pFSH) and, 477, 493, 500 steroid profiles and, 516–17 transfer outcome and, 220 ovarian tissue cryopreservation clinical trials of, 330–32 indications for, 334–35 outcomes, 332–33 patient age and, 333 pros and cons of, 328 protocols for, 330 strategies for, 338 tissue collection for, 334–35 viability assessment in, 334 See also cryopreservation ovarian tumors, 335, 338 ovaries blood flow in, 462, 614, 641–42 cryopreservation of (see ovarian tissue cryopreservation) cysts in, 156, 461, 470, 533, 542, 581, 641–42, 641, 642, 644 (see also polycystic ovaries) egg numbers in, 107 polycystic (see polycystic ovaries) primordial follicle oocytes of, 327, 328, 330 regulation of, 138, 267, 469, 452, 477, 489, 492, 495, 511, 762 reserve of (see ovarian reserve) stimulation of (see ovarian stimulation) transplantation of, 327–32, 329, 332, 333 transposition of, 337–38 xenografting of, 327, 329–30, 337, 338, 339 ovulation induction aromatase inhibitors for, 337, 472 chorionic gonadotropin and, 539, 542, 547, 598, 721, 762 follicle stimulating hormone for, 172, 477, 493, 725 insemination and, 712 luteinizing hormone and, 492, 492, 504, 592 multiple pregnancies and, 795 polycystic ovary and 722–24 (see also polycystic ovaries) procedures for, 494–96, 495, 497, 497, 498–500, 500 stromal blood flow and, 642 tamoxifen for, 335–38 See also superovulation partial zona dissection (PZD), 104, 183, 191, 199, 618, 621 patient history, 39 infertility burden of, 878–79 low responder (see low responder patient) partner relationship with, 878 self-esteem, 798, 863, 867–68, 878, 880 sexuality and, 867, 878 support, 867–72, 871, 873 pelvic inflammatory disease (PID), 460, 673, 787–90 penile vibratory stimulation, 658, 659, 660–61, 665–66 pentoxifylline, 57, 64, 172, 316, 663, 766, percutaneous epididymal sperm aspiration (PESA), 57, 63, 171, 345, 661, 661, 666 perifollicular vascularization, 87, 94 perivitelline space (PVS), 86, 89, 92, 93, 105–7, 132, 183, 195, 209, 269, 359 personnel, 2, 10–14, 11, 12, 13, 14 phosphodiesterases (PDEs), 115, 117, 117, 140, 142 polar body biopsy (see polar body biopsy) first, 86, 92, 103–4, 104, 107, 129, 132, 192, 195, 208, 257, 357–58, 363, 363, 364, 365, 377, formation, 209

oocyte quality and, 208 testing, 371, 372, 373, 374, 622 polar body biopsy advanced maternal age and, 366–67, 367 aneuploidy screening and, 357–58, 361–63, 362, 363 chromosomal disorders and, 375, 377, 378 fluorescent in situ hybridization analysis and, 363–66, 364, 365, 366 (see also fluorescent in situ hybridization) laser-assisted, 366 monogenetic aberrations and, 358–59 procedure, 194–95 single-gene disorders and, 374–75, 376 structural chromosomal aberrations and, 358 techniques for, 359–60, 360, 361 polychlorinated biphenyls (PCBs), 773–76 polycystic ovaries (PCO) diagnosis, 721–22 in vitro fertilization and, 155–56, 640–41, 722–24, 723 ovulation stimulation and, 494–96, 495, 497, 497, 498, 722, 722, 724–26 pregnancy rates with, 157, 722, 723 prevalence of, 721–22 ultrasound of, 462, 640, 641 See also ovaries polycystic ovary syndrome (PCOS), 449, 462, 721, 765, 871. See also polycystic ovaries pregnancy embryonic stage and, 177 endometriosis and, 713, 713, 715–16, 716 extrauterine, 686, 790–91 gonadotropin-releasing hormone antagonist(s) and, 549, 550 hydrosalpinx and, 449, 747–49, 749 iatrogenic multiple (see iatrogenic multiple pregnancy) implantation and, 177, 243, 280, 617 in vitro fertilization and (see in vitro fertilization) maternal age and, 577–78, 738 multiple (see multiple pregnancy) polycystic ovaries and, 157, 722, 723 potential, 280–82 repeated loss of, 391–93, 393 stress and, 177, 704, 879 preimplantation genetic diagnosis (PGD) abnormalities detected by, 375, 385 aneuploidy screening and, 350–51, 391, 618, 622, 626, array comparative genomic hybridization (CGH) and, 408–9, 409 cell choice for, 171, 382–83, 395 cell treatment for, 383–84, 411 comparative genomic hybridization (CGH) and, 393–95 DNA microarrays and, 395 ethical issues in, 896 indications for, 192, 368, 391–93 intracytoplasmic sperm injection and, 348, 664 methods for, 382–86, 388, 410–12 (see also specific method) mosaicism and, 386, 387 mutation analysis and, 407–8, 669 outcome of, 381, 385–86, 386, 387, 388–90, 389, 390, 391, 393, 394 polar body biopsy and, 371, 372, 374 (see also polar body biopsy) polymerase chain reaction and, 403–5, 411–12 polymorphic markers and, 406–7 repeated pregnancy loss (RPL) and, 391–93, 393 spontaneous abortion and, 387–88, 392 trisomic offspring and, 386–87 whole genome amplification for, 405–6, 407, 411–12 primary ciliary dyskinesia, 346, 347 process testing, 10, 19–22

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Index progesterone action, 519, 138, 472, 547, 565, 695 cycles, 277, 331, 519–20, 565–66, 565, 566, 572 receptor, 431 serum levels, 332, 461, 566, 789 synthesis, 592, 596, 597 treatment with, 278, 382, 470, 567, 570–71, 763–65, 807, 812–13, 864 protein profiling, 419, 420–21 proteome complexity, 417 embryonic, 250, 417–20, 419, 420, 421 oxygen effects on, 227 protein profiling and, 419, 420–21 quality assurance, 11, 25, 34–35. See also International Standardization Organization; quality control; quality management) quality control (QC) definition of, 10–11 equipment and, 15–19, 16, 19 (see also equipment) goal of, 11 laboratory personnel and, 11 materials and supplies, 19–20 procedures for, 14–15, 15 record keeping in, 11, 18–19 See also quality assurance; quality management quality management (QM) audits in, 441–42 Deming cycle in, 437 document control in, 440–41, 440 employee training and, 443 forms of, 435–37 implementation of, 439 incidents and complaints and, 442 quality policy and, 437 management and, 437–38, 441–43, 442 processes and, 438–42, 439, 441 total, 437, 437 reactive oxygen species (ROS), 69, 136, 137, 661, 750, 848 repeated implantation failure (RIF), 391, 617–22, 624, 627, 683 repeated pregnancy loss (RPL), 391–93, 393 reproductive aging fertility decline in, 737–38 ovarian reserve and, 737, 737 ovarian tissue banking and, 338 variability in, 737, 738 See also maternal age salpingectomy, 449, 563, 617, 619–21, 626, 643, 750–51, 751, 752, 752, 753–55, 754 salpingostomy, 643, 753–55 semen analysis, 39–44, 40, 43, 44, 172, 466 characteristics, 465 collection, 40, 54, 54, 55–56, 171–72 cryopreservation (see semen cryopreservation) handling, 40–41 laboratory, 3–4 selection, 172 source, 465 See also sperm semen cryopreservation CBS straws and, 313, 316 cross-contamination in, 314 cryoprotectants for, 312 extenders for, 312 history of, 311 methods for, 311–13, 315, 317

911

outcome, 314 patient screening for, 315 physiological effects of, 311 thawing in, 313–14, 317 vapor pressure freezing in, 317 vitrification and, 313 seminal plasma microenvironment, 776–78, 778 single blastocyst transfer (SBT), 205, 220, 231, 708, 765 single embryo transfer (SET), 200, 207, 219–20, 231, 231, 241, 243, 251, 454, 454, 664, 707–9, 795, 797, 800, 887, 892 slow freezing ice crystal formation and, 289–90 outcome, 278–80, 279, 295–96, 298 prevalence of, 289 protocols, 200 solutions for, 277 vitrification and, 296–97, 298, 329 (see also embryo vitrification) sperm abnormalities, 39, 43, 44, 53, 69–70 analyzers, 18 antibodies, 42, 45–46, 56, 460, apoptosis in, 68–69 assessment (see sperm assessment) characteristics, 9, 42, 47, 49–50, 67–68 concentration, 41 cryopreservation (see sperm cryopreservation) damage, 53 evaluation of (see sperm assessment) freezing (see sperm cryopreservation) injection (see intracytoplasmic sperm injection) kinematics of, 44, 45, 45, 53 motility, 41–42, 45, 173, 174 oxidative stress and, 69 preparation (see sperm preparation) recovery (see sperm collection) testicular (see testicular sperm) vitality, 42, 46–47 See also semen sperm assessment assays for, 47–49, 48, 49, 72–77, 77 cancer patients and, 71 for chromosomal abnormalities, 71 diagnosis of male infertility and, 70 dispersion test, 74–75, 75 DNA, 49–50, 67, 70 8-hydroxy-2-deoxyguanosine measurement and, 77–78 embryonal loss and, 70 fluorescent in situ hybridization and, 73 in vitro fertilization and, 70 stains for, 45, 71–72, 72 video recording of, 18 sperm collection electroejaculation for, 658–61, 665 fine-needle aspiration (FNA) for, 661, 661, 666–67 indications for, 658 intracytoplasmic sperm injection for (see intracytoplasmic sperm injection) microsurgical epididymal sperm aspiration (MESA) and, 57, 63, 171, 345, 661, 668–69 penile vibratory stimulation for, 658, 659, 660–61, 665–66 percutaneous epididymal sperm aspiration, (PESA) and, 63, 171, 345, 661, 661, 666 sites for, 54 testicular biopsy for, 63, 662–63, 663, 667–68 (see also testicular sperm: extraction) sperm cryopreservation effectiveness of, 314 history of, 311, 323 packaging for, 313–15

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procedure, 63–64, 172, 267, 324–25 selection for, 311 See also cryopreservation sperm preparation complications in, 59 density gradient and, 56–57, 62 DNA fragmentation and, 67, 78, 316 effectiveness, 59 HIV-infected men and, 57 immobile samples and, 57 media for, 46, 64, 659, 661, 665, 67–68 pentoxifylline and, 64 surgical aspirates and, 57 techniques for, 53, 54, 55, 56, 56, 59, 61–62, 70, 78, 439, 848 stem cells, 93, 827–29, 828, 831, 832–35, 837–39, 899–900 stress alternative medicine and, 881–82 behavioral medicine and, 879–80 embryo and, 221–22, 248, 454, 850–51 osmotic, 258–59, 290 oxidative, 68–69, 94, 421, 750, 854 pregnancy outcome and, 177, 704, 879 psychological, 604, 704, 798, 809, 863, 868–69, 871–72, 877–78 sources, 877–79 subfertility, 40, 67, 78, 451, 466, 471, 479, 598, 716, 713, 738, 775, 779, 781 superovulation, 104, 118–19, 125, 127, 131–32, 312, 466, 500–504, 678. See also ovulation induction tamoxifen, 335–38 testicle(s) atrophy of, 343, 347, 460, biopsy of, 45, 54, 57, 63, 171, 465–66, 661, 662, 662, 663, 663, 667–68 cancer of, 69, 312, 664, 898 failure of, 460, 466, 657–58, 661, 664, 668 hyperthermia of, 70 sperm from (see testicular sperm) testicular fine needle aspiration (FNA), 347 testicular sperm cryopreservation of, 172, 323–35 extraction (TSE), 63, 171, 345, 350, 657–58, 661–63, 662, 663, 861 (see also testicle(s): biopsy of) fertilization with, 59 retrieval (see testicular sperm: extraction) testosterone conversion, 336, 472, 597 follicular, 723 forms of, 596 serum levels, 466, 579, 723, 727 synthesis, 332, 347, 472, 597 thawing. See blastocyst: thawing; embryo: thawing toxicants, 3, 6, 20, 773, 774, 775 transvaginal ultrasound-guided oocyte retrieval (TUGOR), 674, 677, 701 trihalomethanes (THMs), 775–76

trophectoderm sampling. See embryo: biopsy tubal ligation, 624, 711, 753–54, 754 ultrasound complications of, 787–88 embryo transfer and, 696 endometrial assessment by, 642, 648–52, 649, 650 equipment, 124, 461, 464, 561, 562, 640 fallopian tube assessment by, 642–43 fetal evaluation by, 176, 222, 277, 279 follicular development and, 119, 461, 496, 553–55, 644–48, 645, 646, 647, 760 history of, 477, 635, 652 ovary assessment by, 156, 519, 555, 578, 640, 640–42, 641, 642, 643, 721, 722, 748, 760, 762, 820 ovulation and, 553, 643–44 procedures, 22 techniques, 635, 636, 637, 637–38, 638 transvaginal, 124, 140, 461, 554, 559, 563, 652, 674, 675, 677, 787, 809 uterus assessment by, 637–39, 638, 639, 760 vanishing twin syndrome, 708, 795 vascular endothelial growth factor (VEGF), 724, 727, 759–60, 761, 765 vitrification blastocyst, 168 cryoprotectants and, 268, 269, 291, 294, 314 embryo (see embryo vitrification) oocyte (see oocyte: vitrification) semen cryopreservation and, 313 slow freezing and, 200, 261, 268, 296–97, 298, 329 volatile organic compounds (VOCs), 3, 20, 31, 34, 227 xenografting, 327, 329–30, 337, 338, 339 zona pellucida (ZP) binding, 53, 103, 466 composition, 181 fertilization and, 39, 42, 48, 103, 106, 130, 133, 183 glycoproteins of, 18 hardening, 181, 260, 268 hatching and, 168, 181, 602 penetration of, 191, 194–96, 196, 278, 359, 383 structure, 91 thinning, 184–85, 602 zygote intrafallopian transfer (ZIFT) advantages of, 679, 681 clinical issues in, 686–87 history of, 679–81 indications for, 683–85 microinjected oocytes fallopian transfer (MIFT) and, 683 procedure for, 682 success rates of, 680, 681 transcervical tubal transfer procedures for, 682–83 zygote selection for, 682 zygotic splitting, 795–97, 800

d ir on Th iti Ed

Gardner Weissman Howles

Textbook of Assisted Reproductive Technologies

Shoham

Laboratory and Clinical Perspectives

From reviews of previous editions: ‘Sampling liberally from the wealth of knowledge contained between its covers will be rewarded by affirming knowledge already garnered from experience or, better, augmenting knowledge to improve one’s understanding and practice through exposure to a fresh perspective’ Fertility and Sterility ‘The book’s real value is that it is standing on our shelf in the clinic. We discuss a day-to-day problem in the unit and immediately know where to look’ OBGYN Contains sections on: Establishing and Maintaining an IVF Laboratory • Gamete Collection, Preparation and Selection • Micromanipulation • Culture, Selection and Transfer of the Human Embryo • Cryopreservation • Diagnosis of Genetic Disease in Preimplantation Embryos • Implantation • Quality Management Systems • Patient Investigation and the Use of Drugs • Stimulation Protocols • Technical Procedures and Outcomes • Special Medical Conditions • Complications of Treatment • Egg Donation and Surrogate Motherhood • Future Directions and Clinical Applications • The Support Team • Ethics and Legislation

With over 300 color and black-and-white illustrations

David K Gardner DPhil is Chair of Zoology at the University of Melbourne, Australia, and Scientific Director at the Colorado Center for Reproductive Medicine, USA

Ariel Weissman MD is a senior physician at the IVF Unit, Department of Obstetrics and Gynecology, Edith Wolfson Medical Center, Holon, and Sackler Faculty of Medicine, Tel Aviv University, Israel

Colin M Howles PhD, FRSM

is Vice President, Scientific Affairs Fertility, Global Medical Affairs, Merck Serono International SA, Geneva, Switzerland

Textbook of Assisted Reproductive Technologies

A truly comprehensive manual for the whole team at the IVF clinic, this covers both laboratory aspects and their clinical application. Methods, protocols and techniques of choice are presented by eminent international experts. The third edition has been extensively revised, with the addition of important new chapters on developing techniques.

Textbook of Assisted Reproductive Technologies Laboratory and Clinical Perspectives

Third Edition

Edited by

David K Gardner Ariel Weissman Colin M Howles Zeev Shoham

Zeev Shoham MD is Director, Reproductive Medicine and Infertility Unit, Department of Obstetrics and Gynecology, at Kaplan Medical Center, Rehovot, Israel

Third Edition

ISBN 978-0-415-44894-9

Special Edition