Textbook of Assisted Reproductive Techniques: Laboratory and Clinical Perspectives

Textbook of Assisted Reproductive Techniques Textbook of Assisted Reproductive Techniques Laboratory and Clinical Per...

Author: David. K Gardner | Ariel Weissman | Colin M. Howles | Zeev Shoham | David K. Gardner

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Textbook of Assisted Reproductive Techniques

Textbook of Assisted Reproductive Techniques Laboratory and Clinical Perspectives EDITED BY

David K Gardner, DPhil Scientific Director Colorado Center for Reproductive Medicine Denver, USA Ariel Weissman, MD IVF Unit Department of Obstetrics and Gynecology Edith Wolfson Medical Center Holon, Israel Colin M Howles, PhD, FRSM Corporate Medical Vice President, Reproductive Health

Serono International SA Geneva, Switzerland Zeev Shoham, MD Director, Reproductive Medicine Unit Department of Obstetrics and Gynecology Kaplan Medical Center Rehovot, Israel

MARTIN DUNITZ © 2001 Martin Dunitz Ltd, a member of the Taylor & Francis group First published in the United Kingdom in 2001 by Martin Dunitz Ltd, The Livery House, 7–9 Pratt Street, London NW1 0AE Tel: +44 (0) 20 7 482 2202 Fax: +44 (0) 20 7 267 0159 E-mail: [email protected] Website: http://www.dunitz.co.uk/ This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge's collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” 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 drug doses and other information are presented accurately in this publication, the ultimate responsibility rests with the prescribing physician. Neither the publishers nor the authors can be held responsible for errors or for any consequences arising from the use of information contained herein. For detailed prescribing information or instructions on the use of any product or procedure discussed herein, please consult the prescribing information or instructional material issued by the manufacturer. 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. A CIP record for this book is available from the British Library.

ISBN 0-203-42819-6 Master e-book ISBN

ISBN 0-203-44799-9 (Adobe e-Reader Format) ISBN 1-85317-870-5 (Print Edition) Distributed in the USA by Fulfilment Center Taylor & Francis 7625 Empire Drive Florence, KY 41042, USA Toll Free Tel: 1–800–634–7064 Email: cserve@routledge_ny.com Distributed in Canada by Taylor & Francis 74 Rolark Drive Scarborough Ontario M1R 4G2, Canada Toll Free Tel: 1–877–226–2237 Email: [email protected] Distributed in the rest of the world by ITPS Limited Cheriton House North Way, Andover Hampshire SP10 5BE, UK Tel: +44–(0)1264 332424 Email: [email protected]

Contents Preface

xii

Acknowledgments

xiii

List of contributors

xiv

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

1

LABORATORY PROCEDURES Setting up an ART Laboratory 1 2 3

4

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

25

Quality control in the IVF laboratory Klaus E Wiemer, Anthony Anderson, Leslie Weikert

38

Accreditation of the ART laboratory: the North American perspective Brooks A Keel, Tammie K Schalue

49

Accreditation of IVF laboratories: the European perspective Cecelia Sjöblom

66

Handling of the Sperm 5 6

Evaluation of sperm Kaylen M Silverberg, Tom Turner Sperm preparation techniques Gordon Baker, Harold Bourne, David H Edgar

86 112

Handling of the Oocyte 7 8

Oocyte treatment: from egg retrieval to insemination Thomas B Pool, Virginia A Ord

129

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

142

9

Oocyte in vitro maturation Johan Smitz, Daniela Nogueira, Rita Cortvrindt, Daniel Gustavo de Matos

156

Manipulation 10

11

12 13

14

Equipment and general technical aspects of micromanipulation of gametes and embryos Frank L Barnes

214

ICSI: technical aspects Gianpiero D Palermo, Ricciarda Raffaelli, June J Hariprashad, Queenie V Neri, Takumi Takeuchi, Lucinda Veeck, Zev Rosenwaks

230

Assisted hatching Anna Veiga, Irene Boiso

248

Cytoplasmic fragmentation in human embryos in vitro: implications and the relevance of fragment removal Mina Alikani

264

Human embryo biopsy for preimplantation genetic diagnosis Alan H Handyside

291

Handling of the Embryo and Embryo Transfer 15 16 17

Analysis of fertilization Lynette Scott

308

Embryo culture David K Gardner, Michelle Lane

324

Evaluation of embryo quality: a strategy for sequential analysis of embryo development with the aim of single embryo transfer Denny Sakkas

359

Cryopreservation of Gametes and Embryos 18 19 20 21

Oocyte cryopreservation Eleonora Porcu

375

Cryopreservation of human embryos Jacqueline Mandelbaum, Yves JR Ménézo

392

Managing the cryopreserved embryo bank Phillip Matson, Neroli Darlington

416

Cryopreservation and storage of sperm Eileen A McLaughlin

423

Eileen A McLaughlin 22

23

Handling and cryopreservation of testicular sperm William W Lin, Benjamin Hendin, Dolores J Lamb, Larry I Lipshultz

443

Ovarian tissue cryopreservation and transplantation Kutluk H Oktay

450

Diagnosis of Genetic Disease in Preimplantation Embryos 24

25 26 27

Severe male factor: genetic consequences and recommendations for genetic testing Ingeborg Liebaers, André Van Steirteghem, Willy Lissers

459

Chromosome abnormalities in human embryos Santiago Munné, Mireia Sandalinas, Jacques Cohen

481

Genetic analysis of the embryo Yuval Yaron, Ronni Gamzu, Mira Malcov

518

Polar body biopsy Yury Verlinsky, Anver Kuliev

539

Implantation 28

29

Embryonic regulation in the process of implantation Jose Luis de Pablo, Marcos Meseguer, Pedro Caballero-Campo, Antonio Pellicer, Carlos Simón

551

The use of biomarkers for the assessment of uterine receptivity Bruce A Lessey

572

Data Management and Interpretation 30

31

Data management and interpretation—computerized database for an ART clinic: hardware and software requirements and solutions Giles Tomkin, Jacques Cohen

599

Evidence based medicine Salim Daya

623

CLINICAL APPLICATIONS AND PROCEDURES Patient Investigation and the Use of Drugs

32 33 34

35

36

Indications for IVF treatment: from diagnosis to prognosis Nicholas S Macklon, Math HEC Pieters, Bart CJM Fauser

643

Initial investigation of the patient (female and male) Bulent Gulekli, Tim J Child, Seang Lin Tan

657

Drug used for controlled ovarian stimulation: clomiphene citrate and gonadotropins Zeev Shoham

677

Inducing follicular development in anovulatory patients and normally ovulating women: current concepts and the role of recombinant gonadotropins Juan Balasch

698

Use of recombinant DNA technology in ART Colin M Howles

736

Stimulation Protocols 37 38

39 40 41 42 43 44 45

Endocrine characteristics of ART cycles Jean Noel Hugues, Isabelle Cedrin-Durnerin

753

Gonadotropins-only based COH protocols: benefits and drawbacks Kees Jansen, Katherine E Tucker

777

The use of GnRH agonists Roel Schats, Joop Schoemaker

792

Antagonistic analogs of GnRH: preferable stimulating protocol Basil C Tarlatzis, Helen Bili

807

Monitoring IVF cycles Matts Wikland, Torbjörn Hillensjö

821

Follicle aspiration Carl Wood

828

The luteal phase: luteal support protocols James P Toner

844

Evaluation and treatment of the low responder patient Richard T Scott Jr

864

Repeated implantation failure: the preferred therapeutic approach Mark A Damario, Zev Rosenwaks

892

Different Technical Procedures 46 47 48 49 50

Ultrasound in ART Marinko M Biljan

922

Epididymal and testicular sperm extraction: clinical aspects Herman Tournaye

959

Gamete intrafallopian transfer (GIFT) Machelle M Seibel

977

Zygote intrafallopian transfer (ZIFT) Ariel Weissman, Jacob Farhi, David Levran

992

Embryo transfer William B Schoolcraft

1017

Special Medical Conditions 51 52

Endometriosis Mark I Hunter, Alan H DeCherney

1023

Polycystic ovaries and ART Howard S Jacobs, Adam H Balen, Jane MacDougall

1040

Complications of Treatment 53 54 55 56 57

Severe ovarian hyperstimulation syndrome Daniel Navot

1052

Bleeding, severe pelvic infection, and ectopic pregnancy Raoul Orvieto, Zion Ben-Rafael

1069

Iatrogenic multiple pregnancy: the risk of ART Isaac Blickstein

1082

Reducing the incidence of multiple gestation David R Meldrum

1101

Multifetal pregnancy reduction and selective termination Shlomo Lipitz

1109

Egg Donation and Surrogate Motherhood 58 59

Egg donation Mark V Sauer, Matthew A Cohen Gestational surrogacy Peter R Brinsden

1126 1144

Peter R Brinsden The Support Team 60

Patient support in the ART program Sharon N Covington

1165

Ethics and Legislation 61 62

Worldwide legislation Jean Cohen, Howard W Jones, Jr

1182

Times of transition: modern ethical dilemmas Françoise Shenfield

1211

Index

1123

Preface In 1978, Louise Brown, the first baby conceived following fertilization in vitro, was born in a small town in Oldham, Lancashire, England. This is often attributed as the starting point of in vitro fertilization (IVF). However, it was 10 years earlier that two determined men came together and realized that the concept was feasible. In a historic meeting, held at the Royal Society of Medicine in London, Patrick Steptoe, a gynaecologist from Oldham, presented pictures of the ovary and follicles taken via the laproscope. In the audience was Robert Edwards who had been working on human fertilization (see Introduction). In the foyer of the building Robert Edwards came to speak with Patrick Steptoe and their long and revolutionary collaboration started. Since this moment, the field of IVF has been transformed: a steady stream of discoveries and technological progress has led to an expansion of the indications treatable by IVF, such as severe male infertility by intracytoplasmic sperm injection (ICSI) and genetic disorders. Together these discoveries and techniques to treat different disorders are grouped under the term “assisted reproduction techniques” which is the theme of this book. As in 1968, the development and success of this discipline is due to a close collaboration of scientists and clinicians. For the first time, this book has brought together leading medical and scientific experts who describe in a clear and concise manner the “how, why and therefore” of ART. It has been written to be readable and usable by research fellows, who want to get an insight into the technical developments, by a clinical and scientific team who want to know the A to Z of setting up an embryology laboratory, as well as “veterans” in the field who want an up to date review on the newest techniques and advances. We hope that A Textbook of Assisted Reproductive Techniques will benefit all who read it.

Acknowledgments This book is dedicated to our mentors, students and colleagues, who make this such a wonderful discipline to work in, and to our families for their endless support. The editors would like to express their gratitude to Robert Peden and Kate Roberts of Martin Dunitz Publishers and all the contributing authors for their time and enthusiasm in bringing this book to life.

List of contributors Mina Alikani, MSc Institute for Reproductive Medicine and Science Saint Barnabus Medical Center 101 Old Short Hills Road Suite 501 West Orange, NJ 07052, USA Anthony Anderson, BSc Institute for Assisted Reproduction 200 Hawthorne Lane 6th Floor/IVF Charlotte, NC 28233, USA Gordon Baker, MD, PhD, FRACP Department of Obstetrics and Gynaecology University of Melbourne Melbourne IVF, Royal Women’s Hospital 132 Grattan Street Carlton, Victoria 3053, Australia Adam H Balen, MD, MRCOG Department of Obstetrics and Gynaecology The General Infirmary Leeds LS2 9NS, UK Juan Balasch, MD Department of Obstetrics and Gynecology Faculty of Medicine University of Barcelona Calle Casanova 143 E-08036 Barcelona, Spain Frank L Barnes, PhD IVF Labs 2712 East Swasont Way Salt Lake City, UT 84117, USA Zion Ben-Rafael, MD Department of Obstetrics and Gynecology Sackler Faculty of Medicine

Tel Aviv University and Rabin Medical Center Petah-Tikva 49101, Israel Helen Bili, MD 1st Department of Obstetrics and Gynecology Aristotelian University of Thessaloniki 9 Agl. Sophia Street Thessaloniki, Greece Marinko M Biljan, MD, MRCOG, MSc McGill Reproductive Center Department of Obstetrics and Gynecology Women’s Pavilion Royal Victoria Hospital 687 Pine Avenue West Montreal H3A 1A1, Quebec, Canada Isaac Blickstein, MD Department of Obstetrics and Gynaecology Kaplan Medical Center Rehovot 76100, Israel Irene Boiso, BSc Reproductive Medicine Service Institut Dexeus P/Bonanona 67 E-08017 Barcelona, Spain Harold Bourne, PhD Department of Obstetrics and Gynaecology University of Melbourne Melbourne IVF, Royal Women’s Hospital 132 Grattan Street Carlton, Victoria 3053, Australia Peter R Brinsden, FRCOG Bourn Hall Clinic Bourn Cambridge CB3 7TR, UK Pedro Caballero-Campo, PhD Instituto Valenciano de Infertilidad (IVI) Guardia Civil 23 46020 Valencia, Spain Isabelle Cedrin-Durnerin, MD

Hôpital Jean Verdier Division of Reproductive Medicine Ave. du 14 Juillet F-93143 Bondy Cedex, France Tim J Child, MA, MBBS, MRCOG McGill University Department of Obstetrics and Gynecology McGill Reproductive Center Royal Victoria Hospital 687 Pine Avenue West Montreal H3A 1A1, Quebec, Canada Jacques Cohen, PhD Institute for Reproductive Medicine and Science Saint Barnabus Medical Center 101 Old Short Hills Road Suite 501 West Orange, NJ 07052 USA Jean Cohen, MD Clinique Marignan 8 rue de Marignan F-75008 Paris, France Matthew A Cohen, MD Reproductive Endocrinology and Infertility College of Physicians and Surgeons Columbia University 622 West 168 Street, PH16 New York, NY 10032, USA Rita Cortvrindt, PhD, MSc Follicle Biology Laboratory Centre for Reproductive Medicine University Hospital, Free University Brussels Laarbeeklaan 101 B-1090 Brussels, Belgium Sharon N Covington, MSW Psychological Support Services The Shady Grove Fertility Reproductive Science Center 15001 Shady Grove Road Suite 400 Rockville, MD 20850, USA

Mark A Damario, MD Mayo Clinic Assisted Reproductive Technologies Program Mayo Clinic 200 First Street SW Rochester, MN 55905, USA Neroli Darlington Concept Fertility Centre King Edward Memorial Hospital Bagot Road, Subiaco Western Australia 6008, Australia Salim Daya, MB, ChB, MSc, FRCSC Department of Obstetrics and Gynecology McMaster University 1200 Main Street West Hamilton, Ontario L8N 3Z5, Canada Alan H DeCherney, MD Department of Obstetrics and Gynecology UCLA School of Medicine 10833 Le Conte Avenue Los Angeles, CA 90095–1740, USA Jose Luis de Pablo, PhD Instituto Valenciano de Infertilidad (IVI) Guardia Civil 23 46020 Valencia, Spain Nava Dekel, PhD Department of Biological Regulation The Weizmann Institute of Science Rehovot 76100, Israel David H Edgar, PhD Department of Obstetrics and Gynaecology University of Melbourne Melbourne IVF, Royal Women’s Hospital 132 Grattan Street Carlton, Victoria 3053, Australia Robert G Edwards, PhD Duck End Farm Park Lane, Dry Drayton Cambridge CB3 8DB, UK

Jacob Farhi, MD IVF Unit Department of Obstetrics and Gynecology Wolfson Medical Center Sackler Faculty of Medicine Tel Aviv University Holon 58100, Israel Bart CJM Fauser, MD, PhD Department of Obstetrics and Gynecology Division of Reproductive Medicine Erasmus University Medical Center Rotterdam Dr Molewaterplein 40 3015 GC Rotterdam, The Netherlands Ronni Gamzu, MD, PhD Lis Maternity Hospital Tel Aviv Sourasky Medical Center 6 Weizmann Street Tel Aviv 64239, Israel and Sackler Faculty of Medicine Tel Aviv University, Israel David K Gardner, DPhil Colorado Center for Reproductive Medicine 799 East Hampden Avenue, Suite 300 Englewood, Colorado 80110, USA John Garrisi, PhD Institute for Reproductive Medicine and Science of Saint Barnabus Medical Center 101 Old Short Hills Road Suite 501 West Orange, NJ 07052–1023, USA Antonia Gilligan, BS Alpha Environmental, Inc 258 Barrow Street Jersey City, NJ, USA Irit Granot, PhD IVF Unit Kaplan Medical Center Rehovot 76100, Israel Bulent Gulekli, MD

Dokuz Eylul University, School of Medicine Department of Obstetrics and Gynecology Izmir, Turkey and Visiting Professor, McGill University Department of Obstetrics and Gynecology McGill Reproductive Center Royal Victoria Hospital 687 Pine Avenue West Montreal H3A 1A1, Quebec, Canada Alan H Handyside, PhD School of Biology University of Leeds Leeds LS2 9JT, UK June J Hariprashad, BA Weill Medical College of Cornell University 505 East 70th Street New York, NY 10021, USA Benjamin Hendin, MD Baylor College of Medicine Scott Department of Urology Houston, TX 77030, USA Torbjörn Hillensjö, MD Fertility Centre Scandinavia Carlander’s Hospital Carlanderplatsen 1 S-41255 Göteborg, Sweden Colin M Howles, PhD, FRSM Serono International SA 15 bis, chemin des Mines Case Postale 54 CH-1211 Geneva, Switzerland Jean Noel Hugues, MD Hôpital Jean Verdier Division of Reproductive Medicine Ave. du 14 Juillet F-93143 Bondy Cedex, France Mark I Hunter, MD Department of Obstetrics and Gynecology UCI Medical Center

101 The City Drive Orange, CA 92868, USA Howard S Jacobs, MD, FRCP, FRCOG Royal Free and University College London School of Medicine The Middlesex Hospital Mortimer Street London W1N 8AA, UK Kees AM Jansen, MD, PhD Department of Obstetrics and Gynecology and IVF Reinier de Graafgroep loc Diaconessenhuis Voorburg Fonteynburghlaan 5 2275 CX Voorburg, The Netherlands Howard W Jones, Jr, MD Jones Institute for Reproductive Medicine Department of Obstetrics and Gynecology Eastern Virginia Medical School 601 Colley Avenue Norfolk, VA 23507–1627, USA Brooks A Keel, PhD, HCLD Department of Obstetrics and Gynecology University of Kansas School of Medicine The Women’s Research Institute 1010 N Kansas Wichita, KA 67214, USA Anver Kuliev, MD, PhD Reproductive Genetics Institute 836 W. Wellington Chicago, IL 60657, USA Dolores J Lamb, PhD Baylor College of Medicine Scott Department of Urology Houston, TX 77030, USA Michelle Lane, PhD Colorado Center for Reproductive Medicine 799 East Hampden Avenue, Suite 300 Englewood, CO 80110, USA

Bruce A Lessey, PhD, MD University of North Carolina at Chapel Hill Division of Reproductive Endocrinology and Fertility CB #7570 Old Clinic Building Chapel Hill, NC 27599–7570, USA David Levran, MD IVF Unit Department of Obstetrics and Gynecology Wolfson Medical Center Sackler Faculty of Medicine Tel Aviv University Holon 58100, Israel Ingeborg Liebaers, MD Centre for Reproductive Medicine Medical Genetics University Hospital and Medical School Vrije Universiteit Brussel Laarbeeklaan 101 B-1090 Brussels, Belgium William W Lin, MD Baylor College of Medicine Scott Department of Urology Houston, TX 77030, USA Shlomo Lipitz, MD Department of Obstetrics and Gynecology Chaim Sheba Medical Center Tel-Hashomer 52621, Israel Larry I Lipshultz, MD Baylor College of Medicine Scott Department of Urology 6560 Fannin Scurlock Tower, Suite 2100 Houston, TX 77030, USA Willy Lissers, MD Centre for Reproductive Medicine Medical Genetics University Hospital and Medical School Vrije Universiteit Brussel Laarbeeklaan 101

B-1090 Brussels, Belgium Jane MacDougall MD, MRCOG The Reproductive Medicine Unit Addenbrooke’s Hospital Cambridge, UK Nicholas S Macklon, MD Department of Obstetrics and Gynecology Division of Reproductive Medicine Erasmus University Medical Center Rotterdam Dr Molewaterplein 40 3015 GC Rotterdam, The Netherlands Mira Malcov, PhD Sara Racine IVF Unit Tel Aviv Sourasky Medical Center 6 Weizmann Street Tel Aviv 64239, Israel Jacqueline Mandelbaum, MD, PhD IVF Unit Hôpital Tenon 1, rue de la Chine F-75019 Paris, France Daniel Gustavo de Matos, PhD Halitus Institute Médico C1122AAF Marcelo T de Alvear 2084 Buenos Aires, Argentina Phillip Matson, PhD Hollywood Fertility Centre Hollywood Private Hospital Monash Avenue, Nedlands Western Australia 6009, Australia Eileen A McLaughlin, PhD University of Bristol Division of Obstetrics and Gynaecology St Michael’s Hospital Bristol BS2 8EG, UK David R Meldrum, MD Reproductive Partners Medical Group, Inc.

510 North Prospect Avenue, Suite 202 Redondo Beach, CA 90277, USA Yves JR Ménézo, PhD, DrSci, TC(ABB) Laboratoire Marcel Mérieux 1, rue Laborde F-69500 Bron Marcos Meseguer, PhD Instituto Valenciano de Infertilidad (IVI) Guardia Civil 23, 46020 Valencia and Departamento de Pediatría, Obstetricia y Ginecología Facultad de Medicina Universidad de Valencia Valencia, Spain Santiago Munné, PhD Institute for Reproductive Medicine and Science Saint Barnabus Medical Center 101 Old Short Hills Road, Suite 501 West Orange, NJ 07052, USA Daniel Navot, MD Division of Reproductive Endocrinology New York Medical College NY, USA Queenie V Neri, BSc Weill Medical College of Cornell University 505 East 70th Street New York, NY 10021, USA Daniela Nogueira, MSc Follicle Biology Laboratory Centre for Reproductive Medicine University Hospital, Free University Brussels Laarbeeklaan 101 B-1090 Brussels, Belgium Kutluk H Oktay, MD, FACOG Center for Reproductive Medicine and Infertility Weill Medical College of Cornell University 505 East 70th Street New York, NY 10021, USA

Virginia A Ord, MD Fertility Center of San Antonio 4499 Medical Drive, Suite 360 San Antonio, TX 78229, USA Raoul Orvieto, MD Department of Obstetrics and Gynecology Rabin Medical Center Petah-Tikva 49100, Israel Gianpiero D Palermo, MD Institute for Reproductive Medicine Weill Medical College of Cornell University 505 East 70th Street New York, NY 10021, USA Antonio Pellicer, MD Instituto Valenciano de Infertilidad (IVI) Guardia Civil 23, 46020 Valencia and Departamento de Pediatría, Obstetricia y Ginecología Facultad de Medicina Universidad de Valencia Valencia, Spain Math HEC Pieters, MD Department of Obstetrics and Gynecology Division of Reproductive Medicine Erasmus University Medical Center Rotterdam Dr Molewaterplein 40 3015 GC Rotterdam, The Netherlands Thomas B Pool, PhD Fertility Center of San Antonio 4499 Medical Drive, Suite 360 San Antonio, TX 78229, USA Eleanora Porcu, MD Infertility and IVF Centre Department of Gynecology and Obstetrics University of Bologna via Massarenti, 13 40138 Bologna, Italy Ricciarda Raffaelli, MD Weill Medical College of Cornell University

505 East 70th Street New York, NY 10021, USA Zev Rosenwaks, MD Weill Medical College of Cornell University 505 East 70th Street New York, NY 10021, USA Denny Sakkas, PhD Department of Obstetrics and Gynecology Yale University School of Medicine New Haven, CT 06510, USA Mireia Sandalinas, MD Institute for Reproductive Medicine and Science Saint Barnabus Medical Center 101 Old Short Hills Road, Suite 501 West Orange, NJ 07052–1023, USA Mark V Sauer, MD Department of Obstetrics and Gynecology College of Physicians and Surgeons Columbia University 622 West 168 Street, PH16 New York, NY 10032, USA Tammie K Schalue, PhD, HCLD Department of Obstetrics and Gynecology University of Kansas School of Medicine The Women’s Research Institute 1010 N Kansas Wichita, KA 67214, USA Roel Schats, MD, PhD IVF Center Free University Hospital PO Box 7057 1007 MB Amsterdam, Netherlands Joop Schoemaker, MD Subdivision of Reproductive Endocrinology and Infertility Department of Obstetrics and Gynaecology Free University Hospital de Boelelaan 1117 1081 HV Amsterdam, Netherlands

William B Schoolcraft, MD Colorado Center for Reproductive Medicine 799 East Hampden Avenue Suite 300 Englewood, CO 80110, USA Lynette Scott, PhD The ART Institute of Washington DC at Walter Reed Army Medical Center Division of Reproductive Endocrinology 6900 Georgia Ave NW Washington, DC 20307, USA Richard T Scott Jr, MD Reproductive Medical Associates 111 Madison Avenue, Suite 100 Morristown, New Jersey 07962, USA Machelle M Seibel, MD Department of Gynecology and Obstetrics Boston University School of Medicine and Fertility Center of New England 333 Elm Street, Third Floor Dedham, MA 02026, USA Françoise Shenfield, MD The University College London Medical School Reproductive Medicine Unit Huntley Street London WC1E 6AU, UK Zeev Shoham, MD Reproductive Medicine and Infertility Unit Department of Obstetrics and Gynecology Kaplan Medical Center Rehovot 76100, Israel Kaylen M Silverberg, MD Texas Fertility Center 3705 Medical Parkway Suite 420 Austin, TX 78705, USA Carlos Simón, MD Instituto Valencia de Infertilidad (IVI)

Guardia Civil 23 46020 Valencia and Department of Obstetrics and Gynecology Universidad de Valencia Valencia, Spain Cecilia Sjöblom, MSc Fertilitetscentrum Box 5418 S 402 29 Göteborg and Carlanderska Sjukhemmet Carlandersplatsen 1 S 412 55 Göteborg, Sweden Johan Smitz, MD, PhD Follicle Biology Laboratory Centre for Reproductive Medicine University Hospital, Free University Brussels Laarbeeklaan 101 B-1090 Brussels, Belgium Takumi Takeuchi, MD Weill Medical College of Cornell University 505 East 70th Street New York, NY 10021, USA Seang Lin Tan, MD Department of Obstetrics and Gynecology Royal Victoria Hospital, Women’s Pavilion 687 Pine Avenue West Montreal, Quebec H3A 1A1, Canada Basil Tarlatzis, MD Geniki Kliniki Infertility and IVF Centre 2 Gravias Street GR-54645 Thessaloniki and 1st Department of Obstetrics and Gynecology Aristotelian University of Thessaloniki 9 Ag. Sophia Street Thessaloniki, Greece Giles Tomkin, BSc Institute for Reproductive Medicine and Science Saint Barnabus Medical Center

101 Old Short Hills Road Suite 501 West Orange, NJ 07052, USA James P Toner, MD, PhD Doner Egg and Embryo Programs Atlanta Center for Reproductive Medicine 100 Stone Forest Drive Suite 300 Woodstock, GA 30189, USA Herman Tournaye, MD, PhD Centre for Reproductive Medicine Free University Brussels University Hospital Laarbeeklaan 101 B-1090 Brussels, Belgium Katherine E Tucker, PhD, HCLD Department of Obstetrics and Gynecology and IVF Reinier de Graafgroep Ioc Diaconessenhuis Voorburg Fonteynburghlaan 5 2275 CX Voorburg, The Netherlands Tom Turner, MS Texas Fertility Center 3705 Medical Parkway Suite 420 Austin, TX 78705, USA André Van Steirteghem, MD Centre for Reproductive Medicine University Hospital and Medical School Vrije Universiteit Brussel Laarbeeklaan 101 B-1090 Brussels, Belgium Anna Veiga, PhD Reproductive Medicine Service Institut Dexeus P/Bonanona 67 E-08017 Barcelona, Spain Yury Verlinsky, PhD

Reproductive Genetics Institute 836 W Wellington Chicago, IL 60657, USA Lucinda Veeck, MLT, DSc(hons) Weill Medical College of Cornell University 505 East 70th Street New York, NY 10021, USA Leslie Weikert, Bsc Institute for Assisted Reproduction 200 Hawthorne Lane 6th Floor/IVF Charlotte, NC 28233, USA Ariel Weissman, MD IVF Unit Department of Obstetrics and Gynecology Wolfson Medical Center Holon 58100, Israel Klaus E Wiemer, PhD Institute for Assisted Reproduction 200 Hawthorne Lane 6th Floor/IVF Charlotte, NC 28233, USA Matts Wikland, MD, PhD Fertility Centre Scandinavia Carlander’s Hospital Carlanderplatsen 1 S-41255 Göteborg, Sweden Carl Wood, MD 19 Simpson Street East Melbourne, Victoria 3002, Australia Yuval Yaron, MD Prenatal Genetic Diagnosis Unit, Genetic Institute and Lis Maternity Hospital Tel Aviv Sourasky Medical Center 6 Weizmann Street Tel Aviv 64239, Israel and Sackler Faculty of Medicine Tel Aviv University, Israel

Introduction The beginnings of human in vitro fertilization Robert G Edwards

Requests for this topic seem to be never ending. Yet each time I write about it, further slants seem to emerge from those described in previous narratives, and as new perspectives unfold from the massive accumulation of events between 1960 and 2000. There seems little doubt that curiosity about its origins becomes ever deeper as in vitro fertilization (IVF) and its derivatives spread ever wider. I am still stirred by recollections of those early days, and of the days when Bourn Hall was opened in 1980 to become the largest clinic of its kind.

INTRODUCTION So first of all, a few tributes must be expressed to my teachers. In fact, these include investigators from the far off days of earlier centuries when the fundamental facts of reproductive cycles, surgical techniques, endocrinology, and genetics were reported by many investigators. These fields really began to move in the twentieth century, and if one of these pioneers 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 first to show how oocytes aspirated from their follicles would begin their maturation in vitro and a number would complete it by expelling a first polar body. I believe his major work was done in rabbits, where he found that the 10–11 hours 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 fact 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 1 chromosome pairs. Fully mature oocytes would display metaphase 2 chromosomes, and so be fully ripe and ready for fertilization.

Textbook of assisted reproductive techniques

2

Unfortunately, it is well known that oocytes can undergo atresia in the ovary involving the formation of metaphase 2 chromosomes in many of them. These oocytes complicated Pincus’s estimates, even in controls, and was the source of the error leading 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, then remained in abeyance for many years. Progress in animal IVF had also been slow. After many relatively unsuccessful attempts on 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. Later, in 1969 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 reported that rabbit eggs that had fully matured in vitro failed to produce normal blastocysts and none implanted normally.4

MODERN BEGINNINGS OF HUMAN IVF, PREIMPLANTATION GENETIC DIAGNOSIS, AND EMBRYO STEM CELLS When I started my PhD in Edinburgh in 1952, encouraged by Professor Conrad Waddington and supervised by Dr Alan Beatty, capacitation was gaining prominence. I decided to study the growth of mouse embryos with altered chromosome complements. It would be essential to expose mouse spermatozoa to x rays, ultraviolet light, and various chemicals in vitro to destroy their chromatin so they could fertilize eggs but make no genetic contribution to the embryo. These embryos would become gynogenetic haploid embryos. Later, I exposed eggs to colchicine, in order to destroy the second meiotic spindle in mouse eggs. All chromosomes are thus freed from spindle attachment and exit from the egg into tiny artificial polar bodies. A fertilizing spermatozoon would therefore enter an empty egg, to result in 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. My research was successful, and 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. Julio Sirlin and I applied the use of radioactive

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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 gonadotrophin (HCG) with strong luteinizing hormone (LH) activity to induce oestrus and ovulation in immature female mice. Working with Mervyn Runner,5 they 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.6 I was more interested in stimulating adult mice with these gonadotrophins to induce oestrus and ovulation, by now weary of taking vaginal smears. My future wife, Ruth Fowler, and I teamed up to test superovulating adult mice. HCG 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 the adult’s reproductive cycles. The hormone preparations really worked. Doses of 1–3 IU PMS induced the growth of numerous follicles, and similar doses of HCG 42 hours later invoked oestrus and ovulation a further six 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. Multiple implantations occurred just as similar treatments in anovulatory women were to do some years later.7 Oocyte maturation, ovulation, mating and fertilization were each closely timed in all adults, another highly unusual aspect of stimulation.8 Diakinesis was identified as the germinal vesicle regressed, with metaphase 1 a little later and metaphase 2, expulsion of the first polar body, and ovulation at 11.5–12 hours after HCG. 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 UK. The Institute had given me an excellent basis in genetics, but equally in reproduction. I had gained considerable knowledge about the endocrine control of oestrus cycles, ovulation, spermatozoa, and the male reproductive tract artificial insemination and the stages of embryo growth in oviduct and uterus, superovulation and its consequences, and the use of radiolabelled compounds. All this knowledge was to prove of immense value in my later career. Much of it was cemented in California. After California, London beckoned first, to the National Institute for Medical Research working with Drs Alan Parkes and Colin (Bunny)

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Austin. After two intense years on immunology, my curiosity returned to maturating oocytes and fertilization in vitro. It should be easy to stimulate maturation in mouse oocytes in vitro by using gonadotrophins if they matured so easily in vivo. In fact, to my immense surprise, oocytes matured immediately in vast numbers in all groups, exactly on time with those matured in vivo in Edinburgh. Adding hormones made no difference. Rabbit, hamster, and rat oocytes also matured with 12 hours, each at their own species specific rates. Then, again 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 persisting unmoved. 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 gynaecologist who delivered two of our daughters. She accepted to send 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, 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 those oocytes from other adult mammals, as germinal vesicles persisted and diakinesis was absent. This was disappointing, especially as Tjio and Levan, and Ford, identified 46 diploid chromosomes in humans and teams in Edinburgh (Scotland), France, and elsewhere discovered monosomy or disomy in many men and women. XO and XXX women, and XY and XYY in men were identified, and trisomy 21 proved to be the most common cause of Down’s syndrome. The origins of these genetic disorders were hidden in the meiotic chromosomes in maturing oocytes, especially diakinesis, and all this new information reminded me of my chromosome studies on the Edinburgh mice. I had to obtain diakinesis and metaphase I in human oocytes, and continue to metaphase 2 when the oocytes would 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 1962.9 There was so much to write about the animal work, and describing the new ideas then taking shape in my mind. I had heard institute lectures on infertility, and realised 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

Introduction

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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, in mitoses in the excised cells. Choosing female embryos for transfer would avert the birth of boys with various sex linked disorders such as haemophilia. I was becoming totally committed to human IVF and embryo transfer. While reading in the library for any newly published papers relevant for 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 paper9 became very different to 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 to repeat them. Intact ovaries were perfused in vitro with gonadotropin solutions, different culture media failed, joint cultures with mouse oocytes failed, and added hormones had no effect. It began to seem that menstrual cycles affected oocyte physiology. Finally, after two years of fruitless research on the precious few human oocytes available, an idea struck. Perhaps maturation timings differed widely between mice and rabbits compared with 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. The germinal vesicle remained static for 12 hours and then 20 hours in vitro. Three oocytes remained, as I waited for 24 hours. The first contained a germinal vesicle. So did the second. There was one left and one only. I was overjoyed as I gazed down the microscope. Its chromosomes were in diakinesis and its germinal vesicle was regressing. The chromosomes were superb examples of human diakinesis with their classical chiasmata. This was the step I had waited for, a marker that Pincus had missed. He never checked for diakinesis and apparently confused atretic oocytes, which contained chromosomes, with maturing oocytes. Endless human studies were now opening. It was easy now to calculate the timing of the final stages of maturation as ending at about 36 hours, which would be the moment for insemination. All these gaps in knowledge had to be filled, but now we were on the way to human IVF. And at this wonderful moment, John Paul, an outstanding cell biologist, invited me to join him and Robin Cole in Glasgow University to study differentiation in early mammalian embryos. This was exciting, to work in biochemistry with a leading cell biologist. We wanted to grow stem cells from mammalian embryos and study them in vitro. Over 12 months, stem cells migrate out from cultures of rabbit blastocysts, forming muscle and blood islands in vitro.10 A line of embryonic stem cells characterized for biochemical markers was established. Thoughts were strengthened of growing stem cells from human embryos to repair defects in tissues of children and adults. Almost at my last moment in Glasgow, a piece of ovary yielded

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several oocytes and two had reached metaphase 2 and expelled a polar body at 37 hours. The whole pattern of oocyte maturation was increasingly clear even if still in outline. Resuming a mixture of immunology and reproduction remained my dominant themes in the Physiological Laboratory at Cambridge as I rejoined Alan Parkes and Bunny Austin. Thoughts of human oocytes and embryos were never far away. One possible model of the human situation was the cow, 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.11 Human oocytes trickled in, improving my provisional timings of maturation. One or two spare oocytes were inseminated, without signs of fertilization. More oocytes were urgently needed to conclude the timings of oocyte meiosis. Surgeons in Johns Hopkins Hospital, Baltimore (United States), performed the Stein-Leventhal operation, so I could collect the oocytes and send the remaining tissues to pathology if necessary. Victor McKusick worked there and supported my request for a visit to work with the hospital gynaecologists for six weeks. He made laboratory space available and, a wonderful invitation, introduced me to Howard and Georgeanna Jones. This significant moment was equal to my meeting with Dr Rose. The Jones’ proved to be superb and unstinting in their support. Sufficient wedges and other ovarian fragments were available to complete the maturation programme in human oocytes. Within three weeks, every stage of meiosis was classified and timed.12 With Howard and Georgeanna Jones, preliminary studies were made on human fertilization in vitro, using different media or fragments of ampulla in 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.13 During those six weeks, oocyte maturation was fully timed at 37 hours, permitting me to predict that women would ovulate at 37 hours approximately after an HCG injection. For clinical trials, a simple access to aspirate human ovarian follicles in vivo was needed 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 2 in some inseminated eggs. Again, no sperm tails were seen within the eggs. 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

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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 was even stronger. So many medical advantages could flow from it. Human embryos had been very rare, flushed from a human oviduct or uterus after sexual intercourse. It was time to attain fertilization, and 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 were never very far away. I accepted all these issues, remaining confident that a study of human conception was correct ethically, medically, and scientifically even to use the increasing knowledge of genetic and embryology to be applied in such human studies. 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. It was a pleasure to see some fertilized bovine eggs, with sperm tails and characteristic pronuclei, especially using spermatozoa from one particular bull. Things 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. Third, and by no means least, while browsing in the library of the Physiological Laboratory, a paper in the Lancet caught my attention. Written by Dr P C Steptoe of the Oldham and District General Hospital,18 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. This is exactly what I wanted because access to the ampulla was equivalent to gaining access to ovarian follicles. He had worked closely with two pioneers, Palmer in Paris19 and Fragenheim in Germany.20 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.21 Despite advice to the contrary from several medical colleagues, I telephoned him about

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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 began our collaboration six months later in the Oldham and District General Hospital, almost 200 miles north of Cambridge.

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 spermatozoon 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.

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Molly Rose sent me 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 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, strengthening the need to abandon oocyte maturation in vitro and replace it with stimulation of maturation in vivo by means of exogenous hormones. Our paper in Nature surprised a world unaccustomed to the idea of human fertilization in vitro.22 Incredibly fruitful days followed in our Cambridge laboratory. Richard Gardner, another PhD candidate, and I excized 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 sex linked diseases.23 Three years later we tried to repeat this work in human blastocysts, but they failed to express either sex chromatin or the male Y body. Human preimplantation genetic diagnosis would have to wait a little longer. Alan Henderson, a cytogeneticist, and I analysed chiasmata during diakinesis in mouse and human eggs, and explained the high frequencies of Down’s syndrome in offspring older mothers as a consequence of meiotic errors arising in oocytes formed last in the fetal ovary which were then ovulated last at the later maternal ages.24 Dave Sharpe, a lawyer from Washington, joined forces to write an article in Nature25 on the ethics of in vitro fertilization, the first paper ever 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.25 So the first ethical papers were written by scientists and lawyers and not by philosophers, ethicists, or politicians.

THE OLDHAM YEARS By now Patrick Steptoe was waiting in the wings, ready to begin clinical IVF in distant Oldham. We had a long talk about ethics. Work started in the Oldham and District General Hospital and moved later in Kershaw’s Hospital, set up by my assistants, especially Jean Purdy. We knew the routine. I was based on my Edinburgh experiences with mice, and employed to plan our programme. Pietro Donini had purified urinary human menopausal gonadotrophins (HMG) as a source of FSH, and the product was used clinically to stimulate follicle growth in anovulatory

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women by Bruno Lunenfeld.26 It removed the need for PMS, so avoiding the use of non-human hormones. Doses to patients were kept low: to 2–3 vials (a total of 150–225 IU) given on days 3, 5, and 7. 5000 IU of HCG were given on day 10. Initially, oocyte maturation in vitro was confirmed, performing the laparoscopic collection at 28 hours after HCG to check the oocytes were in metaphase 1,27 and then at 36 hours to collect mature metaphase 2 oocytes for fertilization. Those beautiful oocytes were surrounded by masses of viscous cumulus cells and maturing exactly as predicted. We witnessed follicular rupture at 37 hours through the laparoscope. Follicles could be classified from their appearance as ovulatory or non-ovulatory, this diagnosis being confirmed later by assaying several steroids in the aspirated follicular fluids (Fig 2). It was a pleasure and a duty to meet the patients, searching for help to alleviate their infertility. We did our best driving from Cambridge to Oldham 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 the 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, translucent blastocysts (Fig 3).29 Here was my reward—growing embryos was now routine, and examinations of many of them convinced me the time had come to replace them into the mothers’ uteri. I had become highly familiar with the teratological 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. And our human studies had surpassed work on all animals, a point rubbed in even more when we grew blastocysts to day 9 after they had hatched from their zona pellucida (Fig 4).30 This beautifully expanded blastocyst a large embryonic disc was a potential source of embryonic stem cells.

Fig 2 Eight steroids were assayed in fluids extracted from human follicles aspirated 36–37 hours after HCG. The follicles had been classified as ovulating or nonovulating by laparoscopic examination in vivo. Data were analysed 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 HMG and HCG.

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

Introduction

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the zona pellucida. Upper right: a 16–32 cell stage, showing the onset of compaction of the outer blastomeres. Often, blastocoelic 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, blastocoelic cavity, and thinning zona pellucida. Lower right: a fixed preparation of a human blastocyst at five days, showing more than 100 even sized nuclei and many mitoses. There were very very few plaudits for us, criticism being 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 gave us their staunch support. So too did the Oldham Ethical Committee and Bunny Austin back home in Cambridge. Growing embryos became routine, so we decided to transfer one of them to their mothers’ uteri. Here again we were in untested waters. Transferring embryos via the cervical canal, the obvious route was virtually a new and untested method. We would have to do our best with it. And from now on, we worked with patients who had seriously distorted tubes or none whatsoever. We had to do this because no one would have believed us if we had claimed a test tube baby in a woman with near normal tubes. This had to be a condition of our work, yet it led many people to believe we started IVF to bypass occluded oviducts, when 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 an impossibly short time for embryos to

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Fig 4 A hatched human blastocyst after nine 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. 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 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 to 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 progestagen, meant it should be given every five days to sustain pregnancies, since this progestagen supposedly saved threatened abortions. So we began transfers in stimulated cycles, giving this luteal phase support. Even though our work

Introduction

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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 three years. None of our patients was pregnant. Disaster loomed. Our critics were even more vociferous as the years passed, and mutual support between Patrick and I 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 get every scrap of information from our failures. I knew Ken Bagshawe in London who was working with improved assay methods for gonadotrophic hormones. He offered to measure blood samples taken from our patients over the implantation period using his new HCGß assay. He telephoned: three or more patients previously undiagnosed had actually produced shortlived rises of HCGß over this period. Everything changed with this information. We had established pregnancies after all, but they had aborted very early. Today we call them biochemical pregnancies. 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 programme was a waste of time. But we had come through it and now knew exactly what to do next. We reduced levels of Primulot depot, and used HCG and progesterone as luteal aids. We at least now suspected that single embryo transfers could produce a 15–20% chance of establishing pregnancy. Almost instantly, our first clinical pregnancy arose after the transfer of a single blastocyst in a patient stimulated with HMG and HCG.32 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 an ectopic and he had to remove it somewhere 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. And, in any case, ectopic pregnancies are now known to be a regular feature with assisted conception. I sensed 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 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 became

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Fig 5 The first attempts at gamete intrafallopian transfer (GIFT) were called oocyte recovery for tubal insemination (OR+TI). In this treatment cycle, using HMG and HCG, including additional injections of HCG for luteal support, a single preovulatory oocyte and 1.6 million sperm were transferred into the ampulla. 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

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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. 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 long 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 labour 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 the presentation on the whole of the Oldham work at the Royal College of Obstetrics and Gynaecology.

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. 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 it 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 in 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 expanded prodigiously. Thousands of patients queued for IVF. Simon Fishel, Jacques Cohen, and Carol Fehilly joined the embryology team, and new clinicians joined Patrick and John Webster. Patients and pregnancies increased rapidly, and the world was left standing far behind. Howard and Georgeanna

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Fig 6 Recording the progress of the human natural menstrual cycle for IVF. Three patients are illustrated. All three displayed rising 24 hour urinary oestrogen concentrations during the follicular phase, and rising urinary pregnanediol concentrations in the luteal phase. LH levels were measured several times daily and the data clearly reveals the exact time of onset of the LH surge. Jones began in Norfolk using gonadotrophins 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 gynaecologists and others. The low rates of pregnancy, the possibilities of oocyte and embryo donation, surrogate mothers, unmarried parents, one sex parents, embryo cryopreservation, cloning, and endless other objections were raised against the procedures. LIFE even issued a legal action against me for the abortion of an embryo grown for some days in vitro. It was rejected by the senior government lawyer since the laws of pregnancy began after implantation. We fully respected the intense ethical nature of our proceedings and the need to

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protect the embryo even as those not replaced had to be used for research under strict controls, and for open publishing and discussion of our work. Each year, 1000 rising to almost 2000 patients passed through Bourn Hall. Different stimulation regimens or 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, including 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, where men brought 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 BMA officers and for some reason agreed to delay embryo cryopreservation. Apparently, it would be an unwelcome social development. I did not approve: 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 programme. 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 my talk. Next morning, the press furore about my supposed practice of IVF was awful, so bad, 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 BBC, London Times, and other leading newspapers. Seven and another one later. If only one was lost, I could be ruined and disgraced. But they were all won even if it took several years with the BMA and its secretary. These legal actions inhibited our research, the cryopreservation programme being shut down for more than one year. And 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.

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That wretched period passed. Numbers of babies kept on growing, embryo cryopreservation was resumed and Gerhard Zeilmaker in Holland beat the world to the first “ice” baby.35 Colin Howles and Mike Macnamee joined us in endocrinology, 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

Fig 7 A happy picture of Patrick and I, standing in our robes after being granted our Hon. D.Sc. by Hull University. were far in advance of those used elsewhere. Oocyte donation and surrogacy by embryo transfer were introduced. Work on preimplantation diagnosis of genetic disease resumed, and the first papers using new DNA technology were published. But embryo research faltered as all normal embryos were cryopreserved for their parents, so that 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 vitro. 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 7). Clinics sprang up everywhere. Ultrasound was introduced to detect and aspirate follicles by the Scandinavians,37 making laparoscopy for oocyte recovery largely redundant. Artificial cycles were

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introduced in Australia, 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 had the right to at ages still far almost the same as possible by a man. And ethics continues side by side with advancing science and medicine. The UK governmental Warnock report recommending 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 favour, a 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. 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, grey shadows of my earlier times in Glasgow. IVF is now 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. A 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. And new antagonists of GnRH introduced novel ways to control the cycle, enabling many oocytes to be stimulated by HMG and subsequently recombinant 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 that the tight systems place every new gene 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.

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IVF OUTLOOK In one sense, opening 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 500000 babies born worldwide by 2000—yet Louise Brown is only just 21 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 could well combine closely one day with genetics, to eliminate disease or disability genes or lengthen the lifespan. 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. Even as I write this text, I stumbled across a paper in development. Sequencing of the entire mouse genome has enabled genes to be identified that are active during preimplantation stages of mouse embryo. A staggering array of genes operate then—apparently a huge percentage of those acting throughout our complete life. The same must apply to the human embryo. We are indeed enmeshed in some of the most fundamental evolutionary stages of our existence as we pass from oocyte to blastocyst and to implantation.

REFERENCES 1 Pincus G, Saunders B. Anat Rec (1939); 75:537. 2 Menkin MF, Rock J. Am J Obstet Gynecol (1949); 55:440. 3 Hayashi M. Seventh Int. Conf. International Planned Parenthood Federation, Excerpta Medica (1963): 505. 4 Chang MC. J Exp Zool (1955); 128:379–405. 5 Runner M, Gates AH. (1954) J Hered. 45, 51. 6 Gates AH. (1954) Nature. 177:754. 7 Fowler RE, Edwards RG. J Endocrinol (1957); 15:374–84. 8 Edwards RG, Gates AH. J Endocrinol (1959); 19:292–304. 9 Edwards RG. Nature (1962); 196:446–5.

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10 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. 11 Edwards RG. Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature (1965); 208:349–51. 12 Edwards RG. Maturation in vitro of human ovarian oocytes. Lancet (1965); 2:926–9. 13 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. 14 Austin CR. Adv Biosci (1969); 4:5. 15 Chang M. Adv Biosci (1969); 4:13. 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) ii: 913. 19 Palmer R. Acad Chir (1946); 72:363. 20 Fragenheim H. Geburts Frauenheilkd (1964); 24:740. 21 Steptoe PC. Laparoscopy in Gynaecology (1967). Edinburgh: Livingstone. 22 Edwards RG, Bavister BD, Steptoe PC. Early stages of fertilisation in vitro of human oocytes matured in vitro. Nature (1969); 221:632–5. 23 Gardner RL, Edwards RG. Control of the sex ratio at full term in the rabbit by transferred sexed blastocysts. Nature (1968); 218:346–8. 24 Henderson SA, Edwards RG. Chiasma frequency and maternal age in mammals. Nature (1968); 218:22–8. 25 Edwards RG, Sharpe DJ. Social values and research in human embryology. Nature (1971); 231:81–91. 26 Lunenfeld B. In: W Inguilla, RG Greenblatt, RB Thomas, eds. The ovary. CC Thomas: Springfield, 111. (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 preovulator human oocytes. Nature (1970) 227:1307–9. 29 Steptoe PC, Edwards RG, Purdy JM. Human blastocysts grown in culture. Nature (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.

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33 Edwards RG, Steptoe PC, Purdy JM. Clinical aspects of pregnancies established with cleaving embryos grown in vivo. Br J Obstet Gynecol (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 (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.

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 makeshift 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 will not be 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 will discuss the more typical purpose built all inclusive laboratories that are adjacent or in close proximity to oocytes 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 really 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 on the basis of personality and experience, rather than official qualifications and status. The same principle applies to assisted reproductive laboratories, as experience is the key to success and because there are very few standard teaching and

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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 underestimate 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 setup 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. Certain regulatory bodies such as the College of American Pathology and the British Human Fertilization and Embryology Act 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 unproved and the licenses are not interchangeable between countries. Tradition also plays its part, 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 that 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 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

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the types of skills required, the daily case load, and total time. It can be appreciated that 100 cases over one year is a very different matter than the same amount during six 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 high, 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 low to save money, as happens sometimes in commercially oriented clinics. 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. But it should be realized that these tasks seriously detract from their true work. Firstly, the embryo legist’s duty is to perform gamete and embryo handling and culture procedures. Secondly, but equally importantly, the embryologist should maintain full awareness of quality control standards, both by performing routine checks and tests as well as 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 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 only on gamete and embryo handling, and any laboratory with a relatively high number of annual procedures should have additional embryology technicians and assistants that can order and maintain equipment, and properly record laboratory data. In short, skimping on staff can be seriously self defeating in the IVF laboratory.

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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 scanning the content of air samples 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 full fledged design. There are five basic types of design: (I) laboratories using only transport IVF, (II) laboratories adjacent to clinical outpatient facilities that are only used part of the time, (III) full time clinics with intrafacility egg transport using portable breeding chambers, (IV) fully integrated laboratories with clinical areas, and (V) 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, demolition or major changes of any kind, in the foreseeable future. City planning should also be reviewed. 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 air changes (ACH) 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. 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 should not use an open plan design. In the ideal case, 100% outside air with chemical and physical filtration will be used with sealed supply and return ducts. Alternately, the air supply equipment may balance outside air with re-circulated air, with processing to control the known levels of VOCs. Some laboratories will require full time air

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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 degrees centrigrade, 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. A detailed layout and assessment of all laboratory furniture and equipment is therefore essential prior to construction and has many other benefits. 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 three meters in any direction; not only is this efficient, but it also 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 pass through for samples between them. This pass through 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.

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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 continue 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 principal, 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 in order to further limit the number of incubator openings. Strict guidelines must be implemented and adhered to when 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,

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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 nor allow condensation and conserve energy. Medical gases can be directed into the laboratory using prewashed vinyl/Teflon lined tubing. Alternately, 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 non-contaminating 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. 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. Thankfully, these units can be well removed from the laboratory, but must be placed, mind you, in wellventilated 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

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including sterilized disposable items, release multiple compounds for prolonged periods. This “outgassing” 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 firstclass, 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 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 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 to complete reproductive failure. Many of the damaging materials are organic chemicals that are released or outgassed by paint,

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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 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 hopes 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 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. Alternately 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

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(GenX International, Madison, Conn, 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 release alkanes, aromatics, alcohols, aldehydes, ketones, and other classes of organic materials. This section will outline 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 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 like formaldehyde, acetaldehyde, benzene, toluene, styrene, xylenes, and other 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

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temperature of the new area by 10–20 degrees centigrade 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 degrees centigrade, at less than 40% relative humidity. The burn in period can range from 10 days 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 left running the whole time. Naturally, ventilation is critical if redistribution of irritants is to be avoided; the whole purpose is to repeatedly purge the air. 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 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 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 the HEPE system is functional. Particulate sampling can be done by using USA 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 EPA protocols using gas chromatography/mass spectroscopy (GC/MS) and high performance liquid chromatography sensitive at the microgram per cubic meter level.11–13

INSURANCE ISSUES ART has become common practice worldwide, and is regulated by any combination of legislation, regulations or committee based ethical standards. The rapid evolution and progress of ART techniques 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

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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 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 (a) cancellation of cycle prior to egg retrieval, (b) failure to become pregnant, (c) patient identification errors and (d) interrupted cryo-storage events; 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 counter measures.

CONCLUSION All in all, it is surprising 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 approach saturation in most areas. Regardless, this chapter can provide some guidance to those medical professionals aspiring to independence in the world of ART, although it cannot safeguard from disaster 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. Invitro 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 invitro 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 invitro. Hum Reprod (1997); 12:1742–9. 6 Cohen J, Gilligan A, Willadsen S. Culture and quality control of embryos. Hum Reprod (1998); 13(S3): 137–44. 7 Alikani M, Cohen J, Tomkin G, Garrisi GJ, Mack C, Scott RT. 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 laboratories used for human embryo culture. Hum Reprod (1998); 13(S4): 146–55. 10 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. 11 Seifert, B. Regulating indoor air. Proceedings of 5th international Conference on Indoor Air Quality and Climate, Toronto (1990); 5:35– 49. 12 Federal Standard 209 E (1992) General Services Administration USA Federal Government, Washington, DC. 13 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.

2 Quality control in the IVF laboratory Klaus E Wiemer, Anthony Anderson, Leslie Weikert

INTRODUCTION The establishment and maintenance of pertinent quality control protocols, procedures, and the implementation of these issues continues to elude many in vitro fertilization (IVF) programs today. Numerous attempts have been made to establish meaningful quality control (QC) and quality assurance (QA) programs. However, most are impractical, expensive, and shed very little light on the potential pitfalls that may exist in our IVF laboratories. Yet, without some form of QC and QA the ability to trace deficiencies in an IVF laboratory becomes exceedingly difficult. One particular constituent that has been quality assured to extreme measures is culture medium. At some point in time we have decided that optimal culture conditions for mouse embryos would also be acceptable for human embryos. However, considerable evidence and experience now exist that indicate that mouse embryos are not the most appropriate model for humans. In other words, we have erroneously decided that if a particular medium is of good enough quality for mouse embryos then it must be good enough for human embryos. Protein supplementation for embryo culture can be an extremely controversial subject. It can be a source of potential contamination; causing variation in embryonic development and subsequent implantation rates. Protein sources therefore should undergo some form of testing prior to use. Protein sources can be loosely classified under two categories: unprocessed serum and human serum albumin (HSA). Serum may provide numerous beneficial factors to the artificial culture environment created in typical laboratories. These potentially beneficial entities include: amino acids, vitamins, energy substrates, and growth factors. Conversely, serum supplementation can introduce substances that may be highly embryo toxic. HSA may be void of potential embryotrophic cytokines and be a major source of endotoxin contamination. However, the more defined nature of HSA could reduce media batch to batch variations. The general consensus today is that protein supplementation in its various forms and concentrations may be required for proper embryonic development. It is difficult to discuss the

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concept of protein supplementation without also addressing substrate availability. This discussion will deal primarily with various aspects of QC and QA in the laboratory and will attempt to provide some insight in identifying potential hazards that can affect successful outcome.

QUALITY CONTROL AND ASSURANCE A logical beginning to this discussion is to compare and contrast as well as understand the relationship and interdependence between QC and QA. Quality control is defined as the routine monitoring of all important operational aspects directly or indirectly involved with performing IVF. For example, the process of verifying CO2 content and temperature of incubators using an independent instrument is a form of QC, whereas QA entails the evaluation of protocols and establishing a means of identifying problems. For example, suppose that an established minimum fertilization standard for non-male factors is at least 60%; now suppose the rate for a particular patient falls below the accepted level. A QA program would attempt to determine the cause for the lower fertilization rate by following established procedures. QC can be further broken down into long term/continuous (chronic) and short term (acute) formats. The continuous evaluation of supernumerary human embryos to the blastocyst stage represents a chronic form of QC. These continuous evaluations constantly monitor gas environments, incubator settings, general culture conditions as well as the interaction of all these variables, whereas the batch testing of culture media with either mouse embryos or spermatozoa yields information specific to a given time interval and set of conditions. Media testing is an illustration of a short term (acute) test. Obviously, more information and greater insight into the overall conditions that exist within the laboratory are gained when continuous type QC monitoring is strictly followed. We therefore propose that every attempt is made to establish continuous type quality control programs. Monitoring the independent variation of equipment function is a critical aspect of maintaining the highest standards possible in the laboratory. For example, we recommend the daily monitoring of water baths, stage warmers, and other heated surfaces verified by an independent method. Water baths and block heaters are especially notorious for undergoing subtle drifts. The CO2 content as well as temperature of incubators should also be monitored on a daily basis. Water pans within incubators should also be checked for potential bacterial and fungal contamination. Equipment such as balances and other delicate equipment should be calibrated, cleaned and serviced on a routine basis.

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Contaminants can enter and interfere with pre-existing culture environments in many ways. The introduction of potentially cytotoxic material such as syringes, filters (syringe type and receiver type), chemicals for media preparation, embryo culture oils, gases, retrieval needles, plasticware, and glassware can profoundly impact results. For this reason, we have developed an extensive inventory system as well a very strict policy concerning the introduction of these items into our IVF procedures. We leave a comprehensive “paper trail” for each of these items as they become introduced. We have a log book in which we enter all new lot numbers of products as they are introduced into the laboratory for use, as well as dating the package when initially used. This allows us to retrospectively identify any potentially hazardous items. In our experience, the use of plasticware can introduce volatile organic components into the laboratory environment. However, we do not routinely batch test our plasticware since all manufacturers produce plasticware that releases these volatile organics. In order to comply with the College of American Pathologists (CAP) we do periodically test plasticware: especially if we note a severe decrease in sperm survival and embryonic quality. Nonetheless, the continuous assessment of supernumerary embryos in our laboratory indirectly allows us to evaluate the performance of these products. However, we have observed lot to lot variability with other products. For this reason, we perform acute type testing using human spermatozoa for all lots of syringes, as well as syringe type filters, as they are introduced into the laboratory. Particular attention should be paid to the preparation of glassware, spatulas, pasteur pipettes, and any other devices used to manipulate gametes. In our laboratory we soak all glassware in Milli-Q water overnight. The following day, the Milli-Q water is replaced, and the glassware is sonicated for a minimum of 40 minutes. Following sonication all glassware is dried in a dry heat oven. Before packaging, the items are inspected for water spots. All glassware and spatulas are dry heat sterilized for a minimum of eight hours at 123°C. These procedures are followed to minimize the potential presence of endotoxins on glassware. This is especially important with glassware that is used for media preparation. We do not recommend the use of an autoclave to sterilize laboratory materials because of the condensation that forms and may become a source of contamination, namely endotoxins. Recently, water stored in glassware following preparation as described above showed no detectable levels of volatile organic components and heavy metal contaminants following testing. A word of caution should be noted concerning CO2 and triple gas tanks. We have encountered in some instances that the gases expelled by these tanks contained many fine particles of rust. On further investigation we were told by various gas manufacturing plants that if CO2 tanks are stored completely empty and the valves are left open, the interior of these tanks will rust. We have been advised not to allow CO2 tanks to drop

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below 500 psi in order to reduce the likelihood of rust particles leaving the tank. Previously, in-line 0.22µm filters were used to reduce the possibility of introducing rust and similar particles into an incubator environment. Currently, we have added in-line charcoal filters to absorb volatile gases typically found within gas cylinders. We incorporated these charcoal filters because the 0.22µm filters only would filter particulate matter and not gaseous matter. In our IVF program we exchange the 0.22µm gas filters approximately every two months and the charcoal filters with the change of the tank. Furthermore, we use only the highest quality inert plastic tubing to connect gas tanks to incubators. The use of medical grade tubing will assure that no components leach from the tubing into the gaseous stream. The potentially toxic effects of ethylene oxide are well documented.1 The most common form of sterilization of retrieval needles is through the use of this potentially toxic gas. Quite often the manufacturers of these needles will ship to their customers without proper aeration. Therefore it is advisable to allow several weeks to elapse prior to their use. An additional safeguard is to flush these needles with medium at retrieval. We initiated this practice following disappointing results which were attributed to the use of recently gas sterilized retrieval needles. We have currently initiated the policy of allowing off-gassing of all plasticware utilized in the embryology laboratory. Off-gassing refers to allowing volatile gases to escape from polystyrene-based plastics by removing them from their non-permeable packaging. Specifically, we offgas all tissue culture flasks, conical, and test tubes, as well as petri dishes of all sizes for a minimum of 48 hours within the sterile confines of a laminar flow hood. This policy became effective after data were published recently concerning the potential effects that volatile organic components may have on conception.2 Two of many specific compounds used to manufacture laboratory plasticware have been studied in our lab. In brief we found that styrene and toluene had no negative impact on mouse embryo development when embryos were cultured in a micro droplet oil overlay culture system. However, removal of the protective oil overlay and subsequent culture experiments proved that these two components were extremely toxic to mouse embryo development. These subsequent results show the protective nature of an oil overlay system and the preference of these two particular compounds to dissolve in non-polar solvents such as mineral oil. The experience in our laboratory indicates that one of the most common sources of problems is the water used for culture media preparation. After all, water constitutes greater than 95% of its total volume and is very difficult to quality control. Options available to IVF laboratories regarding water and culture media are somewhat limited. Laboratories can purchase ready made media, dry powder components to be dissolved at the time of use and/or prepare media on site from scratch. Each of these various options have their advantages as well as

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disadvantages. Commercially prepared media have a limited shelf life, and the user has no control over the quality of these media, especially concerning the water used in their preparation. Powdered media have a longer shelf life, but the user has no control over the quality of chemicals used in preparation and must purchase or manufacture highly purified water. The purchase of water has no guarantee of high quality since the user has no control over the maintenance of the water purification system. In addition, the water in question is highly dependent upon the initial water source. In our opinion, the preparation of media from scratch allows the user to ensure batch to batch consistency and gives him or her the capability to control quality. However, media preparation is extremely time consuming. Few laboratories have the financial resources, facilities, and technical experience to undertake this tedious task. The solution to this problem is based on one’s philosophy and budgetary constraints. Whichever option a laboratory decides to follow, it is extremely important that all sources of potential contaminants are known and a suitable quality assurance procedure is in place. If a laboratory chooses to manufacture its own water for media preparation, then a very strict maintenance protocol must be developed and followed carefully. CAP also requires that if the laboratory produces its own water, stringent monitoring and protocols are documented. In addition, minimum levels of performance and a course of action if deviation occurs must be in place. In our laboratory we chose to make our media from scratch, using tissue culture tested chemicals and high purity water. In order to ensure that our water is of the highest quality, we monitor the input and output parameters of the reverse osmosis (RO) and Milli-Q system on a daily basis. In addition, we routinely test for the presence of chlorines, chloramines, silica, total organic carbons (TOC), and endotoxins. Our final product water has also been tested at an independent laboratory for the presence of minerals and heavy metals. We reduce the inherent possibility of these contaminants existing by following a very rigorous sanitization and filter exchange policy. We test our final product water for silica and TOC levels daily because the presence of these components are the first indications that the filtration systems in our water system are beginning to break down. We test for the presence of endotoxins every two weeks when culture medium is prepared. The symptoms of endotoxin contamination in IVF culture medium is generally recognized as excessive levels of embryonic fragmentation. Nagata and Shirakawa reconfirmed the widely accepted notion that even low levels of endotoxins can profoundly affect pregnancy rates.3 We also test for the presence of chlorines in our water system after initial filtration to ensure that we are not introducing this ion into our RO system. In addition, we have developed a comprehensive tracking system for all chemicals and products used in culture media preparation. This allows us to easily determine when potentially deleterious variables have been introduced. The major dilemma with this system is that we can only retrospectively

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determine when a potentially hazardous condition developed. It is for this reason that we believe that two types of QC should be performed (continuous and acute). In most instances the quality of culture medium may have a significant impact on pregnancy rates. Therefore, it is justifiable for laboratories to perform batch testing on culture medium prior to its use. The most obvious means of assessing the suitability of culture medium would be by performing some type of bioassay. The most common bioassay in use is the culture of mouse embryos to the hatching blastocyst stage. Mouse embryo bioassay systems have been used for years.4,5 In most laboratories, 1 and 2 cell embryos are cultured for 72 to 96 hours, and the proportion of hatching blastocysts is computed. If less than 75% of the embryos fail to hatch in culture then the medium is deemed unsuitable.6 The appropriateness of this assay system, however, is questioned by many. This is because of the unusual ability of mouse embryos to tolerate adverse culture conditions. Attempts have been made to improve the sensitivity of the mouse embryo bioassay system. Fleethman et al confirmed that the strain of mice chosen as a source of embryos has a tremendous impact on rate of blastocyst development in adverse conditions.7 In their study, 2 cell embryos from CD1 mice developed to the blastocyst stage at exceedingly high rates (>75%) when cultured in medium prepared with Milli-Q water, RO water, or even tap water. Removal of the zona had no significant effect in improving sensitivity. In an attempt to improve this bioassay system, zygotes from B6CBA/F1J were used. These same authors concluded that zygotes from this strain, when allowed to cleave to the 2 cell stage, with subsequent zona removal, offered the highest level of sensitivity. Nonetheless, this assay could not differentiate culture medium prepared in either Milli-Q or RO water. Perhaps a shortcoming in this study was the use of BSA as a protein supplement in the culture medium prepared from all three water sources. BSA may have provided some protective role and thereby reduced the sensitivity of this test (RO water v. Milli-Q water). In addition, the enzymatic removal of the zona pellucida makes this test labor intensive. In an attempt to improve the sensitivity of the mouse embryo bioassay system in our laboratory, we culture zygotes in protein free medium in open dishes. In addition, we evaluate the rate of embryonic development every 24 hours and grade the level of blastocyst formation, expansion and hatching. We perform human embryo culture in an oil microdroplet system routinely. However, mouse embryos are cultured in open dishes so that the oil will not remove any potentially deleterious components. If we are performing QC on oil batches then we routinely run microdroplet systems in conjunction with open dish systems. An alternative to the mouse embryo assay system is the use of hamster epididymal spermatozoa.8 These authors have developed a very sensitive bioassay which can be completed within one working day. However,

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hamster spermatozoa are extremely delicate and require trained personnel to perform this assay without introducing confounding variables. In order to comply with CAP standards, IVF laboratories are required to perform biannual proficiency testing through an approved agency. We have performed these tests using both mouse embryos and human sperm. These blinded tests are performed to determine if IVF laboratories can correctly identify the deficient media. Results from these tests indicate human sperm is sensitive enough to correctly identify the suboptimal media. The use of human sperm may prove to be a more economical bioassay with results that are comparable to mouse embryo data. We have established policies and procedures that allow us to evaluate the overall quality of our laboratory performance. Biannually, using statistical queries, it is determined whether minimum standards are continuously met. Specific minimum standards include: assessing sperm concentration, morphology, and motility, normal fertilization rates, polyspermic rates, embryo cleavage rates, intracytoplasmic sperm injection (ICSI) degeneration rates, cryopreservation survival rates, ongoing pregnancy rates and implantation rates. Specific minimum standards for the aforementioned variables can be found in Table 2.1. We believe that the level of patient care is affected by the level of laboratory cleanliness. This is based on the fact that we maintain embryos outside the incubator for considerable amounts of time in order to perform comprehensive morphological evaluations. The use of a micro-droplet culture system affords us this luxury. To reach these goals of laboratory performance, a weekly cleaning schedule has been established (Table 2.2). Standards have been developed in order to evaluate intertech variation amongst embryologists performing various procedures. Our database allows us to evaluate each embryologists’ impact on pregnancy rates for the following tasks: oocyte retrieval, ICSI, insemination, cumulus removal and fertilization confirmation, culture of zygotes, embryo evaluations, assisted hatching, and embryo replacement. Individual embryologists are compared to the overall laboratory rates for each specific skill (Table 2.3). This evaluation procedure was developed in order to allow each embryologist the opportunity to evaluate their work and make adjustments when appropriate. These evaluations are performed quarterly. We have gone to extreme measures in our IVF laboratory to produce air quality that exceeds levels found in most surgical suites. We have specifically designed our air handling system to not only remove particulate matter but also volatile gases. Our air handling system consists of a dedicated central heating, ventilation, and air condition system (HVAC) system exclusively for the IVF laboratory, which effectively removes 99.995% of 0.3µm and larger particles. For gas contaminant removal, we use a series of AQF 2000 high efficiency gas phase filters. In order to remove large dust particles, we have two inch pleated filters preHEPA, as well as post-carbon filters. This arrangement of filters is designed to effectively remove particulate and gaseous contaminants from

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outside as well as recirculated internal air. In addition, this air handling system provides us the luxury of having frequent air changes per hour. These frequent air exchanges provides a positive pressure in the laboratory relative to the outside surroundings. Prior to installation of our air handling system, we determined the level of outside air pollutants as well as internal air contaminants to be filtered. This is an important factor, since the type and size of carbon

Table 2.1. Biannual verification of embryology procedures. Measurements of embryology procedures Threshold limit Normal fertilization rates >60% Polyspermic rates <10% ICSI degeneration rates <15% Embryo cleavage rates >80% Cryopreservation survival rates >50% Ongoing pregnancy rate >40% Implantation rates >20% Sperm concentration +/− 10% of the mean Sperm morphology +/− 2% of the mean Sperm motility +/− 10% of the mean Table 2.2. Weekly monitoring/cleaning. Week 1 Week 2 Technician: Date: Clean all surfaces HI/LO LN2 levels Order gases Incubator H2O Hood accessories Squeeze bottles Seven X H2O METOH H2O to stage warmers Mop floors (no soaps) Thermometer H2O tubes Biohazard containers Incubator discards

Week 3

Week 4

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Supplies (log all lots) Hot water bath Spordex test dry oven Bacterial test water system Sanitize water system Table 2.3. Embryologists peer review. Embryologists quality measurement Retrieval pregnancy rate Insemination pregnancy rate ICSI pregnancy rate Pronuclear check pregnancy rate Zygote change over pregnancy rate Embryo evaluation pregnancy rate Assisted hatching pregnancy rate Transfer pregnancy rate

Threshold limit >50% +βhCG >50% +βhCG >50% +βhCG >50% +βhCG >50% +βhCG >50% +βhCG >50% +βhCG >50% +βhCG

Table 2.4. Tests carbon filter before and after changes (Institute for Assisted Reproduction, Charlotte, NC). Area tested Gases (ppb) Gases (ppb) 12/20/99 3/8/00 Embryology lab 73 42 Media lab 71 35 Cryo lab 67 34 Procedure room 93 37 Manifold room 100 75 Outside fresh air 69 42 Post return mixed air 68 65 Post carbon filters 64 65 filtration pellets is effected by quantity and type of pollutants. Since there are no standardized levels of air contaminants acceptable for IVF laboratories, each program must establish its own level of maximum air contamination. This is a difficult task since common techniques used for air quality measurement are either not suitable for low level analysis or have the ability to measure the variable composition of the IVF laboratory. Measurements must be sensitive at the microgram per cubic meter level or better; which far exceeds the measurement standards established and used by the environmental protection agency. Therefore,

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exact determination of environmental contaminants can be exceedingly expensive. We randomly selected to have our air monitored on a yearly basis to determine if and when our carbon and HEPA filters became exhausted. The filters of concern were the carbon filters since most HEPA filters will remain efficient for two to three years. Table 2.4 describes the ambient air in our laboratory after approximately one year since our last carbon filter change. The levels found in each room of the IVF laboratory suite represents the presence of gases at the ppb level, but it does not distinguish the particular type of gases present. Results as presented in Table 2.4 indicate a slight improvement in the level of ambient air. This improvement also coincided with an increase in pregnancy rates in our facility despite the presence of construction in the immediate vicinity. We have noted in the past a decrease in pregnancy rates of approximately 5% to 10% when construction was ongoing in the immediate vicinity of the IVF lab. However, other factors such as embryo transfer efficiency and population may account for the difference in pregnancy rates. It is of interest to note that HEPA filtration maintains an efficiency of 99.9% or greater when filters are changed out approximately every two to three years. In addition, based upon this and other gas data analysis we have decided to replace our activated carbon filters every 10 months before complete exhaustion of these filters; this seems to occur 10–12 months after installation. We have yet to determine if we note transient increase in gas contaminants during the summer months when the air quality in our city decreases as a result of ozone effects. This discussion has illustrated that there are many forms of QC/QA that can be implemented to assure an optimum standard of performance is maintained. We believe that the incorporation of two forms of quality control may improve the ability to detect the introduction or presence of harmful contaminants in an IVF laboratory. A continuous type of QC allows for ongoing monitoring of overall present conditions, whereas the acute form of QC allows for the testing of individual components before their use. IVF laboratories should contemplate implementation of these two methods to minimize variation from the accepted standard. Incorporation of a technique to evaluate the impact of individual embryologists on pregnancy rates will further ensure that optimum conditions exist within the embryology laboratory. In fact, very high standards of quality must be maintained in IVF laboratories today. This is imperative given the various advanced techniques available to embryologists. The full beneficial impact of these techniques can only be fully realized in a laboratory that maintains an overall excellent standard of care.

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REFERENCES 1 Schiewe MC, Schmidt PM, Bush M, Wildt DE. Toxicity potential of absorbed/retained ethylene oxide residues in culture dishes on embryo development in vitro. J Anim Sci (1985); 60:1610–8. 2 Cohen J, Gilligan A, Esposito W, Schimmel T, Dale B. Ambient air and its potential effects on conception in vitro. Hum Reprod (1997); 12:742–9. 3 Nagata Y, Shirakawa K. Setting standards for the levels of endotoxin in the embryo culture media of human in vitro fertilization and embryo transfers. Fertil Steril (1996); 65:614–9. 4 Ackerman SB, Swanson RJ, Stokes GK, Veeck L. Culture of mouse embryos as a quality control assay for human in vitro fertilization. Gamete Res (1984); 9:145–52. 5 Boone WR, Shapiro SS. Quality control in the in vitro fertilization laboratory. Theriogenology (1990); 33:23–50. 6 Gerrity M. Mouse embryo culture bioassay. In: Wolf DP, Bavister BD, Gerrity M, Kopf GS, eds. In vitro fertilization and embryo transfer: a manual of basic techniques. New York: Plenum Press (1988):57–76. 7 Fleethman 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. 8 Bavister BD, Andrews JC. A rapid sperm motility bioassay procedure for quality-control testing of water and culture media, J In Vitro Fertil Embryo Transfer (1988); 5:67–75.

3 Accreditation of the ART laboratory: the North American perspective Brooks A Keel, Tammie K Schalue

INTRODUCTION Currently, in the United States, the assisted reproductive technologies (ART) laboratory is subjected to minimal standards and guidelines. Although at first glance it may appear that the laboratory component of ART is heavily regulated in the US, careful scrutiny will reveal that, with exception to a few state regulations, virtually all of the standards put forth for regulating the ART laboratory are voluntary and carry no sanctions for non-compliance. One can avoid regulation by simply choosing to do so. In many regards, the ART laboratory has managed to slip through cracks in the regulatory system that governs all other clinical laboratories in this country. The reasons that ART has avoided regulatory oversight are many and hotly debated, but center around one primary point of contention: the definition of the term clinical laboratory as it relates to ART, and the subtle differences between the practice of medicine (therapy), which is essentially devoid of legislative oversight, versus laboratory testing (diagnosis), which is heavily regulated.1 Few will argue that, for example, a laboratory performing a semen analysis or hormone assays is a “diagnostic laboratory” and should be subject to federally mandated oversight. However, many view the procedures carried out in the embryology laboratory, including oocyte isolation, fertilization, embryo development and transfer, as part of the patient’s treatment and that no useful information is gleaned from these procedures. Thus, the embryologist is involved in the patient’s therapy (the practice of medicine) and the oversight mechanisms which govern diagnostic testing do not apply to this “laboratory.” Some have gone so far as to say that these are not “laboratories” at all, and would prefer the term “embryo culture rooms” or “embryo intensive care units.” The situation regarding the andrology laboratory, in contrast to the embryology laboratory, is somewhat clearer. The testing performed in the andrology laboratory, such as the counting of sperm, the assessment of motility and forward progression, and the determination of morphology, all are clearly diagnostic procedures in nature, and as such, are covered by

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federal mandatory oversight. However, it is when these procedures are performed as a integral part of the ART procedure that the lines delineating what is considered diagnostic testing (mandatory oversight) and therapeutic procedures (voluntary oversight) become blurred. Even though the embryologist may be evaluating sperm using the exact same procedures employed by the andrologist, many feel that these procedures are not covered by existing mandatory federal oversight. Thus, federal oversight of the andrology laboratory is mandatory, while oversight of the embryology laboratory is voluntary, even though these two “laboratories” may be housed in the same room, and the “embryologist” and the “andrologist” are often the same person. Basically, there are two laws that either directly or indirectly regulate the ART laboratory in the United States. The Clinical Laboratory Improvement Amendments of 1988 (CLIA’88)2 is the federal law which sets the standards for almost all laboratories in the United States.1 Although individual states may pass laws which govern laboratory testing within their boundaries, these state laws must be at least as strict as CLIA’88. CLIA’88 rules oversee all clinical laboratory testing in the United States except forensic laboratories, research laboratories that do not report patient results, and drug-testing laboratories. Compliance with CLIA’88 is mandatory, and there are strict penalties for noncompliance. The Fertility Clinic Success Rate and Certification Act (FCSRCA), also known as the Wyden Bill,3 is aimed specifically at “embryo laboratories” and does not address classical laboratory testing. Compliance with this law is completely voluntary, and there are no sanctions for noncompliance. The premise behind the creation of what may appear to be duplicating regulatory laws relates to the above disagreement over whether the activities which take place in the ART laboratory are diagnosis, and therefore come under CLIA’88, or therapy, and therefore require separate and distinct regulatory language (hence, FCSRCA).

THE CLINICAL LABORATORY IMPROVEMENT AMENDMENTS OF 1988 (CLIA’88) CLIA’88 defines a laboratory as “a facility for the biological, microbiological, serological, chemical, immunohematological, hematological, biophysical, cytological, pathological, or other examination of materials derived from the human body for the purpose of providing information for the diagnosis, prevention, or treatment of any disease or impairment of, or the assessment of the health of, human beings.”2 Anything that falls under this encompassing definition must abide by rules of CLIA’88. Under CLIA’88, all laboratories, regardless of size, location, numbers or types of testing must meet minimal standards based on a test complexity model.4 All laboratory tests are categorized as being either waived, moderate complexity, or high complexity.

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Categorization of tests depends upon many factors but primarily relate to the degree of difficulty and interpretation required for successful test performance. In general, CLIA’88 provides standards for six main aspects of laboratory testing: proficiency testing (PT), patient test management, quality control (QC), personnel requirements and responsibilities, quality assurance (QA) and inspections and sanctions. Specific requirements for meeting these standards differ depending upon the complexity of testing being performed. Any testing associated with the ART laboratory falls within the high complexity category, and for this reason, we will limit discussion to this category herein. PROFICIENCY TESTING PT is a process of external, interlaboratory quality control whereby simulated patient samples are tested by participating laboratories, and the performance of the individual laboratory is compared with the collective performance of all participants.5 All laboratories in the United States engaged in high complexity testing are required to enrol in a government approved PT program, if such a program is available, and failure to achieve satisfactory performance in PT may result in sanctions against the laboratory.1 Currently, the American Association of Bioanalysts (AAB) and the College of American Pathologists (CAP) are the only government approved PT programs offering PT in andrology and embryology. The results of the AAB PT program in embryology6 and andrology7 have recently been reported. These results indicate an urgent need for improvement in the quality of andrology testing. PATIENT TEST MANAGEMENT Patient test management is one of the most important aspects of CLIA’88, especially as it relates to the handling of gametes. Standards associated with patient test management help to ensure that a specimen is properly collected, labeled, processed, analyzed, and that accurate results are reported. To do this, CLIA’88 divides clinical testing into three main processes: preanalytic, analytic and postanalytic. Positive identification of the patient and his/her specimen is maintained throughout these three processes to ensure proper chain of custody of the specimen from the time of collection, through testing, to accurate reporting of test results. The preanalytic standards ensure that patients are properly informed about specimen collection; that adequate labeling, preservation, transportation and processing of the specimen takes place; and that the laboratory only performs tests which are requested by authorized individuals. The analytic standards ensure that the actual testing is performed in such a way as to provide accurate and reliable results. A major part of this process is laboratory QC (see below). The postanalytic standards ensure that laboratory tests are reported in a timely manner; that the test report form

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itself is accurate and meaningful; that test results are only reported to authorized individuals; and that the reporting process protects the patients confidentiality. QUALITY CONTROL QC, according to CLIA’88, involves a more broad approach to laboratory testing than merely including a known positive and negative sample in each assay. It is a comprehensive program that covers all aspects of the laboratory, including facilities; test methods, equipment, instrumentation, reagents, materials and supplies; procedure manual; establishment and verification of method performance specifications; equipment maintenance and function checks; calibration and calibration verification procedures; assay control procedures; remedial action; and QC records.1 QC in ART may seem problematic, especially in the andrology laboratory. However, several novel approaches have been proposed.8,9 Another unique aspect of the embryology laboratory involves the need, or lack thereof, for quality control testing of media.10 Controversy exists as to whether media used for oocyte/embryo culture need to be QC tested in light of the fact that most commercial media now are pretested prior to shipment. CLIA’88 does allow the laboratory to use manufacturer’s control checks of media provided that the manufacturer’s product insert specifies that the manufacturer’s quality control checks meet the national standards for media quality control. The laboratory must document that the physical characteristics of the media are not compromised and report any deterioration in the media to the manufacturer. The laboratory must follow the manufacturer’s specifications for using the media and be responsible for the test results. PERSONNEL REQUIREMENTS AND RESPONSIBILITIES Laboratories performing high complexity testing must identify five qualified individuals to assume the responsibilities of the director, clinical consultant, technical supervisor, general supervisor, and testing personnel. One single individual may assume the role of one or more of these positions. In fact, a single individual, if qualified, may assume the role of all five. This is not unusual in small laboratories. Each individual assuming these positions must meet certain well defined qualifications based on formal education, training and experience. In general, to qualify for the director, an individual must either: (1) be a board certified pathologist or a licensed physician with two years’ experience directing or supervising testing; or (2) possess an earned doctoral degree in science with four years’ experience in clinical laboratory testing, two years of which must be at the level of supervisor or director. In addition, as of 31 December 2000, the non-physician director must obtain board

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certification by one of several government approved certification boards.11 Currently, the American Board of Bioanalysis (ABB) is the only approved board offering certifying in the specialities of andrology and embryology. As of 1 January, 2000, the ABB has certified more than 200 high complexity clinical laboratory directors (HCLD) in the specialities of andrology and embryology. In addition, although certification of technical supervisors is not required by CLIA’88, the ABB has certified nearly 190 individuals in these specialities at this level as well. The responsibilities of the director are numerous, broad, and all encompassing.1 In general, the director is responsible for the overall operation and administration of the laboratory, including the employment of personnel who are competent to perform test procedures, record, and report test results promptly, accurately, and proficiently, and for assuring compliance with the applicable regulations.1 Although the duties of the director may be delegated to others, he or she must maintain the responsibility. The director must be accessible to the laboratory at all times, but this accessibility may be achieved through telephone or electronic means. However, a single director may direct up to five individual laboratories, even at distinct and distant geographical sites. The clinical consultant is responsible for assisting the laboratory’s clients in ordering appropriate tests and interpreting test reports. The technical supervisor carries a number of responsibilities including selection and verification of test methodologies, enrollment in PT, establishing QC programs, resolving technical problems, and identifying training needs and evaluating competency of testing personnel. The general supervisor is responsible for providing the day to day supervision of high complex test performance by the testing personnel, and the testing personnel’s primary responsibility is performing accurate testing. QUALITY ASSURANCE CLIA’88 requires each laboratory to establish and follow written policies and procedures for a comprehensive QA program designed to monitor and evaluate the ongoing and overall quality of the total testing process.1 QA attempts to address all aspects of the preanalytic, analytic and postanalytic processes in a continuous fashion. It is a system that monitors not just how well an incubator holds temperature, or the accuracy and precision of an internal assay control, but also considers other relevant things such as communication with physician clients and continuing education of laboratory employees. This process is usually best monitored by a periodic (monthly) QA meeting, which includes the laboratory personnel and, if possible, the ordering physician and his/her staff. Specifically, CLIA’88 requires the laboratory to address several things, including: • monitoring all aspects of patient test management mentioned above, including criteria established for patient preparation, sample collection, quality control, test requisition

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and test reporting; • documenting problems that occur as a result of breakdowns in communication between the laboratory and the authorized individual who orders or receives the results of test procedures. Furthermore, corrective actions taken to resolve the problems and minimize communications breakdowns must be documented; • documenting all complaints and problems reported to the laboratory. Investigations of complaints must be made, when appropriate, and as necessary corrective actions are instituted, with ongoing monitoring to minimize reoccurrences; • documenting of all QA activities including problems identified and corrective actions taken. INSPECTIONS AND SANCTIONS The United States Government Department of Health and Human Services (DHHS) ensures laboratory compliance with CLIA’88 standards by performing on-site inspections at least once every two years. The actual entity which performs the inspection, on behalf of DHHS, depends on the state in which the laboratory is located and the type of certificate the laboratory has requested. Laboratories have the option of being inspected by representatives of their respective State Departments of Health, or requesting that inspections be conducted by one of several private accrediting agencies. The most common of these is the CAP, the Commission on Office Laboratory Accreditation (COLA), and the Joint Commission on Accreditation of Health Care Organizations (JCAHO). For practical reasons, the vast majority of these on site inspections are announced, rather than surprise unannounced visits. These inspectors typically use detailed checklists to determine if the laboratory is in compliance with all of the CLIA’88 standards. If deficiencies are noted, the laboratory is given an opportunity to correct these problems. Failure to correct the noted deficiencies, or other violations of CLIA’88 may result in a range of sanctions, which may include suspension, limitation or revocation of the laboratory’s certificate, civil suit against the laboratory, or imprisonment or fine for any person convicted of intentional violation of CLIA’88 requirements.1 Fines can range from $50 to $10000 per day. In addition, the secretary of DHHS is required to publish annually a list of all laboratories that have been sanctioned during the preceding year. Thus, participation in CLIA’88 is not a matter of choice, and failure to comply with these standards carries stiff penalties.

THE FERTILITY CLINIC SUCCESS RATE AND CERTIFICATION ACT OF 1992 In 1988, in response to consumer concerns about the conduct of ART programs and the apparent lack of uniform information relating to the pregnancy success rates, Congressman Ron Wyden conducted public

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hearings to address these concerns.12,13 Later that same year, a survey was sent to the directors of all ART programs to determine clinic specific success rates, and the results of this survey were released at a second hearing held in 1989. Congressman Wyden held a press conference on 21 June 1990 and introduced the Fertility Clinic Success Rate and Certification Act (FCSRCA), which became widely known as the Wyden bill.13 The final version of the Wyden bill was passed into law on 24 October 1992.3 The FCSRCA was intended to provide the public with comparable information concerning the effectiveness of infertility services and to assure the quality of such services by providing for the certification of embryo laboratories.3,14 Basically, FCSRCA consists of two components. The first component prescribed a mechanism whereby each clinic performing ART in the United States would report its clinic specific pregnancy rates on an annual basis. The Secretary of DHHS charged the Centers for Disease Control and Prevention (CDC) with the responsibility for collecting, analyzing, and reporting these pregnancy data. The CDC contracted with the Society for Assisted Reproductive Technology (SART), an affiliated society of the American Society for Reproductive Medicine (ASRM), to use the registry system15–18 that they had in place to voluntarily collect clinic specific pregnancy data from its member clinics. With the passage of the FCSRCA, SART’s voluntary registry system became mandated by law. Currently, in the United States, all clinics performing ART procedures are required to submit their clinic specific pregnancy data to SART for subsequent reporting to the CDC. The second component of FCSRCA called for the secretary of DHHS, through the CDC, to develop a model program for the certification of embryo laboratories to be administered by the States.3 In developing such a model, the CDC consulted with various consumer (RESOLVE) and professional groups (the ASRM, SART, and the AAB) who had expertise and interest in ART laboratory services. The standards that were to be developed include standards to assure consistent performance of laboratory procedures; a standard for QA and QC; standards for the maintenance of all laboratory records (including laboratory tests and procedures performed, as well as personnel and equipment records); and a standard for personnel qualifications.3,14 Interestingly, these standards were prohibited from establishing any such regulation, standard or requirement that has the effect of exercising supervision or control over the practice of medicine in ART programs.3 Furthermore, compliance with the standards set forth by FCSRCA is completely voluntary, and no sanctions were prescribed for noncompliance. After several years of consultation and planning, the final notice of the model program for embryo laboratory certification was published in July of 1999.14 The model laboratory program defined an Embryo laboratory as a “facility in which human oocytes and sperm, or embryos, are

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subjected to ART laboratory procedures”.14 It further defined ART laboratory procedures as: All laboratory procedures for handling and processing of human oocytes and sperm, or embryos, with the intent of establishing a pregnancy. These procedures include, but are not limited to, the examination of follicular aspirates, oocyte classification, sperm preparation, oocyte insemination, assessment of fertilization, assessment of embryo development, preparation of embryos for embryo transfer, and cryopreservation of specimens. The model program for laboratory certification consists of four main sections: personnel qualifications and responsibilities; facilities and safety; quality management; and maintenance of records. PERSONNEL QUALIFICATIONS AND RESPONSIBILITIES The FCSRCA states as a guideline that the embryo laboratory should employ one fully trained individual for every 90–150 ART cycles performed annually, and at least two qualified individuals should be available to provide appropriate services. According to the model program, the embryo laboratory must identify three individuals: director, supervisor and reproductive biologist. If qualified, one individual may assume the responsibilities and role of more than one position. The qualifications for each position are shown in Table 3.1. In general, the director is responsible for the overall operation, administration, and technical and scientific oversight of the ART laboratory, and is charged with hiring qualified personnel to perform the ART laboratory procedures. The director does not have to be physically present during procedures, but must be accessible to the laboratory to provide on site consultations by telephone or electronic means as needed. The supervisor, as the name implies, is responsible for providing day to day supervision and oversight of the embryo laboratory operation and the personnel performing ART laboratory procedures. The supervisor must be accessible to the laboratory to provide on site, telephone, or electronic consultation to resolve technical problems. It is this individual who is charged with the responsibility of ensuring proper training of all laboratory personnel. The reproductive biologist, synonymous with CLIA’88 defined “testing personnel,” is responsible for performing the ART laboratory procedures and for recording and reporting procedural outcomes. This individual is limited to independently performing only those procedures in which training has been documented. All other procedures must be performed under direct and constant supervision.

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Table 3.1. Qualifications of ART laboratory personnel, according to the CDC Model Program (FCSRCA). Director Supervisor Reproductive biologist State license, if required; State license, if State license, if required; AND required; AND AND Physician or doctoral Qualified as Director; Qualified as director or scientist; OR OR MS or BS in supervisor; OR BS in science; OR science; OR Serving as director on or Serving as Supervisor Serving as reproductive before July 20, 1999; AND on or before July 20, biologist on or before July 1999; AND 20, 1999; AND 2 years’ pertinent experience, Pertinent training (time Pertinent training (time including 6 months’ training not specified); AND not specified); AND in an ART laboratory; AND Personally performed 60 ART Personally performed Personally performed 30 procedures; AND 60 ART procedures; ART procedures; AND AND Document 1.2 CEUs in ART Document 1.2 CEUs in Document 1.2 CEUs in or clinical lab annually; AND ART or clinical lab ART or clinical lab annually; AND annually; AND If doing ART procedures, If doing ART Perform each ART perform at least 20 annually. procedures, perform at procedure at least 20 times least 20 annually. annually. FACILITIES AND SAFETY The model program states that the lab must provide adequate space and appropriate environmental conditions to ensure safe working conditions and quality performance of ART laboratory procedures. These standards not only provide for aseptic conditions required for successful ART procedures, but also ensure a safe working environment for employees. It further mandates that all federal, state, and local regulations be followed regarding the use of human and animal materials and hazardous chemicals. QUALITY MANAGEMENT The model program spells out standards for a comprehensive quality management program that is designed to monitor and evaluate the ongoing ART laboratory procedures performed and services provided. The program contains standards for the make up of the ART laboratory procedure manual, which are similar in scope to those outlined in

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CLIA’88. The program provides standards for proper maintenance and calibration of all equipment used in the ART laboratory, including 24 hour monitoring of applicable equipment and emergency backup in the event of power failure. The standards state that the laboratory must maintain records of the batch or lot number, date of receipt and date placed in use of all reagents and media, and must also verify that materials which come in contact with sperm, oocytes and embryos have been tested and found to be non-toxic either by the ART laboratory or by the manufacturer. The model program standards, in keeping with the spirit of CLIA’88, stipulate that the laboratory must obtain written or electronic requests from an authorized person (usually a physician) before performing any procedure on patients gametes or embryos. Any additional applicable information, including verification of informed consent, must also be obtained before performing procedures. The laboratory must establish written procedures and criteria for: (1) evaluation and assessment of oocyte morphology and maturity, fertilization, and embryo quality; (2) insemination schedule relative to oocyte maturity; (3) the volume, numbers and quality of sperm used for insemination of each oocyte; (4) disposition of oocytes with an abnormal number of pronuclei, as well as disposition of excess oocytes; (5) the time period following insemination for examination of fertilization; (6) micromanipulation of oocytes and embryos; (7) re-insemination of oocytes; (8) length of time embryos are cultured prior to transfer, and medium and protein supplementation used for transfers; (9) types of catheters available, and circumstances when each should be used; (10) methods of transfer and techniques for checking the catheters post-transfer for retained embryos; and (11) disposition of all excess embryos. Similarly, the model plan contains standards requiring detailed records on the outcome of each of the above mentioned steps, including the identity of the individual performing each step. The standards require duplicate log books or files for cryopreserved samples. Prior to implementing any procedure in the ART laboratory, appropriate performance measures of the procedure must be verified and documented. For each batch of culture media prepared in house, the quality of the media, including pH, osmolality, and culture suitability using an appropriate bioassay system, must be confirmed. The model program standards allow the laboratory to accept quality control procedures performed by the manufacturer if commercially prepared media are used. Lastly, the model program stipulates that the ART laboratory must establish and follow a written quality assurance program aimed at monitoring the quality of ART laboratory services provided, and to resolve problems identified. The details of this QA program are virtually identical to that described in CLIA’88, with the inclusion of ART specific standards including the requirement to track and evaluate fertilization rates, cleavage rates, and embryo quality.

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MAINTENANCE OF RECORDS The model program provides standards for the retention of records of all of the laboratory’s policies and procedures; personnel employment training, evaluations and continuing education activities; and quality management activities. The laboratory must maintain these records for a period of time specified by federal, state, and local laws or for 10 years beyond the final disposition of all specimens obtained during each patient’s ART cycle, whichever is later. The standard requires that all records must be maintained on site for at least two years. SANCTIONS AND ENFORCEMENT The secretary of DHHS is instructed to publish annually a list of laboratories who have not complied with the standards of FCSRCA, but the non-compliant laboratory is free to continue to offer ART laboratory services. The final rule stated that while that it was anticipated “that the costs of federal and state monitoring and oversight of embryo laboratories would be covered by the fees paid by participating laboratories, participation by embryo laboratories is voluntary and laboratories not willing to pay these fees would not be limited in their ability to operate.”14 So far, embryo laboratories in the US have “not indicated they would opt into such a voluntary program.”14 The final ruling further stated that “while the model certification program for embryo laboratories does not provide for a federal oversight role, we do believe that this model provides an excellent resource for states that wish to develop their own programs and professional organizations with an interest in establishing or adopting standards for the embryo laboratory.”14 Thus, the major difference between the laboratory standards spelled out in CLIA’88 and the FCSRCA is that compliance with CLIA’88 is mandatory while FCSRCA is voluntary.

THE CAP/ASRM REPRODUCTIVE LABORATORY ACCREDITATION PROGRAM The CAP is a private organization which has offered inspection and accreditation of clinical laboratories in the United States for many years. The CAP is one of several private accrediting agencies recognized by the federal government as meeting or exceeding the standards of CLIA’88. Thus, laboratories who choose CAP as a vehicle for accreditation can meet the standards set down by Federal law. As early as 1991, the members of the ASRM began collaborations with the CAP towards the development of a voluntary accreditation program specific for ART laboratories.13 This collaboration took advantage of the ART expertise of the ASRM along with the years experience of CAP in performing on site inspections and accreditation of clinical laboratories. Out of this

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collaboration developed the reproductive laboratory accreditation program (RLAP). The RLAP also provides a mechanism by which the individual states could, on a voluntary basis, implement the model certification program prescribed by FCSRCA. The CAP/ASRM RLAP provides “an opportunity for laboratory improvement through voluntary participation, professional peer review, education, and compliance with established performance standards.”19 Through the use of an extensive checklist, the RLAP inspects and subsequently accredits ART laboratories through a process involving onsite inspectors who evaluate the laboratory in the areas of test methodologies, reagents, control media, equipment, specimen handling, procedure manuals, test reporting, record keeping, PT, personnel qualifications, facilities, safety, and overall laboratory management. Failure to meet criteria described in the checklist results in a deficiency, and the laboratory is provided with an opportunity to document corrective actions to address these deficiencies. Accreditation is granted by CAP when the laboratory has documented correction of all deficiencies and has responded to all of the recommendations of the CAP. Inspectors are assigned by one of two regional commissioners. Inspections are performed by a peer process. The inspectors, recognized experts in the areas of embryology and andrology and typically practicing laboratory directors and supervisors, are not employed by CAP and volunteer their services for this purpose. The CAP is “deemed” by CLIA’88 as an approved accrediting agency, which is to say that the criteria by which CAP uses to accredit laboratories is at least as strict as the standards set down in CLIA’88. ART laboratories opting to use CAP as its accrediting agency meet the federal mandatory oversight requirement for the andrology laboratory, as well as voluntary oversight of the embryology laboratory. Currently, SART requires the ART laboratories associated with its member clinics to become accredited as a contingency of membership. Currently, the CAP/ASRM and JCAHO are two private accrediting agencies recognized by SART as meeting this membership requirement. This has had a positive effect regarding oversight of ART laboratories in the United States, resulting in a large increase in ART laboratories seeking accreditation. However, although the majority of ART programs in the United States are currently members of SART, the absolute number of centers offering ART services to patients in this country is unknown because registering ART clinics is not required and membership in SART is voluntary.

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JOINT COMMISSION ON ACCREDITATION OF HEALTHCARE ORGANIZATIONS The Joint Commission on Accreditation of Healthcare Organizations (JCAHO), founded in 1951, is a private, non-profit organization dedicated to improving the quality of care in organized healthcare settings. The JCAHO, like CAP, is recognized by the federal government as meeting or exceeding the standards of CLIA’88. JCAHO’s laboratory accreditation program has updated their 2000 survey list to include questions that are specific to the embryology laboratory. With the adoption of these new survey questions ART laboratories now have a choice of two accrediting organizations with inspection processes designed to be specific for the ART laboratory. By choosing either of these programs and successfully obtaining accreditation, ART laboratories can meet the federal standards set down in CLIA’88. There are many similarities between the CAP and JCAHO system of accreditation. They both emphasize professional review for compliance with established performance standards and, through education, seek to improve overall patient care and safety within the laboratory setting. However, there are slight differences in how the two organizations accomplish their goals. The JCAHO uses full time paid inspectors for the inspection process instead of professional peers from within the same field, as does CAP. The reasoning behind this from JCAHO’s prospective is that a professional inspector adds consistency to the inspection process. Like CAP, the JCAHO uses a list of questions to aid the inspector in determining whether a particular laboratory has met all of the requirements. However, unlike CAP where each question will result in an all or none response of a deficiency or no deficiency, the JCAHO system may use several questions to determine the final score in a section. The JCAHO system is divided into two main sections: organization functions and technical functions. Within organization functions there are an additional six subsections: improving organization performance; leadership; management of the laboratory environment; management of human resources; management of information; surveillance, prevention, and control of infection. Within technical functions there are an additional three subsections: quality control; waived testing; and special type I recommendations. Each of the subsections may also have several parts, or grid elements, as termed by JCAHO. Each grid element is then given a score from 1 to 5 on the basis of the answers to the questions relating to that part. The 1 to 5 score is based on degree of compliance within the last inspection period (four months for first time inspections). A rating of 5 would indicate non-compliance, 4—minimal compliance, 3—partial compliance, 2—significant compliance, and a rating of 1 would indicate substantial compliance during the last inspection period. The score in each

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grid element is then used to obtain a percent and it is this score that primarily determines if a laboratory is accredited. A laboratory may receive one or more of what is termed a type I recommendations and still obtain accreditation. All type I recommendations must be addressed and notification of this sent to JCAHO within 30 days of the inspection or the laboratory would be considered not to be in compliance with the accreditation procedure, and measures would be started to revoke their accreditation. The JCAHO, unlike CAP, considers the inspection to be an open public form. The JCAHO requires all laboratories undergoing inspection to post notice of the date of the inspection at least 30 days prior to the inspection to allow for public input. This process allows clients the opportunity to voice any complaints they may have against the laboratory becoming accredited. This complaint process is closely monitored by JCAHO and only fully substantiated complaints are considered in the accreditation process.

THE FOOD AND DRUG ADMINISTRATION (FDA) In September 1999, the FDA proposed new regulations that require manufacturers of human cellular and tissue based products to register with the federal government, and to screen and test donors of cells and tissue for risk factors and for clinical evidence of relevant communicable agents and diseases.20 These proposed regulations will extend to ART procedures in that donor sperm and donor embryos are considered to fall under this rule. The proposed rules state that the human cellular or tissue based product must be quarantined until completion of determination of donor suitability. For reproductive cells, this quarantine is intended to be for at least six months after the date of original donation, and at this time the donor is then to be retested to ensure a disease free state. According to the proposed rules, the donors of reproductive tissue (sperm, eggs and embryos) are to be tested for HIV type 1 and 2, hepatitis B and C virus, and Treponema pallidum. In addition, donors of leukocyte rich cells or tissues (such as semen) are required to be tested for HTLV I and II and CMV. Testing for Chlamydia trachomatis and Neisseria gonorrhea is also required unless the material being donated is procured by a method that ensures freedom from contamination of the cells by infectious disease organisms that may be present in the genitourinary tract (i.e. by laparoscopy). The only exception to the proposed rules for donor testing and quarantine is when the donated material is intended for autologous use, or the material is donated by a sexually intimate partner of the recipient. The total impact of these proposed rules on the field of ART is at present uncertain. Testing and quarantining of donor semen has been the standard of care in the United States for years. However, standards for the testing of embryo donors are not as well defined, although attempts at

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establishing guidelines have been made.21 The possibility of having to subject donor embryos and eggs to a six month quarantine will be problematic, to say the least. However, as mentioned, these rules are “proposed” and not yet final. The final ruling will not be published until public comment on these rules is heard and considered.

THE NORTH AMERICAN PERSPECTIVE The perspective of North America regarding accreditation of the ART laboratory is complex, debatable and varied. This disparity is centered around the very definition of a “laboratory” and what constitutes a diagnostic test versus a therapeutic procedure. If the manipulation of male and female gametes, and the resulting embryos by the ART laboratory truly constitutes therapy only, then the oversight of these procedures comes under the auspices of the practice of medicine, rather than the governance of Federal law. There is a real fear in the United States that obligatory oversight, especially that which is mandated by the federal government, may compromise the practice of medicine and severely limit the ability of physicians to bring state of the art infertility treatments to their patients. With few exceptions, no one in North America argues that oversight of the ART laboratory is necessary. It is, however, the form of this oversight that is contested, and the way in which this oversight should be controlled that is debated. Currently, regulatory oversight of the andrology laboratory is mandatory, whereas regulation of the embryology laboratory is optional. Whether this will change in the future remains to be determined.

REFERENCES 1 Keel BA. The assisted reproductive technology laboratories and regulatory agencies. CLIA’88, CAP, and the Wyden Bill. Infertil Reprod Med Clinic North Am (1998); 9:1047. 2 Clinical Laboratory Improvement Amendments of 1988: Final Rule. Federal Register (1992); 57:7002. 3 PL 102–493. The Fertility Clinic Success Rate and Certification Act of 1992. 102nd Congress, Second Session, 1992. 4 CLIA’88 final rules: a summary of major provisions of the final rules implementing the clinical laboratory improvement amendments of 1988. Northfield, IL: The College of American Pathologists, 1992. 5 Stull TM, Hearn TL, Hancock JS, et al. Variation in proficiency testing performance by testing site. JAMA (1998); 279:462. 6 Quinn P, Keel BA, Serafy NT Jr, Serafy NT Sr, Schmidt CF, Horstman FC. Results of the American Association of Bioanalysts (AAB) embryology proficiency testing (PT) program. Proceedings of the 55rd

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Annual Meeting of the American Society for Reproductive Medicine (1998); S100 (abst). 7 Keel BA, Quinn P, Schmidt CF Jr, Serafy NT Jr, Serafy NT Sr, Schalue TK. Results of the American association of bioanalysts national proficiency testing programme in andrology. Hum Reprod (2000); 15:680. 8 Tomlinson MJ, Barratt CLR. Internal and external control in the andrology laboratory. Keel BA, May JV, DeJonge CJ, eds. Handbook of the Assisted Reproduction Laboratory, Boca Raton, FL, CRC Press (2000); 269. 9 Johnson CA, Kellum TA, Boldt JP. Quality assurance in the embryology, andrology and endocrine laboratories. Keel BA, May JV, DeJonge CJ, eds. Handbook of the Assisted Reproduction Laboratory, Boca Raton, FL: CRC Press (2000); 279. 10 Go KJ. Quality control: a framework for the ART laboratory. Keel BA, May JV, DeJonge CJ, eds. Handbook of the Assisted Reproduction Laboratory, Boca Raton, FL, CRC Press (2000); 253. 11 Extension of Certain Effective Dates for Clinical Laboratory Requirements Under CLIA. Federal Register (1998); 63:55031. 12 Lawrence LD, Rosenwaks Z. Implication of the fertility clinic success rate and Certification Act of 1992. Fertil Steril (1993); 59:288. 13 Visscher RD. Partners in pursuit of excellence: development of an embryo laboratory accreditation program. Fertil Steril (1991); 56:1021. 14 Implementation of the Fertility Clinic Success Rate and Certification Act of 1992-A Model Program for the Certification of Embryo Laboratories; Notice. Federal Register (1999); 64:39374. 15 Society for Assisted Reproductive Technology, The American Fertility Society: Assisted reproductive technology in the United States and Canada: 1991 results from the Society for Assisted Reproductive Technology generated from The American Fertility Society Registry. Fertil Steril (1993); 59:956. 16 Society for Assisted Reproductive Technology, The American Fertility Society: Assisted reproductive technology in the United States and Canada: 1992 results generated from the American Fertility Society/Society for Assisted Reproductive Technology Registry. Fertil Steril (1994); 62:1121. 17 Society for Assisted Reproductive Technology, American Society for Reproductive Medicine: Assisted reproductive technology in the United States and Canada: 1993 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril (1995); 64:13. 18 Society for Assisted Reproductive Technology, American Society for Reproductive Medicine: Assisted reproductive technology in the United States and Canada: 1994 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril (1996); 66:697.

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College of American Pathologists. Reproductive laboratory accreditation program; standards for accreditation. American Society for Reproductive Medicine, 1996. 20 Suitability Determination for Donors of Human Cellular and TissueBased Products. Federal Register (1999); 64:52696. 21 Guidelines for Gamete and Embryo Donation. Fertil Steril (1998); 70 (suppl. 3).

4 Accreditation of IVF laboratories: the European perspective Cecilia Sjöblom

INTRODUCTION Quality assurance and accreditation are concepts that seem to touch on a wide range of functions in our society. Quality control (QC) systems are specially needed in units for assisted reproductive technologies (ART) to assure reproducibility of all methods and 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. In 1996 the Swedish national board of health and welfare raised demands for quality assurance in health care systems.1 To meet these requirements and to establish and maintain high quality in our ART program we developed a system according to international standards. During the development of industrialism at the end of the 19th and the beginning of the 20th centuries, there was a growing need for safety regulations concerning conditions of obvious risk to the citizens. That is how the regulations for occupational safety and health, consumer protection, the handling of explosives, safety at sea, and electrical safety came into existence. These systems were often voluntary systems to begin with, but were later taken over, or given a more or less official status, by society. The first standards for quality systems were drafted within the US defense force, and later by the North Atlantic Treaty Organization (NATO). In Australia and New Zealand this activity was already supported by accreditation systems, mainly for laboratories, in the 1940s. At a later stage accreditation systems in some European countries and the US were established. Such establishment normally occurred in connection with national campaigns to improve the quality status of industry. To begin with, most of these industry supported certification systems aimed at assessing the properties of a product before it was put on the market. QC systems originally created for industry have later also been applied in other types of activities such as management of organizations and services like health care. Over the years different national and international standards for QC systems have been developed. Standards that have been applied for health care includes the ISO 9000 series and the EN 45000 series.2–4 National and international bodies have been established with the purpose to further

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develop the standards to be more general so they can be applied to different activities and try to harmonize standards between different countries. The Swedish board for accreditation and conformity assessment (SWEDAC) is approved by the European co-operation for accreditation (EA) to be the authority responsible for accreditation in Sweden. Over the years that ART has been practiced in both large and small clinics much knowledge has been gained how to run an ART laboratory and what methods to use in order to achieve ultimate success. Facing the new millennium we encounter other variables such as the safety and efficiency of the lab, and quality control 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.5,6 Among others, England and the United States have instituted a system where the ART clinics have to be authorized to practice ART.7,8 In such systems the clinic as well as the laboratory can be audited by this third party authority.9 A system for regular auditing of an ART unit makes it possible for our society to get an insight into how units practice the technique according to legislation and professional guidelines. Furthermore, the audit also provides a possibility for feedback between the authority and the ART clinics. Thus, in general it is positive to have a system, either internal or external, for auditing the work performed in an ART unit. In Sweden there has been an act since 1989 (SOSFS 1989:35) controlling all activities within the field of ART and in 1996 the Swedish national board of health and welfare raised demands for quality assurance in health care systems.10 EN 4500111/ISO 1702512 is an effective means of enabling a clinic to fulfil the national board of health and welfare regulations concerning quality assurance. The guidelines set out in this international standard facilitate the necessary transition from method oriented quality work to more system oriented work. STANDARDS EN 45001 is the required document used within Europe for laboratories. 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 900113 is the document used for a quality system of the whole company or clinic. At an audit the quality system of the different parts of the company/clinic like personnel economy and sales department is audited. The technical part of the activities is not audited. One could say that the audit is broad but not specialized. Most clinical testing laboratories in Europe are accredited according to the European norm EN 45001. This means that there is considerable experience within SWEDAC and other European accreditation bodies for

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auditing according to the EN 45001 in medical disciplines. At seeking internationally approved accreditation for an IVF laboratory this standard is an obvious first choice. Other standards applicable to the activities and quality system in the IVF laboratory are General requirements for the competence of testing laboratories ISO/IEC Guide 25.14 The first drafts of the EN 45001 were originally modeled on the corresponding ISO/IEC guide.14 Today, however, the two standards show a number of differences. The bodies responsible for developing standards in Europe, Comité Européen de Normalisation (CEN) and the International Standardization Organization (ISO), have realized the danger of developing standards which for a given field are not identical. Both organizations are now determined to aim at total harmonization of the existing guides and standards. At the beginning of 2000 the new standard ISO 17025 was issued,12 which covers both the EN 45001 and ISO/IEC Guide 25. This new laboratory standard agrees well with the new ISO 90001:2000. Together with the international standards, SWEDAC has approved minimal guidelines for methods within the field of ART. Since neither SWEDAC nor any other similar authority in Europe had previous experience in accrediting ART laboratories, a group of experts within the field of ART in Sweden was assembled. The guidelines this group developed were issued as an official document approved by SWEDAC in 1999.15

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. These principles needs to be accepted regardless if the activity they carry out is encompassed by the EU or national regulations for conformity assessments or merely by voluntary requirements, when industries or specific buyers require harmonized quality system standards. The European conformity assessment system is based on European standards in the EN 45000 series. These European standards are more or less identical to the ISO/IEC guides for conformity assessment. When the EU’s new 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, certification bodies, 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 sector and within the mandatory sector. The latter should also be based on relevant standards in the EN 45000 series of standards. The European Commission therefore

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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 into EA, the European cooperation for accreditation. EA covers accreditation in all fields of conformity assessment activities.16

Fig 4.1 EA members: nationally accepted accreditation bodies. The European cooperation for accreditation. EA covers accreditation in all fields of conformity assessment activities. At present the EA members represent nationally recognized accreditation bodies from 24 EU and EFTA countries.

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The European Commission states that accreditation must be a transparent, independent, and non-commercial activity. If these requirements can be assured it would ensure that accreditation could 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, and that accreditation bodies should not engage in other conformity assessment activities so as to not compromise their independence and integrity.4 At present the EA members represent nationally recognized accreditation bodies from 24 countries in the EU and the European Free Trade Area (EFTA) countries (Fig 4.1). 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 activities by the other members. 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 will therefore have 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 EA. On the global level, the regional bodies as well as the national accreditation bodies cooperate in the 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 EA as a regional body and national accreditation bodies from all parts of the world.

THE QUALITY POLICY The quality policy is the highest document in a quality system and outlines the company overall objectives and also includes statements of the clinics standard of performance to be obtained and maintained. The policy statement is usually formed by the board of directors and shall be issued under the authority of the chief executive according to ISO 17025 4.2.2. The standard demands a policy for the laboratory but while forming one you can also state a quality policy that covers every part of the clinic, including the laboratory. The policy shall include a statement of the management’s intentions with the operations and actions of the clinic and a statement about the

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importance of a quality system. The quality policy shall also include demands on the personnel concerning their qualifications and education for the job they perform. They have to be familiar with the quality system and its documentation, and the policies and procedures shall be fully implemented with all personnel. The management should state its commitment to good professional practice and quality of its testing and calibration in servicing their clients. Finally, the policy shall include a statement of commitment to compliance with the international standard. The policy should be short, concise and not an essay. The answers to how the statements in the policy shall be fulfilled will be answered in underlying documentation and shall not be dealt with in the policy itself. Most companies today take help from professionals to form an effective and powerful quality policy. The policy is frequently used both in client/patient information and advertising and can usually be found framed in the company/clinic lobby (the quality policy of Fertilitetscentrum, appendix I).

THE QUALITY MANUAL AND BUILDING OF A QUALITY SYSTEM The main purpose of the quality manual is to outline the structure of the documentation used in the quality system.17 It shall 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 insuring 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 is shown in Fig 4.2. A good quality manual shall 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 the system is to make up a table of contents for the quality manual, and there are some crucial chapters and documents that should be found in a manual.

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Fig 4.2 Quality system requirements. The elements of the system are separated into different levels with the quality manual as the highest document. The quality manual and related quality documentation outlines the clinic policies and includes manuals for the standardized routines at all levels of work in the laboratory. IDENTITY This chapter clearly states the identity of the clinic or laboratory, its address and official registration and organization numbers. Since the quality manual usually is an official document shown to both visitors and clients, it is good to include a bit of company history and background in this brief chapter. QUALITY POLICY (ISO 17025 4.2.2) As discussed previously, short powerful and according to the standard. THE QUALITY SYSTEM (ISO 17025 4.2) This chapter should contain a description of the system structure, the different levels and on what standards the system is built in accordance with. An important feature is to outline the areas covered by the certification and accreditation or both. An accreditation according to EN 45001 or ISO 17025 is aimed towards accredited 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

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references or methods for validation. However, in this part of the manual there should be a list of which methods in the laboratory are covered by the accreditation and an authority approved accreditation CV that states the percentiles of variations acceptable within the methods. The laws, regulations, and legislation under which the laboratory and clinic work should be listed with full references. All the documentation of these should be kept as underlying documentation and as controlled documents included in the system and kept accessible for all personnel. This chapter shall also 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. DOCUMENT CONTROL (ISO 17025 4.3) According to EN 45001, ISO 17025, and the ISO 9000 standards the laboratory or clinic shall 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 quality system. Since it is the part 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 laboratory or clinic. Firstly, ensure that the system you choose covers the demands of the standards. A checklist for document control is found in appendix II. The identification of the document should be logical and it is a good suggestion to use numbers. The same identification number could then be used for the file name within the computerized version. The issue number in brackets or after a dash could follow this number. The 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 together with a company logo. AUDITS AND MANAGEMENT REVIEWS (ISO 17025 4.13 AND 4.14) Internal and external audits are tools for improving and keeping your system up to date with the standards. The quality manual shall include specific instructions covering both how and how often they shall be performed (ISO 17025 4.13). The management usually chooses internal auditors, and they should be familiar with both the standards and the

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activities performed in the lab. The manual should include a document describing approach and areas of responsibility for the internal auditors. The national authority for conformity assessment performs the external audits. There are written standards according to which the audits are performed (ISO 10011).18 The quality manual should therefore include a reference to this standard together with a statement of full accessibility to all documentation and localities for the auditors. Together with the audits, the management review is important for the improvement of the system and for long term corrections of errors and incidents that might occur. According to ISO 17025 4.14 the management of the laboratory 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 fulfill the demands in the standard. MANAGEMENT AND ORGANIZATION (ISO 17025 4.1) Every quality system has to have a clear organization plan. This overview is usually made up as a flow chart with the management on top and departments and different sections of the clinic/laboratory under. The chart should, according to ISO 17025 4.1.4, clearly define the organization and management structure of the laboratory, its place in any parent organization (such as a clinic), and the relation between management, technical operations, support services, and the quality system. Apart from the organization plan this document should point out all managers of the laboratory and clinic with executive responsibility (ISO 17025 4.1). There should be defined descriptions on the demands of these managers in respect to education, experience, areas of responsibilities and where they are located in the organization plan. Two managers who play a key part in the accreditation of a laboratory are the technical manager, usually named lab manager or director, and the quality manager. The lab director holds the technical responsibility of the activities in the laboratory, whereas the quality manager is responsible for assuring that the activities are performed in accordance with the laboratory quality system. The quality manager shall have direct access to the highest level of management at which decisions are taken. PERSONNEL (ISO 17025 5.2) There should be defined descriptions on the demands on all personnel groups within the lab in respect to education, experience, areas of responsibilities, 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.

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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 have a goal with respect to further education and training. There should also be a clear statement of secrecy and confidentiality. INCIDENTS AND COMPLAINTS (ISO 17025 4.8; 4.9; 4.10; 4.12) The laboratory should have a policy and procedure for the resolution of incidents and complaints received from patients, clients or other parties. The routines of how these are filed and corrective actions are taken should be documented in the quality manual. When applying a quality system it is important not to hide these incidents and complaints, but to use them as recourses to improve the system. The management reviews shall assure that the collective incidents and complaints lead to long term corrections and improvements of the quality in the lab. METHODS (ISO 17025 5.4) The quality manual should include documentation of the methods used in the laboratory. Usually the description in the manual itself is very brief and refers to underlying documentation as SOPs, method manuals, or working manuals. The quality manual should include the layout of the method manual. An example of layout is found in appendix I. OTHER CHAPTERS OF THE QUALITY MANUAL ACCORDING TO ISO 17025 Laboratory facilities 5.3 Equipment 5.5 Calibration and traceability 5.6, 5.9 Documentation of results 5.10 Insurance Filing 4.12 METHODS AND STANDARD OPERATING PROCEDURES As stated previously, the quality manual should include documentation of the methods used in the laboratory. The description in the manual itself is very brief and refers to underlying documentation as SOPs, method manuals, or working manuals. The quality manual should include the layout of the method manual. According to ISO 17025 5.4 the laboratory shall use appropriate methods and procedures for all tests and or calibrations within its scope. This includes sampling, handling, transport

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storage, and preparation of the items tested. The methods shall include measurement uncertainty as well as statistical techniques for analysis of test and or calibration data. There should be clear descriptions on how to handle and operate relevant equipment and all instructions shall 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 labs the only EA (SWEDAC) approved standard is the minimal guidelines for methods within the field of ART.15 This is a minimal standard for the methods sperm analysis and preparation, analysis, and handling of oocytes and embryos, including scoring of these. When it comes to method such as intracytoplasmic sperm injection (ICSI), freezing, and thawing of embryos there is no existing EA approved standard. These methods should, when accredited, follow the demands of laboratory developed methods or non-standardized methods (ISO 17025 5.4.3, 5.4.4). Since the general requirements of the methods are quite laborious it is a good idea to separate the method descriptions into different levels of the system. The methods manual should be at a high level and the SOP or working manual at a lower level. THE METHOD MANUAL The method manual should be at a high level in the system and include all the demands from the general requirements. As all other documentation the methods manual 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 analytical principles shall include a theoretical description of the method and review of current literature. References are usually put last in the document. The method manual should outline the competence demands on personnel performing the test. All methods have to be validated regularly and the methods manual should include information on how and how often validations are done and should be in accordance with ISO 17025 5.4.5. Sampling and handling of test items should include the sampling procedures and the physical environmental issues such as temperature and pH. Remember that all variables in the manual such as those referring to the measurement of temperature have to be followed by a description of how the temperature is measured, and how often and how the thermometer is calibrated. A part of the methods manual shall deal with the labeling of samples. Considering the risks associated with the work in an IVF lab,19 the marking should be logical and clear to completely eliminate the risk of mixing of samples. External and internal controls should be applied when applicable for the method. Photos could be used for scoring of oocytes and embryos and video together with fixed samples are good controls for sperm analysis.

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The document should state the periodicity of the controls and descriptions of how they are performed. All equipment used for the methods should be listed in the methods manual 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. Reporting of results shall be accurate, clear, unambiguous and objective. Result documentation should follow the documentation procedure set by the quality system and include the requirements of the standards (ISO 17025 5.10). Sources of errors and uncertainty of measurements should be stated and properly calculated for each method. Apart from the issue of documents and document control the methods manuals shall define who in the management has the method responsibility and medical responsibility. The actual operating procedure should just be brief and refer to the lower level working manual or SOP. This is preferable since small changes are frequently made in working descriptions, such as the names of products used, and usually those changes do not effect the method itself; hence the changes are made in a lower level document. SOP OR WORKING MANUAL The SOP or working manual can be a lower positioned document and include the exact work descriptions, material media used and so on. Routines for changes in these manuals should be easy, and usually the laboratory manager, rather than the clinical director, has the responsibility and authority to issue changes in the working manual. This is only achievable if the manual is on a lower level in the system, and it is a good way of avoiding unnecessary paperwork.

EXPERIENCE FROM ACCREDITATION OF THE LABORATORY AT FERTILITETSCENTRUM Introducing and fully implementing a quality system in the laboratory at Fertilitetscentrum meant that some crucial changes have been made within the lab. All testing equipment is calibrated and verified regularly and the calibration certificates indicate the traceability to national standards. The equipment is clearly marked with the date of the last calibration, and certificates are kept in the equipment folders. The test methods follow the guidelines given by the expert group and are annually validated. Internal and external controls are used on applicable methods. The laboratory has a responsibility to keep up to date with the latest technology in the field and one of the goals stated in the quality policy is that new techniques shall be evaluated promptly, methods

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developed efficiently, and outdated and inefficient technologies superseded. Certificates and reports issued by the laboratory are designed to meet the given standards. Each has a given number, and in the footer the issue number and date, the address of the laboratory, and the name and signature of the person who has approved the layout of the certificate or report. The rules for how the reports/certificates are controlled and directed in the quality system are given in the quality manual. All reports or certificates that include an accredited method are marked with the SWEDAC symbol and our accreditation number. The embryologist who has performed the analysis and procedure or both signs the report or certificate. There is also a documented policy and procedure for the resolution of complaints and how to use these for improvement of the system. All personnel have a responsibility to be a part of the improvement and the resolutions of complaints discussed at the weekly laboratory meetings. To ensure a high standard of the laboratory facilities and to get the best environment possible for the patient’s gametes and embryos there are some curtail routines that have been adapted in the lab: All work is done in class I hoods. All culture media, disposals, and lab ware are tested by using mouse embryo assay and endotoxin LAL test. A record of batch number of all culture medium, disposals, and lab ware used for a particular patient are kept in the protocol for each treatment. Incubators are controlled every day with regard to temperature, humidity, and CO2. A measurement of pH of media equilibrated in each incubator is done twice a week to confirm the right CO2 flow. The gas to the incubators is purity class 4.8. The laboratory is under constant over pressure. The hydrocarbon concentration in the air (VOC) shall not exceed 100 ppt. The temperature in the laboratory is registered every day and shall at all time be 20±2°C. MOUSE EMBRYO ASSAY; CLINICAL ASPECTS The goal of every in vitro fertilization (IVF) clinic is to assist those couples with infertility problems who are seeking help, to provide them with the treatment best suited to their needs, and to help them to achieve a pregnancy and birth of a healthy child. The treatment chosen should be the best possible in accordance to every couple’s medical background. Highly competent personnel shall monitor the care of every couple. To achieve this goal it is important to implement a good quality control system to ensure reproducibility of all methods and competence in all duties performed by the personnel.

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The embryo culture environment is an important factor in a successful IVF-ET program. It is the responsibility of the IVF laboratory to establish and maintain a stable, non-toxic, 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. Small variations in temperature, pH, and the physical properties of media such as pH, and osmolarity will inevitably effect the outcome of an IVF cycle. Embryo toxic substances in the media 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, and different assays and variations of them to get increased sensitivity have been suggested.20 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 indicates 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 excess embryos to the blastocyst and hatched blastocyst stages is another way of human embryo assay frequently used. The problem with this assay is that these poor quality embryos might suffer from genetic abnormalities. Conclusions from these culture results therefore are hard to make, 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 lab ware that comes in contact with the patients gametes and embryos. At Fertilitetscentrum we have chosen to use the mouse embryo assay for the testing, since this method, if used properly, provides the best means of testing resembling the human embryo culture system. There are two major parts of our testing system: the assay itself to detect toxicity and our record batch numbers to be able to identify the source of problems. We, like many other smaller privately owned units, have neither the economical nor the laboratory resources to facilitate a standardized mouse embryo assay. Nevertheless, we do have 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 end point 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. It is important to demand a copy of this certificate before the release of a product into the laboratory. If the product fails to meet our testing demands we send them to a test laboratory for testing (Scandinavian QC laboratories, Göteborg, Sweden). This assures that we get our equipment tested in the way that we find proper, and we get a certificate from every test.

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The other part of our testing system is the recording of batch numbers. A record of the batch numbers of material used in a treatment cycle is kept together with the patients case file. We have a computerized case file system and each cycle has a batch record page attached. This page includes a full list of culture media and lab ware and the batches in use. With a simple mouse click we mark what materials we have used in every step of the cycle, from culture media down to pipette tips. Together these two parts creates the foundation of our error search system. In the case of a drop in fertilization rate, embryo quality, and, subsequently, pregnancy rate, we can easily obtain computer lists with these variables connected to each batch. There have been occasions where we have approved a batch with its testing certificate from the producer, which later in our error search system have proven embryo toxic. The first action we take then is to stop the use of that particular batch. We then send the item for a MEA test, and it usually comes back with the result—embryo toxic. This indicates that transport conditions and storage times effect embryo toxicity, especially with plastic ware. 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 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, but at Fertilitetscentrum we test all culture media and lab ware that come in contact with the patient’s embryos and gametes. To test the performance of our incubators, we run mouse embryo assays within them. This is performed during times when no patient material is stored within the incubators. Again, an accredited company does this, and control embryos are cultured in their incubators. We have had this system in practice for three years now, and we conclude that, if done carefully, a good quality control system will prevent fluctuations in results often seen in an IVF lab. This is a costly process and a future goal for IVF clinics worldwide should be to get the producers of media and lab ware to do tests of batches in their product line in a proper way and label them as IVF products. This would mean a sharing of costs and make high quality accessible for all IVF clinics. THE STAFF To maintain a high quality and standard in the laboratory it is important that the staff enjoy the work environment and are offered training to enable them to increase their competence in accordance to their field of work and personal abilities. To ensure this, all personnel employed at the laboratory are educated to achieve competence to perform all methods in the lab. The laboratory director and the clinical director issue a

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competence certificate when their training is finished. This certificate is kept in the personnel folder together with the curriculum vitae and a work and responsibility description for every person. The need for further training is discussed annually at meeting with the personnel manager. The clinic encourages research and participation at national and international conferences.

CONCLUDING REMARKS 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 interlaboratory 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 on uncertainty determination and reporting of results. In February 2000 the European Society of Human Reproduction and Embryology special interest group for embryology issued Guidelines for good practice in IVF laboratories.21 These guidelines partly incorporate standards previously published by the UK Association of Clinical Embryologists.22 A step towards harmonization could be to get these guidelines approved as standards by the EA. Throughout completing the long and work intensive process of applying a QC system in our ART laboratory one might have asked what it has meant for the laboratory. There is no doubt that introducing a fully implemented QC system has standardized the methods and the way in which the embryologists perform their work in the laboratory. The troubleshooting, the maintenance of the equipment, and the milieu improved and were standardized. This guarantees an optimal handling of the couples’ 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 shall guarantee a 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

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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 I QUALITY POLICY The goal of Fertilitetscentrum AB is to assist those couples with infertility problems who are seeking our help, to provide them with the treatment best suited to their needs, and to help them to achieve a pregnancy and birth of a healthy child. The treatment chosen should be the best possible in accordance to every couple’s medical background. Highly competent personnel shall monitor the care of every couple. All samples provided by the couple—their gametes and embryos—shall be handled according to the latest technology available. The samples shall be correctly marked to prevent mistakes and complete confidentiality shall be provided. The personnel shall enjoy the work environment and be offered education to enable them to increase their competence in accordance to their field of work and personal abilities. Fertilitetscentrum AB is striving to be one of the leading fertility clinics in Sweden with respect to clinical practice and research. AMBITION To achieve the goal stated in the quality policy, the company should provide necessary economical recourses so that the work is done according to a quality assured system, with competent ambitious personnel. The content of the quality system and routines shall be implemented among all personnel. The documentation of the quality system shall be available for all personnel. The laboratory shall fulfill the requirements of the EN 45001 accreditation. GOAL The team management group, sets optimal standards for success rates at an annual meeting and is documented in the Minutes. New techniques are evaluated promptly, and methods are developed efficiently. Outdated and inefficient technologies are superseded.

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ASSESSMENT OF GOAL ACHIEVEMENT • The treatment results are continuously presented to the personnel, board of directors, and the national board of health and welfare • a quality report regarding the laboratory performance is annually presented to the board of directors and is available to all personnel • the results of the patient questionnaires are presented annually to the board of directors and all personnel • the results from the quality auditing are presented to the team management group biannually

APPENDIX II

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Document control According to ISO 170 25 4.3 Questions that should have an answer in your document control system: Is ALL documentation in the lab or clinic covered by your doc control system? Who writes or changes the document? Who approves and has the authority to issue documents? Does the document have: a unique identification? issue number and current revision status? date of latest issue? pagination?

• Where can I find the document—physical location, level in the system, and on computer file? • Who assures that only the latest issue of the document is present in the system, removes outdated issues, and files them? • Are amendments to documents clearly marked, initialed, and dated? • How are changes in a document implemented with the personnel?

REFERENCES 1 The Swedish national board of health and welfare act on quality assurance in healthcare systems SOSFS 1996:24.

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2 Haeckel R, Kindler M. Effect of current and forthcoming European legislation and standardization on the setting of quality specifications by laboratories. Scand J Clin Lab Invest (1999); 59:569–73. 3 Libeer JC. Total quality management for clinical laboratories: a need or a new fashion? Acta Clin Belg (1997); 52:226–32. 4 The European quality assurance standards EN ISO 9000 and EN 45000 (1997). European Commission Doc Certif 97.4. 5 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. 6 Clinical and laboratory guidelines for assisted reproductive technologies in the Nordic Countries: NFOG bulitinen supplement 1997:3 7 Dawson KJ. Quality control and quality assurance in IVF laboratories in the UK. Hum Reprod (1997); 12:2590–1. 8 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. 9 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. 10 The Swedish national board of health and welfare act on assisted reproduction. SOSFS 1989:35 11 EN 45001. General criteria for the operation of testing laboratories. 1989. 12 EN ISO/IEC 17025. General requirements for the competence of testing and calibration laboratories. 1999. 13 ISO 9001:2000. Quality management systems. 2000. 14 ISO/IEC Guide 25. General requirements for the competence of calibration and testing laboratories. Third edn, 1990. 15 SWEDAC document ME 46b. Handling, storing, culture and cryopreservation of fertilized eggs and embryos. 1999. 16 Ettarp L. An overview of international conformity assessment systems. WTO Technical Working Group, (1999). 17 Huisman W. Quality system in the medical laboratory: the role of a quality manual. Ann Biol Clin (Paris)(1994); 52:457–61. 18 ISO 10011. Guidelines for auditing quality systems. 1992. 19 Van Kooij JR, Peeters MF, te Velde ER. Twins of mixed races: consequences for Dutch IVF laboratories. Hum Reprod (1997); 12:2585–7. 20 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.

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21 Gianaroli L, Plachot M, van Kooij R, et al. ESHRE Guidelines for good clinical practice in IVF laboratories. Committee of the Special Interest Group on Embryology. Hum Reprod (2000); 15:2241–46. 22 McDermott A, Avery S, Blower J, et al. Accreditation standards and guidelines for IVF laboratories. London: ACE Subcommittee on Laboratory accreditation, Association of Clinical Embryologists (1996).

5 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 has been described and continues to be used in the evaluation of female infertility, the male has been essentially neglected. Most programs offering advanced reproductive technologies (ART) in 2000 apparently employ an only cursory evaluation of the male—rarely extending beyond semen analysis (with or without strict morphology), and antisperm antibody detection. Several factors certainly account for this disparity. First, most practitioners of assisted reproductive technologies (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 (and 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 methods, 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 then penetrate through the cumulus, bind to the zona pellucida, penetrate through the zona, fuse with the oolemma, and then undergo nuclear decondensation. 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

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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 will review the most commonly employed techniques for sperm evaluation, and examine the issues surrounding their utility in the modern ART program. PATIENT HISTORY

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A thorough history of the infertile couple at the time of the consultation will often reveal conditions that could affect semen quality. Some of the important factors to consider are: Reproductive history of the couple, including previous pregnancies with this and other partners. Sexual interaction of this couple, including frequency and timing of intercourse and duration of their attempt to become pregnant. Past medical and surgical history, 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.1 This decreases the diagnostic information that can be obtained from a single analysis, often necessitating additional analyses. What is also apparent from literature analyzing 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 partner. The commonly accepted standard for defining the normal semen analysis is the criteria defined by the World Health Organization (WHO). These parameters are listed in Table 5.1. COLLECTION OF THE SPECIMEN When the semen analysis is scheduled, instructions should be given to the couple to ensure the collection of an optimum semen sample. Written instructions are useful, especially if the patient is collecting the specimen outside the clinical setting. During the initial evaluation, a specimen should be obtained following a two to seven day abstinence from sexual activity.2 A shorter period of time may adversely affect the semen volume and sperm concentration. A longer abstinence may reduce the sperm motility. In light of the natural

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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 a minimum of seven days later can eliminate tensions associated with the initial semen collection and give another count from which a typical set of semen variables can be determined. This second collection may also be used to appropriately adjust the abstinence period. Masturbation is the preferred method of collection. The use of lubricants is discouraged since most are spermicidal.1 However, some mineral oils may be 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

Table 5.1. WHO normal values for semen analysis. Parameter Normal values Liquefaction Complete within 60 minutes at room temperature Appearance Homogeneous, gray, and opaque Odor “Fresh” and characteristic Consistency Leaves a pipette as discrete droplets Volume >2ml pH 7.2–8.0 Concentration 20 million sperm/ml semen or greater Motility 50% or more with forward progression, or 25% or more with rapid progression within 60 minutes of collection Morphology 30% or more with normal forms Viability 75% or greater Leukocytes Less than 1 million/ml Immunobead Less than 20% with adherent particles test MAR Test Less than 10% with adherent particles 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 of previous contents, which can kill or contaminate the sperm. Delivery of the semen to the lab should occur within sixty minutes of collection, and

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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 husband’s name as well as a unique identifying number. Typically, a social security number, birth date, or 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 1. 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 semiliquid to a liquid. Before this process is completed, sperm are contained in a gel-like matrix that prevents their even distribution. Aliquots taken from this heterogeneous distribution of sperm for the purpose of determining concentration, motility, or morphology may therefore not be accurate. As liquefaction occurs over 15–30 minutes, sperm are released and distributed throughout the semen. Incomplete liquefaction may adversely affect the semen analysis in several ways. The coagulum that characterizes newly 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. This highly viscous semen prevents the homogenous 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 occur only after the semen volume has been measured.

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2. SEMEN VOLUME Semen volume is best measured with a serological pipette that is graduated to 0.1ml. This volume is recorded and later multiplied by the sperm concentration in order to obtain the total count. A normal seminal volume is considered to be >2ml.2 3. SPERM CONCENTRATION A variety of counting chambers is available for determining sperm concentration. The more commonly used include the haemocytometer, the Makler counting chamber, and the MicroCell. Regardless of the type of chamber used, an aliquot from a homogenous, mixed semen sample is placed onto a room temperature chamber. The chamber is covered with a glass cover slip, which allows the sperm to distribute evenly in a very thin layer. Sperm within a grid are counted. Then the total number of sperm counted is divided by the number of rows or squares used within the grid. 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. If the sperm are killed and diluted before placing them on a grid, inaccuracy can occur due either to the dilution or to the heterogeneous distribution of the non-motile sperm on the grid. 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, complicating the definition of a normal range for sperm concentration. Demographic studies employing historic controls were used to define a sperm concentration of less than 20 million/ml as abnormal.9,10 Although several investigators observed that significantly fewer pregnancies occurred when men had sperm counts below 20 million/ml, the prognosis for pregnancy did not increase proportionately to the sperm concentration above this threshold. 4. SPERM MOTILITY • • • •

Sperm motility may be affected by many factors including the following: The patient’s age, health, and length of time since 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. This will prevent the influence of the heat from the microscope light source from influencing the results. If a chamber is used to count the sperm, the motility can be determined at the same time as the concentration by using

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a multiple click counter to tally motile and non-motile sperm and then totaling this number to arrive at the 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 sperm, either 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 antibodies.2,3 Motility is one of the most important prerequisites to 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 to penetrate the layers of coronal cells surrounding the egg as well as the zona pellucida and the egg’s cell membrane (oolemma). An exact threshold level of motility required to cause fertilization and pregnancy, however, has never been described.9 This may be due to the variables in equipment and technique used in assessing motility. 5. PROGRESSION

0 1 2 3 4

Whereas sperm motility represents the quantitative parameter of sperm movement expressed as a percentage, sperm progression represents the quality of sperm movement expressed in a subjective scale. A typical scale, such as the one below, attempts to depict the type of movement exhibited by most of the sperm on a chamber grid. With the advent of successful microassisted fertilization, scales such as this have assumed more limited utility. —no motion —motion with no forward progression —erratic movement with slow forward progression —moderate speed with relatively straightforward motion —rapid forward progression3 6. SPERM VITALITY When a motility evaluation yields a low number of moving sperm (less than 50%), a vitality stain may be performed. This is a method used to distinguish nonmotile sperm that are living from those that are dead. This technique is discussed later in the sperm function section.

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7. ADDITIONAL CELL TYPES While observing sperm in a counting chamber or on a slide, additional cell types may also be seen. These include epithelial cells from the urethra, immature sperm cells and white blood cells. Both immature sperm cells and white blood cells are round. In order to distinguish between them, a thin-layer aliquot of semen can be placed on a slide and air dried. The cells are fixed to the slide and stained using a Wright-Giemsa or BryanLeishman stain. These slides may be observed under 400× or 1000× in order to differentiate the cell types, primarily by the appearance of their nuclear morphology. Spermatids may have two or three round nuclei within a common cytoplasm. While polymorphonuclear leukocytes may also be multinucleate, the staining methods will reveal their characteristic nuclear bridges and irregular-shaped nuclei.2 The presence of greater than 1 million white blood cells per ml of semen may indicate an infection, which could also contribute to infertility.1,2 8. SPERM MORPHOLOGY Sperm morphology can be assessed in several ways. The most common classification systems are the third edition WHO standard and the Kruger strict criteria method (fig 5.1). The WHO method requires either a wet slide prep or a fixed, stained slide. A 10–20 microliter drop of semen is prepared on a slide. After placing a coverslip over the specimen, morphology may be determined. Alternatively, the specimen may be mixed with an equal volume of fixative and methylene blue prior to fixing it on the slide. At least 100 sperm must be counted at 400× or 1000× with bright field or phase contrast microscopy. WHO criteria for assessing normal forms include the following: Head • Oval, smooth • Round, pyriform, pin, double, and amorphous heads are all abnormal Midpiece • Straight, slightly thicker than the tail Tail • Single, unbroken, straight, without kinks or coils A normal semen analysis should contain at least 30% normal sperm using WHO criteria.2 In order to perform Kruger strict criteria, sperm morphology is evaluated by placing 5 microliters of liquefied semen on a slide, making a thin smear and air drying at room temperature. The slide is then fixed and stained with a Diff-Quik kit. Slides are read using bright field microscopy under 1000× or higher magnification. At least 100 sperm should be counted for an accurate evaluation.

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The Kruger criteria for assessing normal forms include the following (fig 5.2):11,12 Head Smooth, oval configuration Length: 5–6 microns Diameter: 2.5–3.5 microns Acrosome: must comprise 40–70% of the sperm head

Fig 5.1 Different types of sperm malformations. Reproduced from reference 58.

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Fig 5.2 Diagramatic representation of quickstained spermatozoa. A. Normal form B1. Slightly amorphous head B2. Neck defect C1 and 2: Abnormally small acrosome C3: No acrosome C4: Acrosome >70% of sperm head Reproduced from reference 11. Midpiece • Slender, axially attached • Less than 1 micron in width and approximately 1.5× head length • No cytoplasmic droplets larger than 50% of the size of the sperm head Tail • Single, unbroken, straight, without kinks or coils • Approximately 45µm in length

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As described by Kruger et al, sperm forms that are not clearly normal should be considered abnormal. Normal sperm morphology greater than 14% should be interpreted as a normal result. Normal morphology 4–14% should be considered to be borderline, and normal morphology less than 4% should be considered abnormal.11,12 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 or absent motility. This reduced 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.13,14 In addition to compromised motility, abnormal sperm do not appear to bind to the zona of the egg as well as normal sperm. This has been demonstrated in studies employing the hemizona binding assay.15 In vitro fertilization has helped further elucidate the part that normal sperm morphology plays in the fertilization process and in pregnancy. Both methods of determining normal sperm morphology, the 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.12,16,17 This method of assessing normal sperm morphology, because of its precise, non-subjective nature, establishes a threshold below which abnormal morphology becomes a contributing factor in infertility. COMPUTER ASSISTED SEMEN ANALYSIS Computer assisted semen analysis (CASA) was initially developed to improve the accuracy of the 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 5.2).18 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 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 5.3).18 In this manner, hyperactive motion can also be

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detected and recorded. Hyperactive sperm exhibit a whiplike, thrashing movement, which is thought to be associated with sperm that are removed from seminal plasma and ready to fertilize the oocyte.18,19 Persistent questions about the validity and reproducibility of results have kept CASA from becoming standard equipment 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 oligospermia, counts may be overestimated owing to the machine counting debris as sperm. High concentrations of sperm may be underestimated, as individual sperm cannot be accurately counted in the presence of clumping. Sperm concentration also seems to be closely related to the type of counting chamber employed. Similarly, the process of dilution can interfere with accurate motility determination.19,20 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 the subject of much debate.

Table 5.2. Kinematic measurements in CASA. Symbol Name Definition VSL Straight-line Time average velocity of the sperm head along a velocity straight line from its first position to its last position VCL Curvilinear Time average velocity of the sperm head along its velocity actual trajectory VAP Average path Time average velocity of the sperm head along its velocity average trajectory LIN Linearity Linearity of the curvilinear trajectory (VSL/VCL) WOB Wobble Degree of oscillation of the actual sperm-head trajectory around its average path (VAP/VCL) STR Straightness Straightness of the average path (VSL/VAP) ALH Amplitude of Amplitude of variations of the actual sperm-head lateral head trajectory about its average trajectory (the average displacement trajectory is computed using a rectangular running average) RIS Riser displacement Point to point distance of the actual sperm-head trajectory to its average path (the average path is computed using an adaptive smoothing algorithm) BCF Beat-cross Time average rate at which the actual sperm frequency trajectory crosses the average path trajectory HAR Frequency of the Fundamental frequency of the oscillation of the fundamental curvilinear trajectory around its average path (HAR

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harmonic MAG Magnitude of the fundamental harmonic

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) VOL Area of Area under the fundamental harmonic peak in the fundamental magnitude spectrum (VOL is a measure of the harmonic power-bandwidth of the signal) CON Specimen Concentration of sperm cells in a sample in millions concentration of sperm per milliliter of plasma or medium MOT Percent motility 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) Reproduced from reference 18.

Fig 5.3 Examples of kinematic measurements involved in a single sperm tracing. Reproduced from reference 18.

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In summary, persistent questions about results and their interpretation continue to limit the routine use of CASA. As reproducibility improves over all range of sperm concentration, CASA may become the standard for semen analysis. In addition, as the kinematics of sperm motion become better understood, CASA may play an integral part in determining the optimal method of assisted reproductive technology that should be utilized for specific types of male factor patients. SPERM ANTIBODIES Because mature spermatozoa are formed after puberty, they can be recognized as foreign protein by a man’s 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.21,22 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 contribute to the seminal plasma. They 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 seem 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.22 Several tests are currently employed for detecting the presence of sperm antibodies. The two most common are the following: (1) The mixed agglutination reaction (MAR) This test is performed by mixing semen, immunoglobulin G (IgG) or IgA coated beads or rbcs, and IgG or IgA antiserum on a microscope slide. The slides are incubated and observed at 400×. If antibodies are present, the sperm will form clumps with the coated latex beads or coated rbcs. 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% or greater to be clinically significant. This test is used only for detection of direct antibodies in men, and is not specific for location of bead attachment on the sperm.

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(2) The immunobead binding test This test is performed by combining IgG or IgA coated latex beads and washed sperm on a slide. After incubation, the slides are read at 200× or 400×. If antibodies are present, the small beads will attach directly to the sperm. This test provides potentially greater information, 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. To perform an indirect test, known direct antibody negative sperm are washed and mixed on a slide for a one hour incubation with the bodily fluid to be tested and IgG or IgA-coated latex beads. The test is interpreted by noting the percentage and location of bead attachment. The WHO considers a level of binding of 20% or greater to represent a positive test. Clinical significance is commonly considered to be a level of binding of 50% or more.9,23 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 antibody-positive sperm may have difficulty penetrating cervical mucus. Although, in these cases, intrauterine insemination (IUI) or IVF may improve the prognosis for fertilization, antibody levels exceeding 80% coupled with subpar concentration, motility or morphology may necessitate the addition of ICSI in order to truly make a difference.24 As suggested by the literature, andrology labs may benefit greatly in their preparation of sperm if they are aware of the presence of antibodies. 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. SPERM VIABILITY An intact plasma membrane is an integral component of, and possibly a biologic surrogate for sperm viability. The underlying principle is that viable sperm contain intact plasma membranes that prevent the passage of certain stains, whereas non-viable 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.25 When viewed with either bright field or phase contrast microscopy, these stains allow for the differentiation of viable, non-motile sperm from dead sperm. This procedure may, therefore, play a significant part in selecting appropriate sperm to use for ICSI when only

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immotile sperm are present. Unfortunately, however, dyes such as eosin Y are specific DNA probes which may have toxic effects if they enter a viable sperm or oocyte. Flow cytometry has also been used 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 nucleic acid specific stains. Some techniques employ dual staining, such as the one described by Noiles et al, which can differentiate between an intact membrane and a damaged membrane.26 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 hypoosmotic 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 (2.4um/min/atm at 22°C) of any mammalian cell.27 As originally described, the HOST involves placing a sperm specimen into hypotonic conditions of approximately 150mOsm.28 This environment, while not sufficiently hypotonic to cause cell lysis, will cause swelling of the sperm cells. As the tail swells, the fibers curl, and this change can be detected by phase contrast microscopy. The normal range for a positive test has been described as a score greater than or equal to 60%—60% of the cells demonstrate curling of the tails. A negative test is defined as less than 50% curling.29 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.30,31 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.32,33 It has also been suggested that, owing to sperm morphology changes in response to the test, the HOST may impair an embryologist’s ability to select sperm appropriate for injection. In summary, the HOST lacks sufficient critical evaluation to determine its true role in the evaluation 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.34 The acrosome contains multiple hydrolytic enzymes, including hyaluronidase, neuraminidase, proacrosin, phospholipase, and acid phosphatase, which, when released, are thought to facilitate sperm passage

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through the cumulus mass, and possibly the zona pellucida as well (fig 5.4). 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 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 5.5).

Fig 5.4 Sperm head with intact acrosome. OA=outer acrosomal membrane AC=acrosomal cap ES=equatorial segment SS=subacrosomal space Reproduced from reference 58.

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Fig 5.5 Acrosome reacted sperm IA=inner acrosomal membrane Reproduced from reference 58. Although electron microscopy has produced many elegant pictures of acrosome intact and acrosome reacted sperm, it is not always possible to know if sperm that fail to exhibit an acrosome have truly acrosome reacted, or could possibly be dead. In addition, electron microscopy is not a technique available to all andrologists. This has lead 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 time consuming and difficult to perform.35,36 Contemporary assays for acrosomal status determination employ fluorescent plant lectins or monoclonal antibodies, which can then be detected much more easily with fluorescence microscopy.37,38 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

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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 identify a small sub-population 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.39 These areas certainly await additional study. OTHER BIOCHEMICAL TESTS As noted above, one of the predominant enzymes present in the acrosome is proacrosin. This 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.40 Acrosin activity has been reported to be greater in fertile males than in infertile males,41 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.42 In this same study, researchers found that semen samples in which CK-M ratios exceeded 10% exhibited higher fertilization rates in IVF than specimens with lower ratios. Few other studies have addressed this topic. SPERM PENETRATION ASSAY The sperm penetration assay or hamster egg penetration assay (HEPA) was initially described by Yanagimachi et al. in 1976.43 Oocytes from the golden hamster were treated in order to remove the zona pellucida. One of the functions of the zona is to confer species specificity, therefore its presence would preclude performance of this test. Human sperm were then incubated for 48 hours with the hamster oocytes, and the number of penetrations with nuclear decondensation were 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 in order to assess the ability of sperm to fuse with 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 the inability to assess the sperm’s ability to bind to—and penetrate through—the zona pellucida. This factor continues to be one of the major criticisms that plague this test.

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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.44,45 Published reports demonstrated widely varying conclusions, such as the finding that the SPA could identify 0–78% of men whose sperm would fail to fertilize oocytes in ART procedures.46 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 review46 nor a prospective long term (five-year) follow-up study demonstrate such a correlation.47 In light of these considerations, support for this test has waned over the past several years.

Fig 5.6 Cluster analysis of hemizona assay index and fertilization rate. A: Good fertilization, B: poor fertilization, C: false-positive hemizona assay index. Reproduced from reference 51.

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HEMI-ZONA 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 of Human Reproduction and Embryology (ESHRE) Andrology Special Interest Group to recommend inclusion of such tests in the advanced evaluation of the male.48 Like the SPA, the hemi-zona assay (HZA) employs sperm and non-viable oocytes in an in vitro assessment of fertilization.49 In this test, however, both gametes are human in origin. 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 hemi-zona, while sperm from the subject male are added to the other hemi-zona. Following a four hour incubation, each hemizona is removed and pipetted in order to dislodge loosely attached sperm. A comparison or hemi-zona 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= # test sperm bound/# 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 which suggest that the HZA may help identify individuals with a poor prognosis for success with ART (fig 5.6).50,51 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.52 MANNOSE BINDING ASSAY Another test has been recently 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 which suggest that sperm:oocyte interaction involves the recognition by a sperm surface receptor of a specific complimentary receptor on the surface of the zona pellucida. This zona receptor appears to be a glycoprotein, the predominant sugar moiety of which is mannose.53 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.54 In vitro assays in which labeled probes of mannose conjugated to albumin are coincubated with semen specimens allow for the differential staining of sperm (fig 5.7). Those that bind the probe are thought to

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possess the sperm surface receptor for the mannose-rich zona glycoprotein. Several investigators, including our group, have subsequently demonstrated that sperm from fertile populations exhibit greater mannose binding than sperm from infertile males.55,56,57 This new area shows promise in the area of sperm function testing, but also invites further study.

Fig 5.7 Mannose positive (brown) and mannose negative (clear) sperm. By courtesy of Tammy Dey, Kaylen Silverberg.

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 first line diagnostic tool for all affected males. A more likely scenario will be like that in female infertility, where a battery of tests—each evaluating a specific function—is employed as needed. In the 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

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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 women, it is not likely that micromanipulation will become standard treatment for all infertile men. 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 in order to thoroughly assess these tests. Those tests that demonstrate a 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 sperm-cervical mucus interaction. 3rd ed. New York: Cambridge University Press (1992):3–27. 3 Alexander NJ. Male evaluation and semen analysis, Clin Obstet Gynecol (1982); 25:463–82. 4 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. 5 Overstreet JW, Davis RO, Katz DF, Overstreet JW, eds. Infertility and reproductive medicine. Clinics of North America. Philadelphia: WB Saunders (1992): 329–40. 6 Koren E, Lukac J. Mechanism of liquefaction of the human ejaculate: I. Changes of the ejaculate proteins. J Reprod Fertil (1979); 56:493–500. 7 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. 8 Cohen J, Aafjes JH. Proteolytic enzymes stimulate human spermatozoal motility and in vitro hamster egg penetration. Life Sciences (1982); 30:899–904. 9 Van Voorhis BJ, Sparks A. Semen analysis: What tests are clinically useful? Clin Obstet Gynecol (1999); 42:957–71. 10 Zuckerman Z, Rodriquez-Rigau IJ, Smith KD, Steinberger E. Frequency distribution of sperm counts in fertile and infertile males. Fertil Steril (1977); 28:1310–3. 11 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.

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12 Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF; Oehninger S. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril (1988); 49:112–17. 13 Katz DF, Overstreet JW. Sperm motility assessment by videomicrography. Fertil Steril (1981); 35:188–93. 14 Katz DF, Diel L, Overstreet JW. Differences in the movements of morphologically normal and abnormal human seminal spermatozoa. Biol Reprod (1982); 26:566–70. 15 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. 16 Coetzee K, Kruger TF, Lombard CJ. Predictive value of normal sperm morphology: A structured literature review. Hum Reprod Update (1988); 4:73–82. 17 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. 18 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 (1992); 93:341–52. 19 Irvine DS. The computer assisted semen analysis systems: sperm motility assessment. Hum Reprod (1995); 10 (suppl 1):53–9. 20 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. 21 Marshburn PB, Kuttch WH. The role of antisperm antibodies in infertility. Fertil Steril (1994); 61:799–811. 22 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. 23 Helmerhost FM, Finken MJJ, Erwich JJ. Detection assays for antisperm antibodies: What do they test? Hum Reprod (1999); 14:1669–71. 24 Bronson R. Detection of antisperm antibodies: an argument against therapeutic nihilism. Hum Reprod (1999); 14:1671–73. 25 World Health Organization. Manual for examination of human semen and semen-cervical mucus. Cambridge: Cambridge University Press (1987): 1–12. 26 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. Beltsville, MD: Proceedings of the Second International Conference on Boar Semen Preservation (1991):359–64.

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27 Noiles EE, Mazur P, Kleinhans FW, et al. Determination of the water permeability coefficient and its activation energy for human spermatozoa. Biol Reprod (1993); 48:99–109. 28 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. 29 Zaneveld LJD, Jeyendran RS. Modern assessment of semen for diagnostic purposes. Semin Reprod Endocrinol (1988); 4:323–37. 30 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. 31 Jeyendran RS, Zaneveld LJD. Human sperm hypoosmotic swelling test. Fertil Steril (1986); 46:151–4. 32 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. 33 Joshi N, Kodwany G, Balaiah D, et al. The importance of CASA and sperm function testing an in vitro fertilization program. Int J Fertil Menopausal Stud (1996); 41 (1):46–52. 34 Critser JK, Noiles EE. Bioassays of sperm function. Sem Reprod Endocrinol (1993); 11 (1):1–16. 35 Talbot P, Chacon RS. A triple stain technique for evaluating acrosome reaction of human sperm. J Exp Zool (1981); 215:201–8. 36 Wolf DP, Boldt J, Byrd W, et al. Acrosomal status evaluation in human ejaculated sperm with monoclonal antibodies. Biol Reprod (1985); 32:1157–62. 37 Cross NL, Morales P, Overstreet JW, et al. Two simple methods for detecting acrosome-reacted sperm. Gamete Res (1986); 15:213–6. 38 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. 39 Chan PJ, Corselli JU, Jacobson JD, et al. Spermac stain analysis of human sperm acrosomes. Fertil Steril (1999); 72:124–8. 40 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. 41 Mohsenian M, Syner FN, Moghissi KS. A study of sperm acrosin in patients with unexplained infertility. Fertil Steril (1982); 37:223–9. 42 Huszar G, Vigue L, Morshedi M. Sperm creatinine phosphokinase Misoform ratios and fertilizing potential of men: A blinded study of 84 couples treated with in vitro fertilization. Fertil Steril (1992); 57:882– 8.

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43 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. 44 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. 45 Jacobs BR, Caulfield J, Boldt J. Analysis of TEST (TES and tris) yolk buffer effects on human sperm. Fertil Steril (1995); 63:1064–70. 46 Mao C, Grimes DA. The sperm penetration assay: Can it discriminate between fertile and infertile men? Am J Obstet Gynecol (1988); 159:279–86. 47 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. 48 Consensus Workshop on Advanced Diagnostic Andrology Techniques. ESHRE Andrology Special Interest Group. Hum Reprod (1996); 11:1463–79. 49 Burkman LJ, Coddington CC, Franken DR, et al. The hemi-zona 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. 50 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. 51 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 (1992b); 1760–7. 52 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. 53 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. 54 Mori K, Daitoh T, Kamada M, et al. Blocking of human fertilization by carbohydrates. Hum Reprod (1993); 8: 1729–32. 55 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); 56:113–8. 56 Benoff S, Cooper GW, Hurley I, et al. Human sperm fertilizing potential in vitro is correlated with differential expression of a headspecific mannose ligand receptor. Fertil Steril (1993); 59:854–62. 57 Silverberg K, Dey T, Witz C, et al. D-Mannose binding provides a more objective assessment of male fertility than routine semen

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analysis: Correlation with in vitro fertilization. Presented at the 49th annual meeting of the American Fertility Society. October, 1993. 58 Sathananthan AH, ed. Visual atlas of human sperm structure and function for assisted reproductive technology. Singapore, 1996. LaTrobe and Monash Universities, Melbourne. National University, Singapore.

6 Sperm preparation techniques Gordon Baker, Harold Bourne, David H Edgar

OVERVIEW The aim of sperm preparation for 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 ability to activate the oocyte and form a pronucleus are necessary but morphology, motility, and acrosome status are generally not important.1,2,3,4,5 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.6,7,8 For IVF or gamete intra-fallopian transfer (GIFT), the medium should contain protein and buffers that promote sperm capacitation.1 While serum or high molecular weight fractions from serum appear to be important for sperm motility, more recently comparatively 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 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 30mg/ml, concentrations of around 4mg/ml will support normal sperm function in IVF. Bicarbonate ions are required for capacitation of sperm and are normally present at about 25mmol/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

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glucose, may not be appropriate for fertilization stages of assisted reproductive technologies (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 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 leucocytes or abnormal sperm.6,7 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 that 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 swim up may produce higher yields of good quality sperm.11 However, 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.12 The optimal number of sperm for insemination is poorly defined, but several reviews of results of IVF suggest that there is an increase in fertilization rate with insemination of sperm at between 2000 and 500000 per ml.1 There may be some increase in risks of polyspermy with the higher sperm concentrations thus most groups inseminate oocytes with about 100000 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.13,14 The total volume of sperm suspension added should be minimized to restrict dilution of the oocyte medium.

METHODS Procedures for preparation of the culture media and sperm isolation are given in appendices 1–8 and shown schematically in Figs 6.1–3.

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COLLECTION OF SEMEN OR SPERM While semen for ART is usually collected by masturbation, sperm may be collected by a variety of methods from several sites in the male genital tract (Fig 6.1). The man should collect semen, in a room adjacent to the IVF laboratory, into a sterile disposable plastic jar. The 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

Fig 6.1 Possible sites of collection of sperm or elongated spermatids from the male genital tract for ART. 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.

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The timing of semen collection and preparation does not appear to be critical, especially with good semen samples. In general the oocytes are inseminated four to six hours after collection and the sperm can be prepared during this time. The semen should be placed in a sterile 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. Following mixing, a small portion (~10µl) 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.6,7,15,16 The swim up procedure is now the most commonly used technique (Fig 6.2). 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 6.3). 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. The medium chosen should be equilibrated with the gas mixture and the temperature maintained constant at 37°C. The protein source for the medium needs to be checked for sperm antibodies, and, if pools are used, the donors must be tested for viral illnesses including HIV infection 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

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Fig 6.2 Methods of sperm preparation for ART. 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.

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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, 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 one hour incubation for swim up. They are also relatively simple to perform under sterile conditions (Fig 6.2). The most popular of these is colloidal silica density gradient (CSDG) centrifugation, but other agents have also been used.1,11,17 The colloidal silica particles are coated with polyvinylpyrollidone, for example, PercollTM (Pharmacia AB, Uppsula, 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 including Isolate (Irvine Scientific, Santa Ana, Ca, USA) and PureSperm (Nidacon Laboratories, AB, Gothenburg, Sweden).18 Discontinuous gradients of two or more steps are used. Sperm and other material form distinct bands at the interfaces on the CSDG. It has been claimed that abnormal sperm as

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Fig 6.3 Procedure for seminiferous tubules obtained by fine needle tissue aspiration or open biopsy. 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. Several studies have compared CSDG centrifugation with a swim up and occasionally other sperm preparation techniques. The end points 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

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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.1,8,11,18–20 Some studies suggest that the gradient materials may damage the sperm.21,22 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, fine needle aspiration biopsy, or other techniques (Fig 6.2) and prepared by the methods outlined in appendices 7 and 8.1,2 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– 4,23 Various agents have been reported to enhance sperm motility.1 Pentoxifylline (POF) has been used for ART. The maximally effective dose of POF is between 0.3mmol/l and 0.6mmol/l and many groups use 3.6mmol/l (1mg/ml). POF has been reported to provide greater stimulation of motility and velocity than caffeine or 2-deoxyadenosine. Appendices 10 and 11 give methods for stimulating sperm motility with POF and demonstrating membrane integrity by hypoosmotic swelling.

RESULTS The normal fertilization and embryo utilization rates are compared for swim up and CSDG in Table 6.1. 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.4,24,25

COMPLICATIONS Although there is potential for semen or sperm dependent complications of ART such as infections or allergic reactions these are very rare. Patients should be tested for serious transmissible infections such as HIV infection and hepatitis, and standard precautions for handling biological material must be practised in the embryology laboratory. Transmission of genetic conditions to offspring is possible: suitable counselling and, where

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Table 6.1. Comparison of results with swim up and colloidal silica density gradient (CSDG) preparation of sperm from semen for IVF or ICSI from men with normal semen (sperm concentration ≥20×106ml, progressive motility ≥40% and abnormal morphology ≤85%), abnormal semen (sperm concentration 1–19×104ml or progressive motility 1–39% or abnormal morphology 86–100%) or oligoasthenoteratozoospermia (sperm concentration 1– 19×106ml, progressive motility 1–39% and abnormal morphology 86–100%) from 1990–1999. 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, X2 test). IVF ICSI Oocytes IVF Normal Embryo Oocytes ICSI Normal Embryo collected fertilization utilization collected Fertilization utilization NORMAL SEMEN SWIM 21255 21031 12 286 10520 1396 1113 665 545 UP 99 58 49 80 48 39 CSDG 3319 3298 1833* 1577* 905 685 394 322 99 55 48 76 44 36 ABNORMAL SEMEN SWIM 8826 8733 4236 3513 5718 4664 2804 2367 UP 99 48 40 82 49 41 CSDG 6126 5943 2720* 2338* 6387 5221 3054 2567 97 44 38 82 48 40 OLIGOASTHENOTERATOZOOSPERMIA SWIM 360 354 1328 1072 610 514 97 93 UP 81 46 39 98 27 26 CSDG 1183 1158 416* 358 2436 2016 1142 941 98 35 30 83 47 39

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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.26 In some clinics there appears to be a higher rate of abnormal fertilization with ICSI using testicular sperm.25 The sex ratio is also lower with disproportionately more female babies born after ICSI in some data banks.27,28

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 ART. Improved prediction of results will come from development of new methods of semen analysis: 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.27 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.6,15,16,28 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.29 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.30 Also, approaches will be available for reducing the risk of transmission of infectious agents such as HIV and hepatitis viruses in discordant couples who wish to have children.31

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

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ACKNOWLEDGEMENTS The authors thank Mr G N Clarke for advice about cryopreservation procedures, and Dr C Garrett, Ms A Apedaile and Ms P Sourivong for assistance with the figures.

APPENDICES APPENDIX 1— PREPARATION OF MEDIA • Human tubal fluid culture medium (HTF) and HTF with hydroxyethanepropoxy ethane sulphonate buffer (HTF/HEPES) in 500ml bottles are purchased from Irvine Scientific (Santa Ana, Ca, USA). • As required, 20% human albumin (ALB) solution (pharmaceutical grade) is added (1 part in 50), to give a final concentration of 0.4% (4mg/ml). • Both HTF with albumin (HTF/ALB) and HTF/HEPES/ALB are prepared and stored refrigerated until required (maximum storage time according to the manufacturers expiry date: about six weeks). APPENDIX 2— CHOICE OF METHOD • Patient and sample identity should be checked with another person and recorded as a quality assurance measure. • Examine a drop of undiluted semen (glass slide or haemocytometer). • For standard IVF: if the sperm concentration is >20×106/ml with moderate to good forward progressive motility, swim up can be performed. • Borderline samples may be better prepared by CSDG centrifugation. • If the sample is unexpectedly poor on the day (for example, concentration <10×106/ml, <40% motility and/or poor forward progression), ICSI should be considered. • For ICSI: swim up can be used in most cases. Even samples with severe oligozoospermia (down to ~10000/ml) can be prepared using swim up as long as there are some sperm with good forward progression. • Semen with large amounts of debris, extreme oligozoospermia, severely compromised motility is 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 is little extraneous cellular material and sufficient progressive motility to allow sperm to migrate to the edge of the drop.

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APPENDIX 3— SWIM UP METHOD • After the semen has liquefied (usually 30 minutes at 37°C), aliquots of 1ml of semen are placed in 5ml labelled Falcon tubes and gently overlaid with 2ml of medium, (HTF/ALB for IVF and HTF/HEPES/ALB for ICSI.) • The tubes are incubated at 37°C for 45–60 minutes to allow progressively motile sperm to swim into the overlaid medium. • The medium (up to -90% of the supernatant) is aspirated and placed into a labelled collection tube and centrifuged at 300–400g for 10 minutes. • The supernatant is removed and the pellet is resuspended in 0.3–1.0ml of fresh medium. • To calculate the insemination volume for IVF, sperm count, motility, and velocity are assessed on a sample in the counting chamber of a standard haemocytometer. • The sample is allowed to settle (>3 minutes) and motile sperm in a minimum of five squares from the central 25 squares are counted to give a rough estimate of the motile concentration. • Grade velocity as follows: FP0: no movement, FP1: movement but minimal progression, FP2: slow progression, FP3: moderate to rapid progression. • The insemination volume is adjusted to give a total of 100000 to 200000 FP3 sperm/ml in the medium containing the oocytes.

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APPENDIX 4— WASH AND SWIM UP METHOD FOR SAMPLES WITH POOR MUCOLYSIS Allow semen to liquefy at 37°C for 30 minutes. Mix 1ml of semen with 2–3ml of appropriate medium (IVF or ICSI); mix vigorously if necessary. Allow any particulate matter to settle and transfer supernatant to another test tube. Centrifuge for 10 minutes at 300–400g. Aspirate supernatant. Overlay pellet with 2ml of medium and incubate for 45–60 minutes. Aspirate the sperm rich suspension without disturbing the pellet and place in a labelled collection tube. Centrifuge and resuspend in 0.3–1ml of medium as required. Assess sperm count, motility and velocity. Calculate insemination volume for IVF.

APPENDIX 5— DENSITY GRADIENT METHOD • If the motile sperm concentration is sufficient, apply semen directly to top of gradient. • Alternatively, prepare and centrifuge semen as described for wash and swim method (for severely oligozoospermic samples, increase centrifugation to 1800g). • Resuspend pellet in 0.5ml of medium. • Using “ISOLATE” Sperm Separation Medium, aliquot 0.5ml of ISOLATE LOWER, into labelled 15ml FALCON conical tubes. Overlay with 0.5ml of ISOLATE UPPER.

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• • • •

Overlay ISOLATE layers with the semen or resuspended sperm pellet. Centrifuge at 300–400g for 15–20 minutes. Remove supernatant being careful not to disturb the layers. Transfer the lower portion of the bottom layer and pellet to a clean tube and add 1– 2ml of fresh medium. • Centrifuge at 300–400g (increase to 1800g if needed). • Resuspend the pellet in 0.3–1.0ml of appropriate medium as required. • Assess sperm count, motility and velocity. Calculate insemination volume for IVF. APPENDIX 6— FROZEN SEMEN METHOD • Double check straw code and patient ID. • Thaw straw in air for 10–20 minutes; check integrity of the straw and discard if damaged. • Soak straw in hypochlorite solution (5000 ppm available chlorine) prepared fresh daily (for example a 1:20 dilution of Milton) for at least two minutes to disinfect outside of straw and wipe excess solution from the straw after soaking. • Cut one end and aspirate contents, place into a 5ml Falcon tube. • Assess sperm concentration, motility and velocity. • If the sample is adequate, overlay with 1–2ml of HTF/HEPES/ALB (ICSI) or HTF/ALB (IVF). • Incubate at 37°C for 45–60 minutes. • Remove the medium and place in a collection tube. • Centrifuge at 300–400g for 10 minutes. • Remove supernatant and resuspend the pellet in 0.3–1ml of medium. • Assess sperm count, motility and velocity. Calculate insemination volume for IVF. • If the sample is not adequate for swim up or contains a large amount of debris or round cells, use the density gradient method. APPENDIX 7— MESA/PESA • Expel aspirates into a small Petri dish of warm HTF/HEPES/ALB. • Pool samples and concentrate if necessary. • Depending on concentration, motility and amount of debris, either use directly or separate on a density gradient. • Leave sperm to incubate (up to 24 hours) or prepare plate for ICSI and leave at 37°C to allow sperm to gain motility. • 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 9. APPENDIX 8— TESTICULAR BIOPSY • Place tissue into a small Petri dish of warm HTF/HEPES/ALB.

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• Dissect and squeeze tubules using fine gauge needles (Fig 6.3). • Transfer raw suspension to a test tube. • Depending on concentration, motility and amount of debris, either use directly or separate on a density gradient. • Leave sperm to incubate (up to 24 hours) or prepare plate for ICSI and leave at 37°C to allow sperm to gain motility. • If extra sperm are available, consider freezing the excess (appendix 9).

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APPENDIX 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. If necessary the semen is centrifuged to concentrate the sperm into a minimum volume of 0.4ml. MESA samples or other sperm suspensions are processed in the IVF laboratory by swim up, centrifugation on density gradients, or washing and resuspended in IVF medium. The remaining sperm suspension can be cryopreserved with glucose citrate glycine (GCG) cryoprotectant, glycerol and patient’s serum or 5% albumin solution. GCG cryoprotectant: Dissolve glucose (1.0g) and sodium citrate (1.0g) in 40ml of sterile deionized water and add glycine (1.0g). (pH ~7.5 and osmolality ~500mOsm/ kg). It is stored in 2ml volumes at −70°C. When a sample arrives, thaw a vial of GCG. Add 10ml glycerol. If the sample volume is >2.0ml and contains few motile sperm, centrifuge at 1800g for 5 minutes at room temperature and aspirate the supernatant to leave about 1.0ml and resuspend the sperm. Determine percentage total motile sperm (not just progressing)—if very few motile sperm are present, estimate number motile/coverslip—and record result. Add 0.5–1.0ml of 5% albumin solution to the sperm sample, mix well. Add GCG-glycerol preparation in 1:2 ratio gradually with mixing. Package in straws or cryovials and freeze.

APPENDIX 10— USE OF PENTOXIFYLLINE • Prepare a 10x concentrated solution of pentoxifylline (POF, Sigma) in protein free HTF/HEPES (POF MW=278.3; 10x concentrate=10 mg/ml). • Sterilize through a 0.2µm filter and store at 4°C. • Dilute 1:9 with sperm suspension to expose sperm to a final concentration of 1mg/ml POF (3.6mM). • 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.

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• Aim to collect the motile sperm quickly (<1 hour if possible) as the treated sperm will lose motility with time. • Expel the treated medium from the injection pipette and rinse with the untreated, clean medium in the holding drop. • Immobilize the selected sperm and perform ICSI as usual. APPENDIX 11— USE OF HYPO-OSMOTIC MEDIUM • Prepare a ~100mOsm/kg solution by diluting HTF/HEPES/ALB 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. • Live immotile sperm should coil their tails shortly after contacting the hypo-osmotic medium. • Move the presumptive 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 normoosmotic medium. • 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. 2nd ed. Boca Raton: CRC Press, (1999): 99–126. 2 Nagy Z, Liu J, Cecile J, Silber S, Devroey P, Van Steirteghem A. 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, Van Steirteghem AC. 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, Clarke GN, Harari O, Baker HW. 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, Qian C, Dhont M. Human oocyte activation following intracytoplasmic injection: the role of the sperm cell. Hum Reprod (1995); 10:403–7.

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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 invitro fertilization. Hum Reprod (1991); 6:173–6. 8 Mortimer D. Sperm recovery techniques to maximize fertilizing capacity. Reprod Fertil Dev (1994); 6:25–31. 9 Adler A, Reing AM, Bedford JM, Alikani M, Cohen J. Plasmanate as a medium supplement for in vitro fertilization. J Assist Reprod Genet (1993); 10:67–71. 10 Laverge H, De Sutter P, Desmet R, Van der Elst J, Dhont M. Prospective randomized study comparing human serum albumin with fetal cord serum as protein supplement in culture medium for in-vitro fertilization . Hum Reprod (1997); 12:2263–6. 11 Ng FL, Liu DY, Baker HW. Comparison of Percoll, mini-Percoll and swim-up methods for sperm preparation from abnormal semen samples. Hum Reprod (1992); 7:261–6. 12 De Vos A, Nagy ZP, Van de Velde H, Joris H, Bocken G, Van Steirteghem A. Percoll gradient centrifugation can be omitted in sperm preparation for intracytoplasmic sperm injection. Hum Reprod (1997); 12:1980–4. 13 Gianaroli L, Cristina Magli M, Ferraretti AP, et al. Reducing the time of sperm-oocyte interaction in human invitro fertilization improves the implantation rate. Hum Reprod (1996); 11:166–71. 14 Gianaroli L, Magli CM, Ferraretti AP et al. Prolonged sperm-oocyte exposure and high sperm concentration affect human embryo viability and pregnancy rate. Hum Reprod (1996); 11:2507–11. 15 Twigg J, Irvine DS, Houston P, Fulton N, Michael L, Aitken RJ. Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: protective significance of seminal plasma. Mol Hum Reprod (1998); 4:439–45. 16 Twigg JP, Irvine DS, Aitken RJ. Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection. Hum Reprod (1998); 13:1864–71. 17 Ord T, Patrizio P, Marello E, Balmaceda JP, Asch RH. Mini-Percoll: a new method of semen preparation for IVF in severe male factor infertility. Hum Reprod (1990); 5:987–9. 18 Claassens OE, Menkveld R, Harrison KL, Evaluation of three substitutes for Percoll in sperm isolation by density gradient centrifugation. Hum Reprod (1998); 13:3139–43. 19 Carrell DT, Kuneck PH, Peterson CM, Hatasaka HH, Jones KP, Campbell BE. A randomized, prospective analysis of five sperm preparation techniques before intrauterine insemination of husband sperm. Fertil Steril (1998); 69:122–6.

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20 Centola GM, Herko R, Andolina E, Weisensel S. Comparison of sperm separation methods: effect on recovery, motility, motion parameters, and hyperactivation. Fertil Steril (1998); 70:1173–5. 21 Grab D, Thierauf S, Rosenbusch B, Sterzik K. Scanning electron microscopy of human sperms after preparation of semen for in-vitro fertilization. Arch Gynecol Obstet (1993); 252:137–41. 22 Sterzik K, De Santo M, Uhlich S, Gagsteiger F, Strehler E. Glass wool filtration leads to a higher percentage of spermatozoa with intact acrosomes: an ultrastructural analysis. Hum Reprod (1998); 13:2506– 11. 23 Casper RF, Meriano JS, Jarvi KA, Cowan L, Lucato ML. The hypoosmotic swelling test for selection of viable sperm for intracytoplasmic sperm injection in men with complete asthenozoospermia. Fertil Steril (1996); 65:972–6. 24 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. 25 Watkins W, Nieto F, Bourne H, Wutthiphan B, Speirs A, Baker HW. Testicular and epididymal sperm in a microinjection program: methods of retrieval and results. Fertil Steril (1997); 67:527–35. 26 Bonduelle M, Wilikens A, Buysse A, et al. A follow-up study of children born after intracytoplasmic sperm injection (ICSI) with epidymal and testicular spermatozoa and after replacement of cryopreserved embryos obtained after ICSI. Hum Reprod (1998); 13:196–207. 27 de Kretser DM, Baker HWG. Infertility in men: recent advances and continuing controversies. J Clin Endocrinol Metab (1999); 84:3443– 50. 28 Baker HW. Marvellous ICSI: the viewpoint of a clinician. Int J Androl (1998); 21:249–52. 29 Antinori S, Versaci C, Dani G, Antinori M, Selman HA. Successful fertilization and pregnancy after injection of frozen-thawed round spermatids into human oocytes . Hum Reprod (1997); 12:554–6. 30 Fugger EF, Black SH, Keyvanfar K, Schulman JD. Births of normal daughters after MicroSort sperm separation and intrauterine insemination, in-vitro fertilization, or intracytoplasmic sperm injection. Hum Reprod (1998); 13:2367–70. 31 Marina S, Marina F, Alcolea R, et al. Human immunodeficiency virus type 1—serodiscordant couples can bear healthy children after undergoing intrauterine insemination. Fertil Steril (1998); 70:35–9.

7 Oocyte treatment: from egg retrieval to insemination Thomas B Pool, Virginia A Ord

OVERVIEW The mature follicle provides a discrete environment in which the final stages of oocyte maturation either occur or are initiated in preparation for ovulation. Likewise, the ampulla of the fallopian tube at midcycle presents both chemical and physical conditions favorable, not only for the viability of both sperm and eggs, but for their successful union and subsequent embryogenesis. But the conditions encountered in vivo by the oocyte between follicular rupture and the eventual entry into the distal tube, through the ciliary activity of the fimbrial epithelium, are transitional ones. During this process, the oocyte escapes the chemical influences of follicular fluid, passes into the rectouterine pouch, a collection point for blood, follicular fluid and peritoneal exudates and traverses the anatomical interface between peritoneal cavity and tubal lumen, a destination that is topologically external to the interstitium. The oocyte is well equipped for this journey with surrounding investments, the cumulus coronal complex complete with a newly secreted hyaluronan extracellular matrix that essentially “seal” the oocyte from direct contact with the transitional environment. Exposure to inappropriate conditions during this transitional period could, however, potentially disrupt fertilization and early development. Assisted reproductive procedures in humans are undertaken with a reliance upon the embryology laboratory to provide an efficient and safe transitional environment for oocytes from follicular aspiration until insemination. This chapter describes simple, reproducible procedures for ensuring oocyte health during oocyte retrieval, evaluation and transitioning into culture for insemination. The goal of the laboratory during the ovum pickup procedure is to recover all oocytes in a minimum amount of time, to evaluate their meiotic maturity and quality in preparation for insemination or sperm injection, to evaluate the investing layers in the oocyte-cumulus-coronal complex (OCCC) for maturity, to examine the remainder of the follicular aspirate for signs of potential physiological or pathological significance, such as a premature luteinization or endometriosis, and to communicate all information helpful to the clinical team in completing the egg retrieval procedure expeditiously. At the same time, it is crucial that rigorous

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patient test management procedures be followed which ensure proper patient sample identification and a documented chain of custody for all laboratory activities throughout an ART procedure. At the heart of the retrieval procedure, however, is the use of appropriate media, supplies, equipment, and methods that maintain the prospective developmental potential of each oocyte. To this end, the laboratory should identify both media and protocols that fulfill the specific needs of their clinic, taking into account such variables as proximity of the laboratory to the retrieval room, caseload, and staffing. For this reason, a laboratory may select one of any number of materials and approaches for egg retrieval, but the selection should include a consideration of availability, convenience, flexibility, and cost effectiveness. Above all, the media, supplies and methods should be the simplest that meet the physiological requirements of the oocyte, given the location of the laboratory, the instrumentation available, and the technical proficiency of the laboratory staff. Overly complicated methods and culture systems lend nothing to outcome but make troubleshooting and quality assurance difficult, at best.

METHODS The responsibility of the laboratory to provide a nurturing environment for the oocyte extends into the retrieval room. For example, the temperature of any heating devices used to warm media or aspiration tubes must be monitored using a calibrated thermometer. Vacuum for aspiration should be generated by a calibrated pump and must be monitored and recorded before and during the retrieval procedure. The laboratory should either test all contact materials used in the retrieval room or provide pretested supplies to the clinical staff, documenting lot numbers and dates of use for quality control purposes. We provide plastic tubes for follicular aspiration, plastic specimen containers containing aspiration medium and 10 cc Luer lock plastic syringes for needle flushing, all mouse embryo tested and issued in dated, defined lots. This facilitates traceability of all contact materials where performance characteristics can be verified with specific bioassay and biocompatibility data, an essential quality improvement activity. SELECTION OF TRANSITIONAL MEDIA Table 7.1 gives the composition of several of the most popular transitional media used in human ART. The media used for the transitional activities of follicular aspiration, ovum holding, and ovum maturational grading must meet the minimal nutritional requirements of the oocytes in transition, provide appropriate pH stability through buffer action, meet the osmotic needs of the oocyte, and be compatible with the culture system selected for insemination and embryogenesis. Although the emerging

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nutritional requirements of the early human embryo are gaining better definition through direct experimentation, much less is known about those of the mature oocyte prior to fertilization. Furthermore, the requirements can change depending on the status of final meiotic maturation of the oocyte at the time of retrieval. It has long been known in mice, for example, that only pyruvate and oxaloacetate can support oocyte development in the absence of cumulus cells but in their presence, lactate, phosphoenolpyruvate, and glucose are able to support maturation and the first cleavage division.1 This finding was followed by direct demonstrations of the ability of cumulus cells to produce pyruvate by using glucose as a substrate.2,3 Much recent work has shown that fertilization and early development in humans require little to no glucose,4,5 but oocytes collected from a cohort of follicles, produced for ART by controlled ovarian hyperstimulation, represent a spectrum of maturational states, even if subtle and not morphologically apparent. Cumulus cells can number as high as 20,000 in the OCCC6 and can persist for as long as 72 hours.7 Therefore, the inclusion of glucose in the range of 2.0 to 5.5mM as a glycolytic substrate for cumulus cells is reasonable for collection, holding and grading media. The nutritional requirements for oocytes in transition from the follicle to the incubator are minimal, or are at least induced to be minimal by methodology. There seem to be no special ionic or amino acid requirements for oocytes in this transitional state, and the electrolytic as well as the osmotic needs are met by most balanced salt solutions. Perhaps the single most important part played by transitional media is the prevention of pH shift. There has not been, to date, an unequivocal experimental demonstration of a pH optimum for human oocytes and early embryos, but excursions in extracellular pH, most often into the alkaline range, can prove costly to subsequent embryogenesis. The selection of an appropriate buffer is, therefore, the most critical decision in selecting transitional media. The three most popular buffers used in ART transitional media are bicarbonate, phosphoric acid, and the zwitterion N-2hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), and each has its advantages and limitations. An equilibrium of H2CO3 and HCO3− is reached at pH 7.4 when bicarbonate is included in medium in the presence of elevated atmospheric CO2. This buffer system has the advantages of being physiological and of low toxicity. Furthermore, it ensures that metabolic requirements for CO2 are met, such as that seen in the synthesis of malonyl CoA and in the carboxylation of 5-aminoinidazole ribonucleotide to produce carbon 6 of the purine ring, although the amount required for

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Table 7.1. Composition of transitional media. Concentration (mM) Component Dulbecco’s Earle’s Modified PBS8 balanced HTF22 21 salts Sodium chloride 137.00 116.40 101.60 Potassium chloride 2.70 5.40 4.69 Magnesium chloride 0.50 – – (hexahydrate) Magnesium sulfate – 0.65 0.20 (anhydrous) Calcium chloride 0.90 1.80 – (dihydrate) Calcium chloride – – 2.04 (anhydrous) Sodium phosphate – 1.00 – (monobasic) Sodium phosphate 8.00 – – (dibasic) Potassium phosphate 1.50 – 0.37 (monobasic) Sodium bicarbonate – – 4.00 HEPES – – 21.00 Glucose – 5.55 2.78 Sodium pyruvate – – 0.33 Sodium lactate – – 21.40 Penicillin -G – – 100units/ml Streptomycin sulfate – – 50µg/ml Gentamicin sulfate – – – Phenol red – 0.011g/l 0.01g/l

TALPHEPES (TH)14 114.00 3.20 0.50 – 2.00 – 0.40 – – 2.00 10.00 5.00 0.33 10.00 – – 10µg/ml 0.01g/l

these events is small. It has significant limitations as well. Although the actual expense for bicarbonate and CO2 is modest the cost for the equipment needed to generate and maintain an elevated CO2 environment outside of the incubator can be high, particularly if one elects to use a modified pediatric isolette as a work station where the entire chamber is maintained at 5% CO2. Even if one uses a simpler and cheaper system, such as flowing gas to an inverted funnel placed over a oocyte holding dish, a stable concentration of dissolved CO2, and ultimately extracellular pH, is difficult to obtain. For this reason, many ART programs elect to use

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buffer systems that are not dependent on elevated gas concentrations. Phosphoric acid is a tribasic acid with three pK’s, the middle dissociation from H2PO4 occurring at a pH of 6.8. An appropriate concentration of phosphoric acid that provides buffer capacity in the physiological range is obtained by including both monoand dibasic phosphate, as the sodium and potassium salts, in the transitional medium. The most widely used phosphate buffered saline (PBS) is that described by Dulbecco and Vogt8 (Table 7.1). Like bicarbonate, phosphate buffered media are inexpensive, but they are much simpler to use. Also like bicarbonate, phosphoric acid is a physiological buffer but is of little importance in the bloodstream because the concentration of phosphate is normally quite low in plasma. This highlights one of the major drawbacks to phosphate buffer systems; in order to produce even modest buffer capacity, the concentrations of monoand dibasic phosphate must be relatively high. Good et al9 developed HEPES buffer largely in response to many of the drawbacks of phosphate buffer, such as poor buffer capacity above pH 7.5, a tendency to precipitate most polyvalent cations and the observation that at buffer concentrations of phosphate, it is either a metabolite or an inhibitor in many systems. We compared the buffer capacity of Dulbecco’s phosphate buffered saline to that of 10mM HEPES in Tyrode’s solution by direct titration assays with NaOH. The amount of added base required to increase the pH of PBS from 7.2 to 7.4 was 0.34meq/ml compared to 0.53meq/ml for 10mM HEPES. Thus, HEPES has 35% more buffer capacity than PBS over the pH range considered by many ART laboratories to be physiological. HEPES has also shown superior properties as a biological buffer, compared with phosphate and other organic buffers, in its ability to give active and stable mitochondrial preparations in cell fractionation studies and to support high rates of protein synthesis in cell free bacterial preparations.9 Finally, HEPES does not impair the buffering capacity of bicarbonate and can even be used in conjunction with media containing bicarbonate and requiring elevated gas concentrations. The limitations to HEPES as a buffer for transitional media are few and mostly theoretical. Firstly, there have been reports of a photo-induced toxicity of HEPES in medium used for somatic cell culture. Spierenburg et al10 demonstrated the phototoxicity of HEPES in both a leukemic cell line and a normal B cell line. The compounds involved in phototoxicity with HEPES, however, include riboflavin, riboflavintryptophan and riboflavin-tyrosine, which unite with oxygen to form toxic peroxides. None of these compounds are present in transitional media, and no reports exist documenting this effect in simple media. Further, pyruvate eliminates this effect, albeit at concentrations higher than are employed classically in most embryo media. Secondly, there are many individuals who dislike the idea of using HEPES since, unlike bicarbonate and phosphate, they do not envision it as being physiological. Physiological, in this context, means “natural” To the contrary, the chemistry of HEPES is remarkably physiological since it buffers by

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zwitterionic action. This property of HEPES, the same which imparts buffer capacity to amino acids, is due to the fact that HEPES is chemically a substituted taurine, a naturally occurring amino acid found in high concentrations in the fallopian tube and of demonstrated nutritional benefit to human embryos. There are two medium supplements that are of value in specific transitional media; albumin and heparin. Albumin, added in the form of human serum albumin (HSA) at concentrations of from 2 to 10mg/ml, suppresses stickiness and can provide osmotic stability by elevating the oncotic pressure of holding media. Media supplied to the retrieval room for follicular flushing should be devoid of all protein as rare instances of anaphylaxis relating to HSA have been reported. Heparin is added to media used to flush the aspiration needle and ancillary tubing, but it is not included in follicular flushing medium since it could impair clotting at the site of entry into the ovary made by the aspiration needle. In all cases, the heparin must be free of preservatives. There are two final technical considerations to be made regarding the use of aspiration and holding media in ART, both relating to temperature. Raising the temperature of flushing and holding media to 37°C prior to use is considered essential by most ART centers, although we do not find this to be either the case or even necessary. It should be noted that dissolved gases, including the CO2 that has equilibrated into the medium from ambient atmospheric conditions, are driven out of media by time and heat. This, if overlooked, can produce two suboptimal conditions. First, pH can change by as much as 0.2 units as media is warmed to 37°C from 4°C, so that technicians may not be working near the pH they have so diligently worked to achieve when preparing the transitional media. Secondly, oocytes, like all eukaryotic cells from homeothermic organisms, are thermodynamic metabolic engines and reach maximal metabolic rates at 37°C. As indicated earlier, transitional media are not intended to support full metabolic activity, but a potentially dangerous condition, one that can affect the developmental potential of an oocyte, can be established by the prolonged exposure of oocytes to elevated temperatures in simple transitional media. One of the earliest balanced salt solutions to be used widely for handling extirpated tissues and cells is Tyrode’s solution.11 Not only is it a simple but effective balanced salt solution, but it was used successfully in some of the earliest efforts to fertilize human oocytes in vitro under the name “Bavister’s medium.”12 It was further modified in later years by the addition of albumin, lactate, and pyruvate and was named TALR13 Bavister et al14 added 10mM HEPES to TALP to produce a transitional medium that was used successfully to recover and hold oocytes from the rhesus monkey, a modification called TALP-HEPES. We incorporated a modification of this medium into our ART program over 15 years ago and have used it successfully, with various supplements, as aspiration medium, oocyte holding/grading medium, sperm preparation medium,

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intracytoplasmic sperm injection (ICSI) medium, embryo transfer medium and embryo freezing medium. It is inexpensive and easy to prepare in large lots of 10 or 20 liters using positive pressure filtration with an inert gas such as nitrogen. We store the medium at 4°C in sterile 1 liter bottles that have been sealed with heat-shrink wrappers. This serves to ensure that a single bottle is used completely before another is opened and contributes to the long shelf life of the medium. For practicality and labeling purposes, we have shortened the acronym of TALP-HEPES to simply TH. Further, we indicate the amount of protein added, when the medium is supplemented with HSA, by indicating the mg/ml present following the acronym “TH.” Medium supplemented with 3mg/ml of HSA, for example, is called “TH3.” As a general rule, we adjust the pH of TH to 7.20 to 7.25, but this can easily be adjusted to suit the needs of the individual laboratory. PERFORMING THE OVUM RETRIEVAL Many centers prefer to equip the retrieval room with a waterbath or block heater that is used to house and warm aspiration tubes before and during the retrieval. When a waterbath is used, sterile water is added, warmed and a stainless steel, sterile test tube rack is placed into the bath. The tubes are then loaded into the rack and, in many clinics, sterile aspiration medium (approximately 1ml) is dispensed into each tube. A sterile drape surrounding the bath or heating block is essential. We have compared outcomes in our center and have seen no difference in fertilization, embryogenesis and pregnancy rates when aspiration tubes are used dry, thus simplifying the setup of the retrieval room. To this end, we prepare the back table by laying out a heating pad, set on high, and covering it with two layers of sterile table drapes. A test tube rack filled with empty tubes (17×100mm, Falcon 2057) is placed next to the heating pad. TH medium is supplied for use as aspiration medium, but an additional aliquot of TH is prepared by supplementation from a preservative-free stock solution of 1000 USP units/ml Na heparin, which is added to TH to a final working strength of 40 USP units/ml. The heparin supplemented TH is used to flush the aspiration apparatus only and is not used to flush follicles. The TH is prewarmed to 37°C in a warming chamber the morning of the case and is then poured into plastic sterile specimen containers on the heating pad. Six Luer lock syringes, 10 cc, are filled with the prewarmed TH and are laid flat on the heating pad. Using a microthermocouple and electronic thermometry, we have determined that the media within the syringes remain at 35–36°C throughout the retrieval. We have also found that oocytes remain as healthy during retrieval, holding and grading when held at 32–35°C as they do at 37°C. For this reason and to slightly reduce metabolic rate while oocytes are in transition, we hold oocytes at an average of 34°C instead of 37°C.

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Follicular aspirates are passed to the embryologist by the circulating nurse and the contents are poured into a 100×15mm plastic Petri dish, swirling the aspiration tube if necessary to dislodge any cell clumps or blood clots. All blood clots and any excess cumulus masses are dissected thoroughly using a sterile insulin syringe fitted with a 28 ga, 1/2 inch needle. The more acute bevel of insulin needles makes them easier to use for microdissection than those on standard tuberculin syringes. All OCCCs are transferred, using a sterile glass Pasteur pipette, to a 60×15mm dish containing 8 ml of TH3. If the retrieval is of short duration, oocytes are held collectively in this dish until scoring. An additional holding dish, consisting of an organ culture dish (Falcon 3037) containing 1ml of TH in the center well with a 1 ml mineral oil overlay, is used to hold oocytes if the retrieval is expected to take longer than usual. Both dishes are maintained at 34°C on a block heater. OOCYTE GRADING Oocyte scoring for maturity can be performed during the ovum pickup as OCCCs are identified from aspirates, but this may slow the retrieval process and thus prolong anesthesia time unnecessarily. For this reason, oocytes are usually collected for maturational grading until the end of the retrieval process. The oocyte and investing layers can be examined in solution using Hoffman modulation contrast optics at 100 to 200× magnification. In this manner, it is the maturity of the cumulus coronal complex that is scored, not the meiotic stage of the oocyte. This method is rapid and is largely successful in that there is usually a high degree of concordance between the maturational state of the cumulus/corona and meiotic maturity of the oocyte, with a highly dispersed cumulus and radiating coronal layer signifying the arrival of the oocyte at metaphase II. Conversely, a tightly packed cumulus with dense coronal layers indicates meiotic immaturity. Another approach that provides direct visualization of the oocyte and prospective first polar body, along with giving information about cumulus/coronal morphology, cytoplasmic clarity, zonal thickness, and extent of the perivitelline space is to spread the OCCC on the moist bottom of a culture dish, in essence flattening the OCCC into a more singular focal plane. With this, good visualization can be obtained with a stereomicroscope operated at 30–40× magnification. The addition of three to four OCCCs to a 30×15mm dish containing about 700µl of TH3 will produce this effect. Scoring can be performed quickly, and the complexes can be transferred rapidly into culture vessels in the incubator. Knowledge of the meiotic state of oocytes allows one to determine the timing of insemination or ICSI in a more precise manner. Veeck15 suggests that oocytes in metaphase II (M II) at scoring should be inseminated or injected 3–5 hours after collection, whereas those in metaphase I (M I) should not be injected until 1–5 hours after first polar body extrusion. She

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further suggests that oocytes containing germinal vesicles (prophase I; P I) should be inseminated or injected 26–29 hours after collection. A more detailed grading system for human oocytes has been described by Bongso and Trounson16 that incorporates a germinal vesical stage (grade 1), a metaphase I stage (grade 2), two metaphase II stages (grade 3, mature; grade 4, very mature) and a postmature stage (grade 5). We have adopted most of the elements of this grading system into our laboratory practice with minor modification. First, we have extended the system to include a grade 6 for mature oocytes with regions of darkened cytoplasm. Secondly, we have expanded the mature oocyte grades of 3 and 4 by using intermediate grades signified with a+sign (for example, grade 3+). It is also very helpful to the clinical staff to make additional notes about the ease of obtaining oocytes in the initial aspirate or the necessity of examining additional follicular flushes to find the OCCC. Lastly, it is crucial after scoring to rapidly move the OCCCs to pregassed culture medium for preincubation prior to insemination. INSEMINATION MEDIUM AND INSEMINATION Insemination can be performed conveniently in one of several configurations. Multiple oocytes can be preincubated and inseminated in organ culture dishes (Falcon 3037) containing 1ml of insemination medium and 1ml of oil overlay. Individual oocytes can be inseminated in 30–50µl drops in a 60×15mm dish with oil overlay, thus reducing the actual number of sperm needed for insemination. Other laboratories use multiwell dishes or microtiter plates in much the same manner as microdrop culture. Finally, it is both inexpensive and very effective for moderate male factor patients to inseminate oocytes in 75×12mm tubes (Falcon 2003) containing 1ml of medium. Two to three OCCCs are inseminated in each tube, and this approach has the advantage of concentrating the gametes to the bottom of the tube via gravity, yet still providing 1ml of buffer and nutritional capacity. The drawback is that the complexes must be removed and transferred to a separate dish for evaluating fertilization, a technical operation that can require special training. INSEMINATION AND ICSI Oocytes are inseminated or injected four to six hours after collection, unless overt immaturity is noted during scoring. This simply creates a convenient time interval from insemination to the fertilization check of 15 hours and oocytes inseminated at 4 pm can be evaluated for fertilization at 7 am the next morning. Insemination medium consists of P-1 medium4 (Irvine Scientific, Santa Ana, CA, USA) supplemented with 3mg/ml of HSA that has been pregassed overnight in the CO2 incubator. With normospermic males, the insemination concentration is equivalent to

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100000 sperm per ml of insemination medium, but the actual number of sperm is adjusted downward if microdrop culture is employed. With the advent of ICSI, little is gained in cases of male factor by grossly overinseminating oocytes with large numbers of sperm. When ICSI is indicated, it is performed in drops of TH3 medium overlaid with Dow Corning silicon oil in the lids of Falcon 1007 dishes. The TH3 is prewarmed, but ICSI is done at room temperature as described previously.17 A summary of the media, supplements and plasticware used during retrieval, grading and insemination is given in Table 7.2.

RESULTS AND COMPLICATIONS Oocytes collected in TH medium have fertilized, cleaved, and implanted at high rates following IVF-ET.4,18,19 Similarly, oocytes treated in the manner described here have produced high continuing pregnancy rates with the tubal transfer procedures of gamete intrafallopian transfer (GIFT) and zygote intrafallopian transfer (ZIFT).20 As with any medium, the major complications arise from the quality of components used to formulate the medium, including water, and the technical execution of the formulation process. A rigorous quality control program is essential and must include careful archiving of lot numbers of all expendable supplies used while the oocyte is in transition from retrieval to culture. Positive pressure filtration with an inert gas should be used to sterilize media, as opposed to suction filtration, which can draw in unsterile air during processing. In centers that elect to maintain a temperature of 37°C in aspirates and media during all

Table 7.2. Summary of treatment during oocyte retrieval, holding, grading, and insemination. Activity Medium Supplement Plasticware (vol) Flushing of aspiration TH (60ml) 40 USP units/ml Na – needle and tubing heparin Flushing of follicle TH (200ml) – – Oocyte identification – – 100×15mm dish Oocyte holding and TH3 (8ml) HSA, 3mg/ml 60×15mm dish rinsing Extended oocyte holding TH3 (1ml) HSA, 3mg/ml organ culture dish Falcon 3037a 30×15mm dish Oocyte grading TH3 (700µl) HSA, 3mg/ml Insemination P–1 HSA, 3mg/ml mediumb

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Falcon 3037a 60×15mm dish 75×12mm tube Falcon 2003a ICSI TH3(20µl) HSA, 3mg/ml lid of 60mm dish Falcon 1007a a Falcon Plastics, Becton Dickinson Labware, Franklin Lakes, NJ, USA. b Irvine Scientific, Inc., Santa Ana, CA, USA –organ culture –microdrop –tube

–1ml oil overlay –30 to 50µl oil overlay –1ml –

transitional activities it is essential that all heating devices be calibrated, stable, and monitored during use. Oocytes whose metabolic activity has been maximized by exposure to 37°C during transition should be moved to incubator conditions as quickly as possible since most transitional media are not designed to be nutritionally complete.

FUTURE DIRECTIONS AND CONTROVERSIES Quality management philosophies and assurance programs are changing with the manufacture of commercial media, medium components, and ART products. Classically, the quality of an ART product has been tested for post-manufacture, giving rise to “embryo tested” categories and the inclusion of bioassay data when products are shipped to the end user. New certification procedures, such as ISO certification in the United States and CE marking in Europe, are examining and verifying an ongoing quality process, not only in acquisition of components but in manufacturing as well. With these, quality is built into the product, not simply demonstrated by testing after production. This should optimize the development of quality procedures in the IVF laboratory by eliminating much of the effort that is directed currently in troubleshooting.

CONCLUSIONS The laboratory should seek, define, and employ techniques and materials, including media, that preserve the developmental capacity of oocytes while minimizing the amount of time required for egg retrieval and transitioning into culture. Single medium formulations are available that can be supplemented uniquely for each step of transition, including retrieval, holding and grading, and are fully compatible with contemporary embryo culture media used for IVF and embryo culture. Although quality materials and procedures are the foundations for eventual quality outcomes, they must be applied using rigorous quality assurance procedures and strict patient test management.

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REFERENCES 1 Biggers JD, Whittingham DG, Donahue RP. The pattern of energy metabolism in the mouse oocyte and zygote. Proc Natl Acad Sci USA (1967); 58:560–7. 2 Donahue RP, Stern S. Follicular cell support of oocyte maturation: production of pyruvate in vitro. J Reprod Fertil (1968); 17:395–8. 3 Leese HJ, Barton AM. Production of pyruvate by isolated mouse cumulus cells. J Exp Zool (1985); 234:231–6. 4 Pool TB, Atiee SH, Martin JE. Oocyte and embryo culture: basic concepts and recent advances. Infert Reprod Clinics of North America (1998); 9:181–203. 5 Graham M, Pool T. Evolution of energy substrates in the culture of human embryos. Assist Reprod Rev (1997); 8:65–8. 6 Ortiz ME, Salvatierra AM, Lopez J, Fernandez E, Croxatto HB. Postovulatory aging of human ova: I. Light microscopic observations. Gamete Res (1982); 6:11–17. 7 Austin, CR. The egg. In: Austin CR, Short RV, eds. Reproduction in mammals, germ cells and fertilization. Cambridge: Cambridge University Press, 1982:46–62. 8 Dulbecco R, Vogt M. Plaque formation and isolation of pure lines with poliomyelitis viruses. J Exp Med (1957); 106:167–9. 9 Good NE, Winget D, Winter W, Connolly TN, Izawa S, Singh RMM. Hydrogen ion buffers for biological research. Biochemistry (1966); 5:467–77. 10 Spierenburg GT, Oerlemans FTJJ, van Laarhoven JPRM, de Bruyn CHMM. Phototoxicity of N′-2hydroxyethylpiperazine-N′-2ethanesulfonic acid-buffered culture media for human leukemic cell lines. Cancer Res (1984); 44:2253–4. 11 Tyrode MV. The mode of action of some purgative salts. Arch Internal de Pharmacodyn et de Therap (1910); 20:205. 12 Edwards RG, Bavister BD, Steptoe PC. Early stages of fertilization in vitro of human oocytes matured in vitro. Nature (1969); 221:632–5. 13 Bavister BD, Yanagimachi R. The effects of sperm extracts and energy sources on the motility and acrosome reaction of hamster spermatozoa in vitro. Biol Reprod (1977); 16:228–37. 14 Bavister BD, Boatman D, Leibfried L, Loose L, Vernon MW. Fertilization and cleavage of rhesus monkey oocytes in vitro. Biol Reprod (1983); 28:983–99. 15 Veeck LL. An Atlas of Human Gametes and Conceptuses. New York: Parthenon Publishing Group, 1999. 16 Bongso A, Trounson AO, Gardner DK. In vitro fertilization. In: Trounson AO, Gardner DK, eds. Handbook of In Vitro Fertilization. 2nd edn Boca Raton: CRC Press LLC, 1999:127–43. 17 Atiee SH, Pool TB, Martin JE. A simple approach to intracytoplasmic sperm injection. Fertil Steril (1995); 63:652–5.

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18 Pool TB, Martin JE. High continuing pregnancy rates after in vitro fertilization-embryo transfer using medium supplemented with a plasma protein fraction containing a- and b-globulins. Fertil Steril (1994); 61:714–9. 19 Weathersbee PS, Pool TB, Ord T. Synthetic serum substitute (SSS): a globulin-enriched protein supplement for human embryo culture, J Assisted Reprod Genet (1995); 12:354–60. 20 Pool TB, Ellsworth LR, Garza JR, Martin JE, Miller SS, Atiee SH. Zygote intrafallopian transfer as a treatment for nontubal infertility: a 2-year study. Fertil Steril (1990); 54:482–8. 21 Earle WR. Production of malignancy in vitro. IV. The mouse fibroblast cultures and changes seen in the living cells. J Natl. Cancer Inst (1943); 4:165–9. 22 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.

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 decondensed and a nuclear structure known as germinal vesicle (GV) is present (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 chromosomes recondense and 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 mass. 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 glucoseaminoglycans, 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

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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

Fig. 8.1 Morphological markers characterizing the meiotic status of oocytes (a) Immature GV oocyte—Meiosis has not been reinitiated and the typical nuclear structure is visible. (b) GVB oocyte (MI)—Meiosis has been reinitiated, the GV has disappeared but the first polar body is still absent. (c) Mature oocytes (MII)—The GV has disappeared and the first polar body has been extruded. 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.20–22 This enzymatic action is assisted by mechanical force generated by vigorous tail beatings, that facilitate the penetration of the sharp sperm head.18,19 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

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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 and 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 layed 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. The in vitro fertilization (IVF) regimens of treatment, which are continuously improving, allowed the birth of hundreds of thousands of babies all over the world. 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),28,30–33 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 Steirteghen at The Free University in Brussels, Belgium and 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 a faulty interaction between sperm and egg 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 clinical section). 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 the section about egg retrieval. 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. The 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

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have reached 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. Best optical conditions are also necessary for the positioning of the mature oocyte in the right orientation for injection (see chapter 10 on micromanipulation). The preparation of the retrieved mature oocytes for ICSI should be carried out under conditions of constant pH of 7.3 and stable temperature of 37°C. In order to maintain the appropriate pH, HEPES-buffered culture media are used. The right 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, CO2-equilibrated paraffin/mineral oil that prevents evaporation of the medium and minimizes the fluctuations of both the pH and the temperature. Temperature fluctuations 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 The susceptibility of the microtubules to temperature variations has been also shown in mature mouse oocytes.40 Interference with spindle organization can disturb the orderly segregation of the chromosomes resulting in uneupolidity. LABORATORY PROCEDURES The 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 three hours between oocyte retrieval and removal of the cumulus cells was recommended by one study.41 This recommendation was challenged by other studies, which did not demonstrate differences in ICSI outcomes correlating with the time interval between egg aspiration and microinjection.42,43 On the other hand, preincubation time that exceeded nine hours resulted in embryos of lower quality.42 Since oocyte denudation cannot be carried out before some preliminary laboratory preparations that are described below are completed, a preincubation period of at least one hour is unavoidable. During this period the retrieved mature cumulus-oocyte complexes are kept in the incubator at 37°C with 5% CO2.

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PRELIMINARY PREPARATIONS FOR OOCYTE DENUDATION Injecting dish—A special shallow Falcon dish (type 1006) is used for placing the denuded eggs. Nine small droplets of HEPES-buffered culture media, 5µl each, are arranged in a square of 3×3 within this dish. An additional tenth droplet serves for orientation. The middle droplet, in which the sperm will be placed, contains 10% polyvinilpyrrolidone (PVP). The droplets are then covered with paraffin or mineral oil that was CO2equilibrated, and the dish is placed in the incubator to warm up before removal of the cumulus cells. 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 320 IU/ml, Sigma Chemical Company, St Louis, MO, USA) is dissolved in HEPES-buffered Earle’s medium. A high concentration of 760IU/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 80IU/ml, which is being commonly used, significantly decreased the rate of parthenogenesis.44 A concentration as low as 10IU/ml has also been shown to efficiently denude mature oocytes.45 Denuding dish—A droplet of 100µl of hyaluronidase solution and five droplets of HEPES-buffered medium covered with CO2-equilibrated oil are placed in a large culture dish and incubated to warm up for 30 minutes. REMOVAL OF THE CUMULUS CELLS Cumulus-oocyte complexes are transferred into the droplet of hyaluronidase solution and repeatedly aspirated through a hand drawn Pasteur pipette for up to one minute. 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 a mouth-controlled fire-polished Pasteur pipette with an inner diameter of approximately 200µM. The oocytes are then transferred through the droplets of enzyme free medium, until all coronal cells have been finally removed. 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.46,47 Finally, the denuded oocytes are placed in the droplets of the injecting dish and their meiotic status and morphology are evaluated.

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EVALUATION OF DENUDED OOCYTES FOR ICSI 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.48–51 These oocytes can be divided into two categories: (1) GV oocytes in which meiosis has not been reinitiated and the typical nuclear structure is visible (Fig 8.1a); and (2) 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 rate of fertilization and embryo development between these and the other oocytes retrieved at MII. However, only one pregnancy was achieved following transfer of embryos obtained from fertilized MI oocytes that had matured in vitro.52 A more recent study demonstrated that 26.7% of MI oocytes extruded the first polar body in vitro within four 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 compared with those oocytes 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.51 The 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 one pregnancy resulted from oocytes that were retrieved at the GV stage from a patient administered with human chorionic gonadotropin (hCG) 36 hours prior aspiration.53 Because of the poor results, these GV oocytes, are usually discarded. Only in cases of very few or no MII oocytes, GV oocytes are rescued for fertilization, provided that they have completed their maturation. Immature GV oocytes can also be retrieved from the small (3–13mm) ovarian follicles present in nonstimulated patients.54–58 These oocytes,

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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. However, even though a fertilization rate of 46% by ICSI of such in vitro matured GV oocytes was obtained,59 only a few cases resulted in pregnancy. 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, are associated with a lower fertilization rate, embryos of poor quality, and, consequently, a lower pregnancy rate.60–62 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.63–66 However, the transfer of these seemingly normal embryos resulted in a poor implantation rate65 and a high incidence of early pregnancy loss.64 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.61 This laboratory reported that evaluation of oocyte quality based on these criteria correlated well with the rate of fertilization and to embryo quality after ICSI. 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 associate with granularity of the perivitelline space.50 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.67 Therefore, the relatively high concentration of LH in HMG effectively promotes oocyte maturation but is insufficient to stimulate ovulation. A delayed administration of hCG in these patients entraps the mature

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oocytes in the follicle leading to egg aging. One notable morphological marker in this case is the fragmentation of the first polar body.68 The presence of aged eggs can also explain the decreased quality of oocytes and lower fertilization rate in polycystic ovarian syndrome (PCOS) patients69 who exhibit relatively high serum concentrations of LH throughout their menstrual cycle.70

Fig. 8.2 Various morphological abnormalities exhibited by oocytes (a) Granulated perivitelline space (b) A fragmented polar body (c) Thickened and darkcolored zona pellucida (d) Cytoplasmic inclusions (e) Enlarged and granulated perivitelline space (f) A large cytoplasmic vacuole.

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 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

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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.

APPENDIX LABORATORY PROTOCOL

1.

2.

3.

4.

PRELIMINARY PREPARATIONS FOR OOCYTE DENUDATION Injecting dish—Place nine, 5µl each, droplets of HEPES-buffered culture medium arranged in a 3×3 square within a shallow Falcon dish (type 1006). Place one additional droplet for orientation. Cover with CO2 equilibrated oil. Replace the middle droplet, with a solution of 10% polyvinilpyrollidone (PVP). Place the dish in the incubator to warm up. Enzymatic solution—Dissolve 1 mg of hyaluronidase (type III, specific activity 320 IU/ml, Sigma Chemical Company, St Louis, MO, USA) in 4ml of HEPES buffered Earle’s medium to obtain a final concentration of 80IU/ml. Denuding dish—Place a droplet of 100µl of the above hyaluronidase solution and five droplets of enzyme free HEPES buffered medium in a large culture dish. Cover with CO2 equilibrated oil and place in the incubator to warm up. Prepare fire polished Pasteur pipettes with an inner diameter of approximately 200µM.

REMOVAL OF THE CUMULUS CELLS 1. Place the cumulus-oocyte complexes into the droplet of the hyaluronidase solution (up to five complexes at a time) and aspirate repeatedly through a hand-drawn Pasteur pipette for up to one minute. 2. Transfer the cumulus-oocyte complexes to an enzyme free HEPES buffered medium droplet and aspirate repeatedly through a mouth controlled fire polished Pasteur pipette. Repeat this procedure through the other four droplets of the medium, until all coronal cells have been totally removed. 3. Transfer the denuded oocytes to the droplets of the injecting dish. MICROSCOPIC EVALUATION 1. Place the injecting dish containing the oocytes on the heated stage of an inverted microscope equipped with DIC. 2. Evaluate oocytes’ morphology and meiotic status at a ×200 magnification.

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REFERENCES 1 Dekel N, Aberdam E, Goren S, Feldman B, Shalgi R. Mechanism of action of GnRH-induced oocyte maturation. J Reprod Fert (1989); (Suppl 37):319–27. 2 Lindner HR, Tsafriri A, Lieberman ME, et al. Gonadotrophin 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: Biochemical Action of Hormones. Litwack G. (ed.), Academic Press, Orlando, Florida, Vol. 13, pp. 57–90, (1986). 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 expansionenabling 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 PH-20 protein on acrosome-intact and acrosome-reacted spermatozoa of cynomologus macaques. Biol Reprod (1995); 52:105–14. 11 Sabeur K, Cherr G, Yudin A, Primakoff P, Li M, Overstreet J. The PH20 protein in human spermatozoa. J Androl (1997); 18:151–8. 12 Meyers SA, Rosenberger AE. A plasma membrane-associated 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.

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14 Beaver EL, Friend DS. Morphology of mammalian sperm membranes during differentiation, maturation, and capacitation. J Electr Microscop 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: Fertilization and Embryonic Development in vitro. Mastroianni L, Biggers JD. (eds.), Plenum Press, New York, pp. 81–187, (1981). 19 Yanagimachi R. Time and process of sperm penetration into hamster ova in vivo and in vitro. J Reprod Fertil (1966); 11:359–70. 20 Dunbar BS, Budkiewicz AB, Bundman DS. Proteolysis of specific porcine zona pellucida glycoproteins by boar acrosin. Biol Reprod (1985); 32:619–30. 21 Brown CR, Cheng WTK. Limited proteolysis of the porcine zona pellucida by homologous sperm acrosin. J Reprod Fertil (1985); 74:257–60. 22 Dunbar BS, Prasad SV, Timmons TM. Comparative overview of mammalian fertilization. New York: Plenum Press (1991). 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. 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.

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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); 340:17–8. 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. Reprod of 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 Magistrini M, Szollosi D. Effects of cold and isopropyl-Nphenylcarbamate on the second meiotic spindle of mouse oocytes. Eur J Cell Biol (1980); 22:699–707. 41 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); 13:1014–9. 42 Yanagida K, Yazawa H, Katayose H, Suzuki K, Hoshi K, Sato A. Influence of preincubation time on fertilization after intracytoplasmic sperm injection. Hum Reprod (1998); 13:2223–6. 43 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. 44 Joris H, Nagy Z, Van de Velde H, De Vos A, Van Steirteghem A. Intracytoplasmic sperm injection: laboratory setup and injection procedure. Hum Reprod (1998); 13 (Suppl 1):76–86. 45 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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46 Iritani A. Micromanipulation of gametes for in vitro assisted fertilization. Mol Reprod Dev (1991); 28:199–207. 47 Flaherty SP, Payne D, Swann NG, et al. Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection. Hum Reprod (1995); 10:2629–32. 48 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. 49 Mandelbaum J, Junca AM, Balaisch-Allert J, et al. Oocyte maturation and intracytoplasmic sperm injection. Contracept Fertil Sex (1996); 24 (7–8):534–8. 50 Hassan-Ali H, Hisham-Saleh A, El-Gezeiry D, Baghdady I, Ismaeil I, Mandelbaum J. Perivitelline space granularity: a sign of human menopausal gonadotropin overdose in intracytoplasmic sperm injection. Hum Reprod (1998); 13:4325–30. 51 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. 52 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. 53 Nagy ZP, Cecile J, Liu J, Loccufier A, Devoroey P, Van Steirteghem A. Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal-vesicle stage oocytes: case report. Fertil Steril (1996); 65:1047–50. 54 Jaroudi KA, Hollanders JMG, Sieck UV, Roca GL, El-Nour AM, Coskum S. Pregnancy after transfer of embryos which were generated from in-vitro matured oocytes. Hum Reprod (1997); 12:857–9. 55 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. 56 Edrishinghe WR, Junk SM, Matson PL, Yovich JE. Birth from cryopreserved embryos following in-vitro maturation of oocytes and intracytoplasmic sperm injection. Hum Reprod (1997); 12:1056–8. 57 Trounson A, Anderiesz C, Jones GM, Kausche A, Lolatgis N, Wood C. Oocyte maturation. Hum Reprod (1998); 13(Suppl 3):52–62; (discussion) 71–5. 58 Russel JB. Immature oocyte retrieval with in-vitro maturation. Curr Opin Obstet Gynecol (1999); 11:289–96. 59 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. 60 Sousa M, Tesarik J. Ultrastructural analysis of fertilization failure after intracytoplasmic sperm injection. Hum Reprod (1994); 9:2374–80.

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61 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. 62 Loutradis D, Drakakis P, Kallianidis K, Milingos S, Dendrinos S, Michalas S. Oocyte morphology correlates with embryo quality and pregnancy rate after intracytoplasmic sperm injection. Fertil Steril (1999); 72:240–4. 63 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. 64 Alikani M, Palermo G, Adler A, Bertoli M, Blake M, Cohen J. Intracytoplasmic sperm injection in dismorphic human oocytes. Zygote (1995); 3:283–8. 65 Serhal PF, Ranieri DM, Kinis A, Marchant S, Davis M, Khadum IM. Oocyte morphology predicts outcome of intracytoplasmic sperm injection. Hum Reprod (1997); 12:1267–70. 66 Balaban B, Urman B, Sertac A, Alatas C, Askoy S, Mercan R. Oocyte morphology does not affect fertilization rate, embryo quality and implantation rate after intracytoplasmic sperm injection. Hum Reprod (1998); 13:3431–3. 67 Dekel N, Ayalon D, Lewysohn O, Nevo N, Kaplan-Kreicer R, Shalgi R. Experimental extension of the time interval between oocyte maturation and ovulation: effect on fertilization and first cleavage. Fertil Steril (1995); 64:1023–8. 68 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. 69 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. 70 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.

9 Oocyte in vitro maturation Johan Smitz, Daniela Nogueira, Rita Cortvrindt, Daniel Gustavo de Matos

THEORETICAL OVERVIEW THE RELATION BETWEEN OOGENESIS, MEIOTIC MATURATION, AND DEVELOPMENTAL COMPETENCE In the human species oocyte growth from a diameter of 20–30µm up to a variable final oocyte diameter of 115–120µm is achieved mainly during the preantral follicle phase. Regulation of the preantral growth phase is still not very well studied, and by using fertility drugs we cannot intervene with these growth stages. Knowledge on early follicle growth stages was mainly gained from studies in rodent and domestic animal species. These studies showed that 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 directing part in the follicular environment, for example by preventing premature luteinization by regulating 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 2–12mm follicles and have not completely fulfilled their growth and final maturation. Previous work has shown that, during the period just preceding the final meiotic maturation stage, synthesis and packaging of RNA and translational products are a very important process 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 responsiveness.7

THE REGULATION MECHANISMS GOVERNING MEIOTIC ARREST It is the somatic environment which holds the oocyte in nuclear arrest. The exact molecular nature by which this happens is still incompletely understood. The inhibitory signals are originating in theca and granulosa cells, are positively influenced by follicle stimulating hormone (FSH), and are conducted via the gap junctions and the follicular fluid into the oocyte (Fig 9.1).

Fig 9.1 (a) The oocyte is surrounded by a compacted mass of granulosa cells which holds the oocyte in the 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 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

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junction between the transzonal projection of a granulosa cell and the oolemma. The meiosis “arrestor,” named oocyte meiotic inhibitor (OMI), present within the somatic compartment of the follicle has not been fully characterized yet.8 Several candidate molecules, peptides, have been showing a meiosis arresting activity such as TGF-β, anti-Mullerian hormone (AMH), activin, inhibin, or follistatin. This meiosis arresting activity is rather the result of the contribution by many factors originating in theca interna, granulosa, and follicular fluid. Purine bases such as hypoxanthine and adenosine present in follicular fluid inhibit phosphodiesterase activity and maintain by this a high intra-oocyte cyclic adenosine monophosphate (cAMP) concentration.9–10 Recently there have been experimental data in rat oocytes suggesting 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.11 The relation between oocyte volume and the competence to reinitiate meiosis has been well established for the 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.12 This oocyte volume has been related to a certain follicular diameter in the different species: 1 to 2mm in mice;13 2 to 4mm in cattle;14–16 and 10mm in humans.17–19 By aspirating the oocyte granulosa cell complexes (COC) from the follicle, connections to surrounding cells are broken. The factors from theca and granulosa responsible for keeping meiosis arrested are not transferred to the oocyte via the junctional contacts any more, 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 the folding of the oocytes’ nuclear membrane instead of the rupture of the gap junctional processes (Fig 9.2).20 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. Meiotic competence is the result of the activation of the two components of M-phase promoting factor (MPF), a protein kinase: p34cdc2, a serine-threonine kinase, and cyclin B, a

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Fig 9.2 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.

Fig 9.3 Maturation promoting factor (MPF) activation: activation of MPF by complex formation of p34cdc2 and cyclin B and dephosphorylation. protein of the cyclin family. Activation of MPF requires complex formation of these two components and the dephosphorylation of threonine residue 14 and tyrosine 15 of p34cdc2. This last step of

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dephosphorylation is under the control of the gene products wee1 and cdc25. There exists furthermore an autocatalytic amplification of MPF (Fig 9.3).21 Activation of MPF in human requires protein synthesis like in other domestic mammalian species. Recent work from Crozet et al in goats 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.22 Experiments in bovine oocytes showed that cyclin B plays a major part for initiating p34 activation and that this protein represents the limiting factor for meiotic resumption.23 This 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 ending with the first polar body (PB) extrusion. In anaphase I there is a decrease of MPF (owing to cyclin degradation) to a lower level of activity, which is sufficient for 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 an 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 (Fig 9.4).24 The reinitiation of meiosis can be provoked by a transient fall in cyclic adenosine monophosphate (cAMP) concentrations within the oocyte.25–26 The delicate equilibrum in cAMP concentrations within granulosa cells and oocyte is maintained by inflow of cAMP driven by the gonadotropin environment and the degradation of cAMP by phosphodiesterases (PDE) (Fig 9.5). The cAMP stabilizes interphase microtubules27 and the relevant mediators for MPF activation are retained in a cortical cytoskeletal scaffold.28 The PDEs exist under several isoforms that are differently expressed in somatic cells and oocyte. The effectors of cAMP are the protein kinases of which there is also a compartmentalization of different isoforms (Fig 9.6).29 In-vivo the primary stimulus for meiosis resumption is the rise of LH which generates (1) an initial cAMP increase and (2) the liberation of intracellular calcium stores (via phospholipases and phosphoinositol) which will stimulate protein kinase C (PKC).30 PKA and PKC exert their action via a cascade of phosphorylations and dephosphorylations, which finally lead to MPF activation. The LH trigger propagates a microtubule labilizing factor (calcium perhaps) which provokes intermixing of cell cycle molecules and by such activation of MPF (Fig 9.7).31–33

OOCYTE MATURATION AFTER SUPEROVULATION THE RELATION BETWEEN OOCYTE MATURITY AND FOLLICLE DIAMETER IN SUPEROVULATED CYCLES FOR IVF/ICSI 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 to optimize

Fig 9.4 Evolution of MPF activity during oocyte meiosis. During anaphase/telophase MPF remains elevated above baseline; this causes the extension of the condensed state of the chromatin.235

Fig 9.5 Hypothetical molecular mechanisms of reinitiation of meiosis. Hypothetical links between signal transduction factors and MPF activation.

Fig 9.6 Upon a similar stimulus cumulus cells and oocyte react differently due to compartmentalization of the regulatory proteins PKA and PDE.

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Fig 9.7 Effects of luteinizing hormone (LH) action on the cascade of second messenger systems in follicle cells. ovarian superovulation treatments for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).34 The small antral follicles (4–8mm diameter) present at the onset of the menstrual cycle are receptive to the gonadotropins and can develop up to a range of small to large follicular volumes. The size of the cohort of follicles that is aspirated is largely dependent on the moment of appearance of the spontaneous LH surge or on the decision of the time to inject the ovulatory dose of human chorionic gonadotropin (HCG). Very commonly, in protocols without suppression of gonadotropin releasing hormone (GnRHa), the clinician decided to administer HCG when at least three large follicles reached a mean follicular diameter of 17mm 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) (including ours) prefer to use as criterium for HCG injection: the presence of a majority of follicles with diameters between 17mm and 22mm. On the day that is decided for HCG injection aspirated follicles have generally been showing a progressive growth profile for six to eight days. It seems from a study by Tan et al that a window of decision for HCG injection of three days can be tolerated without influencing pregnancy outcomes.35 Some researchers evaluated the outcome of IVF or ICSI in relation to the different classes in 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 6.5ml or >23mm diameter.36–37 Studies from Nayudu

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et al34 and others38 found that most normal pregnancies after IVF came from follicles in the 2.5–6.5ml volume or 15–23mm diameter ranges. Studies were conducted by us to analyse the relation between fertilizability, embryo cleavage, and clinical pregnancy rates in GnRHa and gonadotropin stimulation cycles showing a superior developmental capacity from oocytes aspirated from large follicles with a diameter over 20mm (Smitz, unpublished personal observations). All IVF studies consistently show that follicles ≤2ml (volume) or ≤14mm (diameter) generate a very low proportion of clinical pregnancies. Most commonly, oocytes from small follicles do not cleave after fertilization, and even if they succeed to implant early abortion occurred.34,38 When oocytes were stripped from their densely packed surrounding granulosa cumulus, it was found that a higher proportion of immature (germinal vesicle, GV) oocytes were recovered from these small follicles.39 When immature COC (GV oocytes from ICSI cycles) are injected, fertilization fails, and when denuded oocytes are injected 24 hours after an in vitro maturation period most preimplantation embryos are of poor quality, have a high aneuploidy rate, and yield karyotype anomalies.40 Retrospective analysis on a large number of cycles from couples undergoing ICSI testified that after a GnRHa and gonadotropin stimulation about 80% of the follicles aspirated yielded a metaphase II oocyte (the remaining 20% of the cohort were either MI or GV). The cycles that yielded a higher proportion of immature oocytes had experienced a poor stimulation management with an aspiration of smaller follicle diameters (<17mm). 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.39 This suggests that intrinsic meiotic maturation defects in infertile couples are probably very rare events. Figures on the failure in 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. 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 PROGRAMME FOR IVM Although there is a tendency today to apply superovulation drugs more cautiously, the serious complication, severe ovarian hyperstimulation syndrome (OHSS), cannot be completely prevented.41–43 Considering the long treatment time (several weeks),44 the development of a viable and efficient IVF system without stimulation may

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reduce the patients’ burden by shortcutting both the duration and the costs of the treatment. Although already pioneered by several well experienced ART clinics for many years, there is as yet no consistent IVM treatment programme that could substitute for the classical superovulation protocols in terms of acceptable pregnancy rates (Fig 9.8).45–47 An analysis from Plachot revealed that approximately 15% of the oocytes collected after superovulation for ICSI were still in prophase or metaphase I.48 Although the metaphase I oocytes matured within a short incubation period of four hours, embryos obtained were of a lower developmental potential.48,49 It should be made clear that perhaps results from these immature oocytes are disappointingly low because they represent an already compromised group of follicles not responding to an HCG stimulus. These follicles might suffer from intrinsic anatomical (vascularization) or local metabolic (paracrine) defects.

Fig 9.8 Comparative outcomes in human oocytes of in vivo and in vitro oocyte maturation in relation to gonadotropin stimulation. Immature oocytes from superovulated cycles for IVF or ICSI are therefore not comparable to immature oocytes resulting from unstimulated or slightly stimulated cycles. Moreover, the manoeuvre of enzymatic decoronization of immature oocytes in view of ICSI is probably also compromising further maturation progression. Granulosa cells are the production site of steroids, growth factors (IGF I, EGF), proteins that have

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as yet not been characterized, and other compounds that contribute to cytoplasmic maturation of oocytes.50,51 Immature oocytes—considered as a side-product in ART cycles— among most 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. PREVIOUS EXPERIENCE FROM THE CULTURE PROCEDURE FOR IMMATURE OOCYTES IN HUMAN Pioneering groups have shown that COC can be retrieved in a reproducible way from small antral follicles. In unstimulated or stimulated normo-ovulatory women or polycystic ovary (PCO) patients 10–15 COC were obtained.45,46,52 Two thirds of the aspirated COC showed spontaneous nuclear maturation within 36–38 hours and were fertilized (rate: 30–45%). Although the first cleavage divisions were apparently morphologically normal (reported rates: 30–40%), clinical pregnancy rates remained low.45,46,52,53 Very limited clinically significant information could be drawn from these studies on immature human oocyte culture. The patients scheduled for IVM were pretreated with several stimulation drug regimens. Various reports are anecdotal, and the culture material (COC) was always poorly characterized as to its follicular origin. The COC 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 concentration and nature of the protein source differed. Some researchers used coculture on a variety of primary feeder cells. In a large prospective group of 75 normo-ovulatory infertile women who were not stimulated, immature CCOC were aspirated from ovaries, showing a leading follicle of 10mm diameter.54 After maturation in vitro followed by ICSI, ongoing pregnancy rates were 14% per embryo replacement. All children born from this group were reported to be normal (A L Mikkelsen, personal communication). Although this last series of patients showed promising outcomes, it would be safe to analyse the in vitro matured oocyte cytogenetically to detect possible increases in aneuploidy rates before this technique becomes clinically available. OOCYTE RETRIEVAL FROM IMMATURE FOLLICLES THE ENDOCRINE MILIEU AT OOCYTE RETRIEVAL Early in the follicular phase small antral follicles of between 2mm and 8mm diameter can be observed by vaginal ultrasound. These antral

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follicles are recruited by the rising FSH concentrations that follow the regression of the corpus luteum of the preceding cycle. These 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 the recruited antral follicles. During an immature oocyte aspiration procedure some follicles from this cohort might be at the edge of undergoing atretic changes. Part of oocyte granulosa complexes from these small follicles might already have activated signals that lead to reactivation of meiosis I as part of the process of atresia. A study by Yuan and Guidice analysed human ovaries from normal cycling women and quantified the atretic changes in the different classes of follicles.55 Their observations are exposed in Table 9.1. These data suggest that when aspirating small (2.1–9.9mm) follicles about half of these might have initiated a cell death programme. These findings in human are similar in this respect to domestic animals, in which 85% of follicles found in an ovary at any time of the cycle are atretic.56–57 However, early stages of follicular atresia may not be an impediment to full meiotic maturation and further embryonic development as was shown in sheep50 and cattle.58 The first signs of atresia in antral follicles are manifested by pyknosis in the granulosa cell compartment whereas the oocyte is affected last by atresia.59 In cattle, Blondin and Sirard surprisingly found that slightly atretic or non-atretic follicle status had no impact on the further developmental competence of the oocyte.58 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 punction. In humans there are many conflicting data in relation to IVM from natural or from gonadotropin-stimulated cycles. Cha and Chian illustrated a more rapid reinitiation of meiosis I and first polar body extrusion rate in stimulated cycles compared with unstimulated cycles.60 Data from Gomez et al61 and Toth et al62 showed a higher MII maturation yield after gonadotropin priming. More recent prospective work from Wynn et al63 showed improved meiotic maturation rates after priming with a short course of FSH, whereas Mikkelsen et al64 could not find any beneficial effect on oocyte maturation, fertilization and preimplantation embryo development. An overview of the numbers of oocytes retrieved per procedure is shown in Table 9.2. In a search for the optimal moment during the unstimulated cycle for immature oocyte aspiration, Cobo et al65 showed that follicular diameter at the time of oocyte pickup plays an important part in further development. Oocytes with the best developmental competence originated when patients were aspirated before the leading follicle reached 10mm diameter (earlier than day 6 of the cycle). Similar observations were reported by Smith et al66 who found that 62% of all aspirated COC were suitable for maturation and 28 hours after collection 73% were metaphase

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II. Clinical implantation rates were not different whether oocytes matured in vitro for 28 hours (10% implantation) or 36 hours (12% implantation).

Table 9.1. Frequency of follicular apoptosis in human ovaries (adapted from Yuan and Giudice, 1997)55. Follicle size (histological Follicle Frequency of follicular apoptosis sections) numbers (%) Primordial 35 0.00 Primary 25 0.00 Secondary 7 0.00 Pre- and small antral (0.1– 13 38 2mm) Small antral (2.1–5mm) 22 50 Preovulatory (5.1–9.9mm) 11 27 Dominant (≥10mm) 3 0.00 Table 9.2. Reports on immature oocyte retrieval in relation to endocrine profile and type of treatment (ovarian stimulation or natural cycle). Author Patients Cycle Mean number oocytes aspirated Cha1991 normal natural 12 Cha1996 PCOD natural 12.6 Trounson 1994 PCOD natural 12–15 normal natural 2.8 Barnes 1996 PCOD natural 16.5 normal natural 4.9 Russell 1997 mixed natural 11.5 Wynn1998 normal natural 3.7 normal stimulated 7.7 Mikkelsen 1999 normal natural 3.7 normal stimulated 3.7 Cobo 1999 normal natural 4.9–6.8 Smith 2000 normal natural 5–6

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METHODS THE TECHNIQUE OF IMMATURE OOCYTE RETRIEVAL Initially aspiration of small follicles was attempted under laparoscopic view. A special needle with a reduced length, a short bevel, and a more rigid structure was developed for aspiration of small follicles. Fixation of the ovary during puncture by using holding instruments introduced through two 5mm incisions in the lateral part of the lower abdomen was helpful.46 The aspiration technique can nowadays also be performed via the transvaginal ultrasound guided way. The ultrasound machine should have a very good resolution to permit a clear detection of 2–4mm follicles. A double lumen needle offers the possibility for flushing in case COC are not easily found. A prewarmed Hepes buffered solution containing heparin is used for the flushing of the punctured follicles. Reduction of the aspiration pressure used in the needle (reduction from 15 kPa to 7.5 kPa) is absolutely necessary.46 IDENTIFICATION AND TYPING OF THE CUMULUS CORONA OOCYTE COMPLEXES (COC) After transvaginal aspiration of the contents of large follicles (>15mm diameter) obtained by superovulation in view of IVF, COC can be easily recognized floating in between the other parts of the mural granulosa cells. The COC is recognized as a mucified clump of cells under the stereomicroscope (Fig 9.9). Owing to the more traumatic procedure of aspirating small follicles, the search for the unexpanded COC is more difficult than in IVF. Careful inspection of the bloody aspirates under the stereomicroscope or the use of filters might enhance the recovery of the densely enclosed oocytes. The more time consuming aspiration procedure and the prolonged handling of the COC during the searching procedure necessitates strict control of temperature and pH conditions. The COC from large follicles are graded on the basis of the expansion of the surrounding cells from corona and cumulus. In routine IVF practice, grading of the nuclear maturation stage is approximated by the degree of expansion of the surrounding cumulus and corona cells.67 Early work of Testart et al established that in natural cycles there is a good synchrony between the development of the cumulus cells and the nuclear maturation stage.68 This synchrony in maturation is observed less often in COC from superovulation cycles for ART.69

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In the aspirates from smaller follicles (<12mm diameter) it is less easy to recognize the oocytes, which are trapped within a very dense mass of surrounding granulosa cells. Often the clumps of COC are floating in between more compact masses of mural granulosa cells. The aspiration fluid is generally very bloody because successive small follicular structures are punctured subsequently, leading to more damage to the well vascularized theca cell compartment. As the mucification process has not yet taken place within the ovarian follicles of this size, detachment of COC from the follicle wall is more difficult, and multiple flushing cycles have to be applied.

Fig 9.9 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 in the different pictures from low to large magnification as visualized under the stereomicroscope.

The degree of expansion of the COC will be dependent 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 the mucification of the differentiated granulosa cells neighboring the oocyte. A classification of aspirated COC from small follicles is proposed in Table 9.3 and illustrated in Fig 9.10. For an easy search and accurate manipulation of the COC we operate on a stereomicroscope Wild M-8 (Wild Heerbrugg) at magnifications from 5× to 60×. At first the entire petri dish is overviewed at a magnification of 5×, going field by field. Tissue clumps suspected to contain an oocyte are sucked up in the pipette and transferred in 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. The selected pieces are placed in culture dishes (Primaria, Falcon 3801 Becton Dickinson), evaluated and classified following the cumulus and

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corona expansion criteria. The COC are distributed among culture conditions depending on their maturation stage. A more profound evaluation can be performed on the inverted microscope (Nikon Diaphot, Tokyo, Japan) 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. During all manipulation steps extreme attention is given to maintain the physiological temperature (37°C) by adapting heating stages on stereomicroscope and on inverted microscope. In order to keep a stable pH during the multiple manipulations a Hepes buffered Earle’s medium is used. Within the laminar flow a gassed table mini-incubator can preserve optimal temperature and pH of the culture medium outside the incubator.

Fig 9.10 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 an excentric position, (b) CU1, CE2, CO2:

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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) There are no granulosa cells which surround the oocyte. These oocytes have a degenerative aspect and show extrusion of the polar body.

Table 9.3. Maturity grading of COC during prematuration days in culture. 1. Grading of granulosa cell mass: 3. Assessment of oocyte cumulus expansion and oocyte morphology coverage Oocyte diameter (µm) Cumulus mass (CM): Oocyte cytoplasm 3 or less layers of cumulus cells (CM presence of 0) more than 3 but less than 10 layers (CM inclusions : vacuoles / of cumulus cells 1) refractile bodies 10 or more layers of cumulus cells (CM darkness : clear / dark 2) Cumulus expansion (CE): granularity : homogeneous / granular tight, dense cells (CE Zona : normal / abnormal 0) moderate expansion of cells (CE Perivitelline : normal / enlarged 1) space fully expanded cells (CE Oocyte shape : regular / irregular 2) Contact (CO) between cumulus Polar body : intact / cells and oocyte: fragmented naked (CO 0) partially naked (CO 1) fully enclosed (CO 2) fully enclosed and part of follicle (CO wall 3)

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2. Assessment of oocyte nuclear maturation stage GV GVBD PB For further maturation, COCs are placed one by one in microdroplets of culture medium which have been pre-incubated for at least three hours. The petri dishes (Falcon) contain 12 droplets of 10µl media covered with 2ml of paraffin oil (Sigma). The culture dishes are kept in a 5% CO2 and 100% humidified incubator maintained at 37°C. The COCs are cultured and oocyte maturation is scored for cumulus expansion, the nuclear maturation and oocyte morphology (Table 9.3). NON-INVASIVE TECHNIQUES TO EVALUATE COC CULTURE PROCEDURES An optimal culture medium has not been described yet; a suitable strategy for culturing immature oocytes has yet to be developed. Depending on the grade of oocyte immaturity a differential approach in culturing technique could lead to improved oocyte quality. The grade of maturity of an individual COC can be defined using morphological criteria. Making use of the inverted microscope equipped with a Hoffman Modulation can allow the grading of the nuclear maturity when either the germinal vesicle or the first polar body (PB) can be visualized. On the same microscope the degree of mucification of the cumulus cells can be recorded and semiquantified. The grading of the cumulus mucification which in the natural cycle has an evolution paralleling the nuclear maturation has its theoretical limits in FSH stimulated cycles. The induction of supraphysiological levels of FSH dissociate nuclear and cumulus maturity.69 When puncturing COC from unstimulated, FSH stimulated, or HCG induced patients the morphology of the COC is already very different at the start of the IVM procedure. In Fig 9.11 examples are shown. INVASIVE TECHNIQUES FOR OOCYTE EVALUATION OOCYTE NUCLEAR MATURITY 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 period in culture, the oocyte has not only extruded its PB but has as well formed a well aligned second metaphase plate (2 hours after PB extrusion). The most convenient way to assess nuclear maturation and

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abnormalities is to identify the oocyte’s meiotic stage by staining the DNA with Hoechst 33342 (Molecular Probes, Leiden, Netherlands) and visualizing the nuclear stage with UV light (Fig 9.12). After first polar body extrusion it is possible to observe a physically unarranged chromosome plate (pro-metaphase II), or a disarrangement of the chromosomes. If abnormal, some chromosomes are not well aligned on the metaphase plate, or there might be even a dislocation of chromosomes in the cytoplasm

Fig 9.11 Aspect from the cumulus corona oocyte complex (COC) immediately after retrieval throughout the culture period. (a) COC punctured from an unstimulated patient: more than five layers of granulosa cells are tightly surrounding the oocyte (CM2, CE0, CO2). Fig (b) shows the same oocyte after one day in culture. The distal granulosa cells are expanded and the proximal granulosa cells are still attached to the oocyte (CM1, CE1, CO2). Figs (c) and (d) show representative examples of oocytes cultured for 2 days. Fig (c) shows that some oocytes can still have some proximal

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granulosa cells attached (CM2, CE1, CO1). (GC) (d) The oocyte has lost connections with the surrounding cells which form a clump. The oocyte has extruded its polar body. 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. 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 (CG) originating in the Golgi apparatus during the late preantral and antral stages. The cortical granules in mammalian eggs are electron-dense small spherical vesicles

Fig 9.12 (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 M II plate. (a) The arrow shows the DNA content within the polar body. In (b) the arrow shows the well aligned M II chromosome plate.

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Fig 9.13 Electron-micrographs taken from cultured oocytes. (a) Immature oocyte at GV-stage with few cortical granules (CG) aligned (arrow). Clumps of mitochondriae are indicating the immature stage of the oocyte (arrow head). (b) Oocyte after 48 hours culture: although the mitochondriae have spread over the cytoplasm (arrow head) as is typical for a meiotically competent oocyte, a complete cytoplasmic maturation has not occurred and double layers of CG are found at the periphery (arrow). (c) Oocyte with polar body extruded after 48 hours culture. CG are well-aligned under the oolemma forming a single layer (arrow). Star in figures represent the zona pellucida. (300–500nm diameter) surrounded by a single membrane. CG contain mucopolysaccharides, proteases, tissue-type plasminogen activator with serine-protease activity, acid phosphatase, and peroxidase enzyme activity. CG 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.70–71 During the transition of GV to MII, migration and dispersal of CG take place in such way that in a mature egg CG they are lined up just below the oolemma. Incomplete dispersion of CG is a reliable marker for a disturbance in cytoplasmic maturation. Localization of CG can be done by EM analysis (Fig 9.13) and/or by staining CG with lectins labelled with a fluorescent marker and analysis on a confocal laser microscope (Fig 9.14).

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Cytoskeleton: actin and tubulin microfilaments Mammalian oocytes possess cortical filaments of actin that play an important part in CG migration during oocyte maturation, polar body extrusion, and cell division.72 Cortical microfilaments are also involved in the peripheral migration of the first meiotic spindle. Analysis using staining for actin and microfilament inhibitors have 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 the rotation of the second meiotic spindle after fertilization. In primates the second meiotic spindle is localized radially to the cell surface,

Fig 9.14 Confocal images representing the distribution of cortical granules (CG) in bovine immature (a) and in vitro maturing (b) oocyte. CGs display a green fluorescence owing to FITCpeanut 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. Chromosomes are still at

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prometaphase stage revealed by unaligned chromosome configuration (arrow).

Fig 9.15 Immunofluorescence image of a human oocyte shows the second meiotic spindle localized paratangentially to the cell surface after in vitro maturation. The microtubules display green fluorescence owing to FITC-conjugated anti-IgG anti-αtubulin. The chromosomes are stained with propidium iodide. In the insert the images of the MII plate and the chromosomes are separated for better visualization. thus avoiding this specific function of the microfilaments (Fig 9.15). 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.73,74

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Microtubules in human meiotic spindles are highly sensitive to temperature variation75 and environmental perturbances.76 Perturbances on the spindle microtubules are irreversible and affect the genetic balance since the segregation and alignment of the chromosomes during meiosis involves a complex interaction between chromosomes and cytoskeleton.77 Therefore, evaluation of abnormalities of the spindle structure after oocyte maturation might be important for control of the manipulations during culture and a checkpoint for assessment of the microenvironment surrounding the developing oocyte (Figs 9.16 to 9.19). Mitochondriae 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.78 Mitochondrial redistribution was also remarked in ova of primates and cattle prefertilization during IVM. In cattle GV oocytes mitochondriae are arranged in a cortical distribution but relocate during IVM.79 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 mitochondriae can be stained by fluorescent dyes, such as Rhodamine 123 or Mitotracker (Molecular Probes, Lüden, The Netherlands), and scanning of the entire oocyte can be done by confocal laser microscopy. By image processing the distribution patterns can be shown and interpreted. High resolution two dimensional (2D) gel electrophoresis The mature human oocyte has a diameter of 110–120µm and contains some 150ng of total protein. During mammalian oocyte growth and maturation there are transcriptional processes that lead to the synthesis and accumulation of proteins necessary for

Fig 9.16 Single sections of confocal images representing the microfilaments (actin) in human MII oocytes 44 hours after culture. In (a) and (b) left and right are the same oocyte at different levels of the MII plate. At the level of the first polar body F actin displays an increased red staining, which is more predominant than at the level of the metaphase plate, (b) on the left some granulosa cells transzonal processes are stained. The chromosomes are stained with picogreen.

Fig 9.17 Optical sections on confocal microscope representing the microfilaments in a human oocyte at MI stage. F actin displays red staining predominant at the border where the first polar body will be extruded. Chromosomes are stained with picogreen.

Fig 9.18 Overlap sections of confocal images representing the microfilaments of a human oocyte denuded after 44 hours of culture. F actin displays red staining predominant on the border of the oocyte. Note the transzonal processes from granulosa cells (GCs) also stained red. This testifies the

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maintenance of connections between GC and oocyte during culture.

Fig 9.19 Confocal image representing the microfilaments and a chromosomal disarrangement in a MII human oocyte. On the right, sections are overlapping and the polar body is clearly visualized as well. Chromosomes are stained with picogreen. regulation of nuclear and cytoplasmic maturation.80 Sensitive 2D gel electrophoresis techniques allow us to distinguish nuclear and cytoplasmic maturation by analysing the difference in protein neosynthesis over an arbitrarily chosen time interval (such as the in vitro maturation 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, from in vitro matured oocytes for 48 hours (collected in unstimulated cycles) that were compared to 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.81 The missing protein spots in IVM matured oocytes were probably a result of the shortened oocyte growth phase of oocyte retrieved from small follicles and may explain the poor developmental capacity of embryos obtained from small follicles. Collaborative work between CRRA (A Goff, St Hyacynthe, Quebec, Canada), and our laboratory analysed protein profiles of 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.82 Analysis of protein neosynthesis in the cumulus cells from corresponding oocytes did not show major changes in relation to duration of follicle culture.

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2D-SDS-PAGE analysis was used to evaluate protein profiles of aspirated immature human oocytes from small follicles (2–8mm). Goff et al reported that in a serum free culture system addition of 10ng/ml EGF induced the synthesis of at least 12 proteins.83 These data further showed that EGF supplementation (but not FSH+LH supplements) yielded similar results to 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. FERTILIZATION Insemination by conventional IVF of in vitro matured oocytes has been performed with lower success compared with in vivo matured oocytes (45% v 73%, respectively45). Hardening of the zona pellucida of the oocyte caused by a prolonged culture period could be responsible for the decreased fertilization.84,85 ICSI is used to overcome the problem of zona hardening and could in these cases lead to results superior to IVF. Since there are still very few studies on the timing of oocyte maturity, and as optimal culture condition has not been well defined yet, we may assume that insemination time after IVM has to be programmed by the exact moment at which the oocyte extruded its first PB in order to avoid senescence of oocytes. The time of maturation influences the fertilization and developmental capacity of the oocyte and depends on the conditions in which the oocyte has been cultured.86 Others have shown that the PB extrusion rate of cumulus enclosed oocytes cultured for at least 36 hours was improved over denuded ones and had fertilizability and cleavage rates that were comparable to fresh MII oocytes.46,47,87 In IVF it was shown that a prematuration time before insemination is beneficial for the outcome.46,67 Similarly for ICSI the injection procedure has to be performed within two to six hours after oocyte retrieval. After PB extrusion, the oocyte needs a short time to rearrange its meiotic spindle in preparation for sperm entrance. The most appropriate time for microinjection of IVM oocytes is probably between two to six hours after PB extrusion. This has, however, never been studied prospectively. 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 the patients.60,63 In gonadotropin primed patients (normo-ovulatory women) at least 24% of the oocytes extrude the first PB after 23 hours.66 In a first group of patients COC were denuded at 28 hours; 73% of oocytes showed presence of a PB. In a second group denudation was performed at 36 hours and showed that 77% of the ova were MII.

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IN VITRO OOCYTE AGEING In postmature oocytes, changes in localization of some cytoplasmic organelles may occur. Numerous CG may conglomerate beneath the oolemma, or they may migrate centripetally.88 After fertilization, postmature oocytes may have inhibition of cortical granule release or poor zona reaction.89 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 deepseated spindles with fewer microtubules or spindles with attenuated poles. Chromosomes may clump together or scatter in the cytoplasm.90 Fragmentation of the polar body may be a sign of ageing in human oocytes. Fukuda et al compared short and long culture of mouse oocytes after subzonal sperm injection with reference to spontaneous zona hardening.91 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 long oocyte culture, which seems to indicate that hardening of the zona is an indicator of oocyte ageing. Calcium signaling is involved in important events in oocytes, such as meiotic competence acquisition92 and oocyte activation.93 Modification in the regulation of intracellular Ca2+ is one of the major changes taking place during oocyte maturation.94 Normally Ca2+ releases from the inositol 1,4,5-triphospate (InsP3) channels of the ER membrane, which affect Ca2+ oscillations in fertilized oocytes. In vitro ageing related changes in Ca2+ release after fertilization have been recently shown. Microinjection of InsP3 into the cytoplasm of eggs demonstrated a lowering in the maximum rate of increase in Ca2+ in aged compared to fresh mouse oocytes. This is due to a depletion of the ER Ca2+ stores with ageing.95 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 ova and provide the framework for the chromosomal movement and cell division.96,97 In porcine oocytes IVM, the involvement of microtubules and microfilaments in chromosomal dynamics was proven during transition from GV to MII.98 Thus, disturbances in cytoskeleton organization may

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result in abnormal development patterns and lower incidence of embryonic development. Damiani et al compared IVM of cow and calf oocytes. They demonstrated some indicatives of intrinsic ooplasmic deficiencies causing an abnormal fertilization characterized by lack of sperm aster formation, asynchronous development of pronuclei, or extrusion of maternal chromatin.99 All those features can be investigated by using Hoechst and immunofluorescent techniques already described in this chapter. Even simple observation under light microscope can reveal eventual defects induced by culture. Morphology of pronuclear pre-embryos can be used as a first step in the evaluation of fertilization after IVM. Scott et al, in a retrospective study, described characteristics of the pronuclear morphology of pre-embryos in relation to their development to term.100 After fertilization (16–17 hours) a good pre-embryo possesses close pronuclei, aligned nucleoli, and a heterogeneous cytoplasm with a clear halo present. It might be possible to apply the same criteria to analyse 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. Several studies showed a high incidence of cleavage arrest after IVM and an increased multinucleation owing to cell division arrest. Possible causes of cleavage arrest include inadequate culture conditions,101,102 inherent or induced abnormalities,103,76 and failure of embryonic gene expression.104 Trounson et al obtained low cleavage rate after IVM retrieved from PCO patients (54% cleavage).46 Consequently, the incidence of polyploidy and mosaicism is also increased owing to the cell division arrest.105,106 It has been shown that multinuclear blastomeres are related at some extent to chromosomal abnormalities.107,108 Another interesting way of evaluating embryo quality is the spreading technique for fluorescence in situ hybridization (FISH) method. Analysis of the chromosomes by FISH is straightforward to obtain information on the nuclear status of IVM embryos.40 Finally evaluation of blastocyst formation rate might be an important alternative for evaluation as well. In large mammals it is known that in vivo matured oocytes fertilized and cultured in vitro gave almost twice as many blastocysts than in vitro matured oocytes fertilized and cultured in the same conditions.109–111 THE BASAL MEDIUM FOR IN VITRO MATURATION The type of media and laboratory conditions used for oocyte maturation can affect nuclear and cytoplasmic maturation of oocytes after in vitro maturation. 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,112 and changes in pH during isolation of

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mammalian oocytes can alter their developmental capacity.113 Changes in pH from 6.8 to 7.4 during in vitro maturation of mouse affected the response of the oocytes to meiotic inhibitors and inducers.114 During in vitro maturation of mouse oocytes, the use of different culture media, or even minor changes in culture conditions can lead to a significant variation in spontaneous oocyte maturation, in ability of meiotic inhibitors to suppress GVB or in the efficacy of meiosis inducing ligands.114 Numerous media have been formulated for the purpose of somatic cell culture that support the spontaneous in vitro maturation of oocytes. For human IVM following basal media have been used by different authors: TCM 199, alpha MEM, synthetic oviduct fluid (SOF), or Ham’s F-10. 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.115,116 Many different media are available for oocyte maturation, and whether or not they are inducing or arresting oocyte maturation does not necessarily depend on their composition or complexity. Addition or extraction of certain compounds from a particular medium can sometimes have different effects that could be interfering with the experimental design. Some additives or compounds that have a proven effect on in vitro maturation will be described, and the knowledge of these requirements is essential to determine optimal in vitro maturation 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 in vitro maturation117,118 while cumulus cells mediate glucose utilization. Cumulus cells metabolize glucose to pyruvate that will be used by the oocyte.119,120 In denuded oocytes (DO) cultured with pyruvate meiotic arrest is maintained in the presence of dibutyryl cAMP (dbcAMP). Moreover, the arrest is not maintained in cumulus surrounded oocytes (COCs),121 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.114 Absence of pyruvate in different in vitro maturation media decreases the maturation potential.

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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 substances122 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 cumulus oocyte complexes and was enhanced by LH.123 Glutamine plays a primary part in oocyte maturation. Glucose is essential for acquisition of developmental competence in cattle and is metabolized by cumulus oocyte complexes but not by denuded oocytes or cumulus cells alone.124 There is a need for more fundamental studies to assess possible energy sources during IVM of human COC. 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.125,126 Furthermore 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 preparation 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 gamete and embryo. Serum contributes to ammonia formation in culture, which can damage the mitochondria.127 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 can protect the embryo. When serum is replaced by albumin or another macromolecule (polyvinylpyrolidone, 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 out 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 preparation are recombinant albumin or inert matrices such as hyaluronan (HA), PVA or PVP.

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GLUTATHIONE METABOLISM The oxidative modification of cell components via reactive oxygen species (oxidative stress) is one of the most potentially damaging processes for the proper cell function. In most cells, efficient antioxidant systems can attenuate the effect of oxidative stress by scavenging reactive oxygen species.128 Glutathione (GSH) is the major non-protein sulphydryl 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,129 hamsters,130 pigs,131 and cattle.132,133 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 to oocyte activation, and in the transformation of the fertilizing sperm head into the male pronucleus.129–131,134,135 Glutathione is synthesized by the γ-glutamyl cycle,136,137 and its synthesis is dependent on the availability of cysteine in the medium (Fig 9.20). In a recent 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.138,139 These changes are essential for normal fertilization and embryo development. Funahashi et al suggested that intracellular GSH content of porcine oocytes at the end of IVM appears to reflect the degree of cytoplasmic maturation.140 Moreover, results obtained with bovine oocytes are in agreement with the hypothesis that measurement of GSH after IVM may be a valuable indicator of the cytoplasmic maturation.141–143 It has been shown that β-mercaptoethanol and cysteamine reduce cystine to cysteine and promote the uptake of cysteine, enhancing GSH synthesis.144–147 It was shown that

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Fig 9.20 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 (γglutamylcysteine 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 dependency expressed in oocyte and early embryo. cysteamine, β-mercaptoethanol, cysteine, or cystine supplementation of IVM medium increased the intracellular GSH content of oocytes after in vitro maturation and improved embryo development and quality.148 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.149 The availability of cysteine in the IVM medium seems to be the limiting factor for glutathione synthesis in mammalian oocytes.150 The concentration of cysteine in TCM-199 used for routine IVM of bovine oocytes is very low (0.6µM) compared with that of cystine (83.2µM), and because of auto-oxidation, essentially no cysteine is present.

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Consequently, GSH synthesis may be impaired owing to the lack of substrate, generating suboptimal culture conditions for in vitro maturation. It is possible that the cystine generated by auto-oxidation is converted into cysteine by cumulus cells and then incorporated into GSH synthesis.142 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.148 MEIOSIS ACTIVATING STEROLS Several factors, such as high concentrations of cAMP and hypoxanthine (Hx) in the compartment surrounding the oocyte, prevent meiotically competent oocytes from resuming meiosis spontaneously.151–153 Keeping the concentration of cAMP in the oocyte high inhibits resumption of meiosis.153,154 Byskov discovered a group of endogenous meiosis activating sterols (MAS) occurring naturally in the biosynthetic pathway between lanosterol and cholesterol.155 One of these sterols was isolated from human follicular fluid and was named FF-MAS (4,4-dimethyl-5αcholesta-8,14,24-trien-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), or dbcAMP.156 An almost identical sterol (T-MAS) with similar meiosis activating effect has been found in bull testes.155 The reversal of the Hx induced meiotic block by FF-MAS is dependent on protein synthesis.156 The dynamics of microtubule and actin mediated organelle movement is well established for processes such as extension of endoplasmic reticulum,157 changes in mitochondrial distribution,158 and movement of the cortical granules to the cell surface.159 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.160 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.160 These studies suggest that FF-MAS may have beneficial effects on in vitro maturation, although it may not be an indispensable component at the initiation of meiotic resumption in vivo.161

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HORMONAL AND GROWTH FACTOR REQUIREMENTS GONADOTROPINS Follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) are generally included as components of in vitro maturation medium. In vitro maturation 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.162 A delay in resumption of meiosis in the presence of FSH has also been reported in rodent oocytes.116,163,164 In the presence of FSH a transient rise of cAMP was observed in COC,165 and this can stimulate oocyte maturation.116,166 There is experimental evidence that FSH induces cumulus expansion in bovine oocytes.167 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.115,168 On the other hand, resumption of meiosis has been seen in many species before reduction of functional communication.169–172 Although LH induces both maturation of oocytes and progesterone production in intact follicles in vitro, LH apparently does not induce maturation via progesterone synthesis. Earlier studies showed that high concentrations of LH during IVM of bovine oocytes improves embryo development up to the 4 to 8 cell stages.173 There exist evidences that LH acts via the cumulus cells to increase glutamine metabolism within intact cumulus cell enclosed oocytes.123 Bovine antral follicles smaller than 8mm possess only FSH receptor mRNA and no LH receptor mRNA in mural granulosa cells. Van Tol, using small to medium sized follicles, showed the presence of FSH receptors in granulosa cells, cumulus cells, but not in oocytes.174 LH receptors were only present 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,174 it is possible that the effect of FSH on oocyte maturation is exerted through the cumulus cells. Izadyar162 and Alberio and Palma175 found that FSH added during bovine oocyte IVM improved fertilization and had beneficial effects on embryo development.

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GROWTH FACTORS: INSULIN AND INSULIN-LIKE GROWTH FACTOR I Various paracrine actions for insulin-like growth factor I (IGF-I) and insulin have been demonstrated in the ovary. IGF-I stimulates rat granulosa cells mitogenesis, and its effect is made more potent by FSH.176 FSH and IGF-I act synergistically, as evidenced by the maximal estradiol secretion in cultured human granulosa cells.177 IGF-I is a potent mitogen for granulosa cells178 and enhances nuclear maturation in oocytes surrounded by compact cumulus cells both in cattle,179 humans,61 rats,180 and rabbits.181,182 It was reported that FSH stimulates intraovarian IGF-I production.183 In the buffalo it was suggested that IGF-I was effective in stimulating nuclear maturation but not cumulus expansion, without an impairment of fertilization and embryo development. IGF and FSH stimulate progesterone secretion in granulosa cells184,185 and their synergistic coupling induces a steroidogenic activity in both bovine cumulus and granulosa cells.186 IGF-I possesses multifunctional role in follicle development and oocyte maturation. 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 in vitro maturation in rats,187 mice,116 cattle,179 pigs,188 and humans.189 EGF can stimulate cumulus expansion of bovine oocytes in serum free conditions.179,190 In pigs, in the presence of gonadotropins and serum, EGF did not stimulate cumulus expansion.191 In cattle, the presence of EGF during in vitro maturation influences the protein neosynthesis pattern in the oocyte.125 It has been proposed that some of the proteins synthesized during oocyte maturation may be essential for normal oocyte maturation process while others may be required for fertilization and embryo development.192 GROWTH HORMONE The addition of growth hormone (GH) during in vitro maturation of bovine oocytes accelerates nuclear maturation, induces cumulus expansion, and promotes subsequent cleavage and embryonic development.193 Moreover, it also improves the cytoplasmic maturation as testified by improved migration of cortical granules and sperm aster formation leading to improved fertilization of bovine oocytes.162 Culture of rat and porcine oocytes in the presence of GH also showed an acceleration of the process of nuclear maturation.194,195 In human IVM there has been no systematic in vitro study done to evaluate whether GH could improve cytoplasmic maturation.

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ACTIVINS AND INHIBINS In different animal species and in the human cumulus oocyte complexes obtained from small and medium sized follicles expression of activin A, inhibin, follistatin, and activin receptor type II proteins have been shown both in the cumulus cells and oocyte during in vitro maturation.193 These 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,196 humans,197 pigs,198 and cattle.199 It functions as a high affinity binding protein of inhibins and activins.200 Within the ovary, these peptides have both autocrine and paracrine functions during folliculogenesis,201 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,202–204 as well as in cumulus cells of rat and mouse.205,206 Hulshof demonstrated the presence of activin receptors in granulosa cells and oocytes of bovine antral follicles.207 Experimental work from Van Tol208 and Izadyar193 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.209,210 A study from Alak et al in nine women found a stimulating effect of activin (100ng/ml) on oocyte maturation.211 These data still need confirmation by others before it 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.212,213 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.214 In our laboratory, as a standard procedure, in vitro maturation of mouse40 and bovine142 oocytes is made in 20% oxygen. In a recent collaborative study by Hu et al the maturation rate, spindles and chromosome alignment in oocytes were analysed after exposure of in vitro cultured mouse follicles to low (5%) or normal oxygen tension in the atmosphere during the final in vitro maturation period (after HCG/EGF administration).215 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

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important in relation to the oxygen concentration which is provided to the cultured tissues and might cause oxidative damage.216 It is possible that the presence of several layers of cumulus cells around the maturing oocyte mitigates 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 towards the oocyte,217 so that the oocyte is located in a relative anoxic environment.

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 are gradually acquired during oocyte growth.6,16,218 During the last phase of its growth the oocyte produces different RNA species of a prolonged stability, with average half life of 28 days.219 These RNA species are functional in different levels at different times.220 This is made possible by the different degrees of polyadenylation (poly-A). Long poly-A tails (~150 A residues) are for immediate use (during maturation), whereas shorter poly-A tails (<90 A) will have to be polyadenylated later to become functional.221 These mRNAs are stockpiled within specialized microribonucleoparticles within the cytoplasm for later use after fertilization, during early embryonic development. The reduced potential to produce clinical pregnancies after in vitro maturation of oocytes can be explained by shortcomings of the higher 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 a normal pre-implantation development. 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. Experimental evidence in large mammalians showed that nuclear and cytoplasmic maturation have to go hand in hand to enable normal further development. Delays in cytoplasmic maturity emanate 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.222–224 By the act of retrieval of meiotically competent oocytes, the spontaneous reinitiation of meiosis is triggered leading to an almost immediate arrest of transcription.

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Perhaps a prematuration phase of immature oocytes could be beneficial to enable the biochemical processes accompanying the cytoplasmic rearrangements to develop in a more physiological way. Several possible strategies could be adopted to reach this goal either by co-culture of follicle shells or by using pharmacological agents interfering with cAMP metabolism, the generation of pre-MPF, or inhibiting MPF activity. FUTURE STUDIES ON IN VITRO MATURATION 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. When immature oocytes are retrieved from small follicles, the optimal maturation (cytoplasmic maturation) is truncated by the arrest of all transcriptions once nuclear maturation is reinitiated. In order to obtain an optimal cytoplasmic maturation, the reinitiation of meiosis should be inhibited and the correct culture environment provided for a critical period. The correct achievement of these principles is only possible if technical developments could provide the essential non-invasive tools to objectively 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. 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 coculturing COC with granulosa or theca cells. This system, although quite effective, would not be suitable for human ART routine practice owing to its heaviness.225 The in vitro methods to control meiosis progression in rodent COC such as hypoxanthine, dbcAMP, and IBMX are easier to use but not completely effective in COC from large species.226 Some researchers suggested to instore prolonged inhibition of meiosis (>24 hours) by applying substances involving inhibition of protein synthesis or phosphorylation processes.227 After the use of cycloheximide (1µg/ml), a protein synthesis inhibitor, or 6-dimethylaminopurine (2mM), an inhibitor of phosphorylation approximately 80% of temporarily (for a minimum of 24 hours) blocked oocytes developed to MII (after washout of inhibitors). Some 10–20% of these oocytes developed to blastocysts, showing that this option is feasible but should be further improved.228 Inhibition of MPF kinase activity by roscovitine (25µM) could also reversibly keep H1

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kinase activity very low after which a comparable blastocyst formation rate (36% v. 40% in controls) was observed as without roscovitine but matured in TCM 199 plus 10ng/ml EGF. This experiment proves the feasibility of arresting nuclear maturation for 24 hours to allow for cytoplasmic maturation without compromising the resulting developmental potential.229 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.230 Similar work was done in humans by Anderiesz et al81 involving 6dimethylaminopurine (DMAP), a serine threonine protein kinase inhibitor of histone H1 kinase, but not interfering with protein synthesis.231 DMAP could temporarily (at least 24 hours) inhibit human meiotic maturation without effecting subsequent maturation to MII 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 COC 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 6–12mm diameter follicles for IVM, these follicles would in vivo still undergo a growth phase of four to five days before ovulation. Hence, a prolonged “prematuration system” could be beneficial for IVM. Oocyte nuclear and cytoplasmic maturation, which can proceed as independent processes, need to be coordinated to ensure developmental competence. Therefore the intimate transzonal connections between granulosa cells and oocyte must be kept patent for transfer of regulatory substances (proteins, mRNA) between the two cellular compartments. Keeping the oocyte meiotically arrested (by either using a coculture approach or pharmacological agents that arrest the meiosis process) and providing growth factors and hormonal supplements to sustain completion of oocyte’s cytoplasmic maturation could theoretically lead to an improvement of development after fertilization. This strategy is actually pioneered in bovine and human.81,229,230 Safety restricts use of protein sources from other species in human IVM media. Serum substitutes might be found necessary to cover the “serum functions.”

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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 proven whether r-HCG or r-FSH in IVM medium are essential components during the in vitro prematuration phase. Additions of a meiosis activating sterol,155 growth hormone,232 activin/inhibin ratios,211,233 or substances that increase GSH in the cytoplasm140,148 might be a possible approach towards an increased cytoplasmic maturation. A more futuristic approach to oocyte maturation in vitro is the transfer of ooplasm. “Ooplasmic transplantation” has even already been applied to humans in cases where ooplasmic rather than genetic anomalies were considered to be at the origin of a repeatedly poor embryo viability.234 There should be an emphasis to check for the safety of all these procedures before they can be proposed for routine clinical practice.

ACKNOWLEDGEMENTS The authors acknowledge the Fund For Medical Research Flanders (FWO Grant n° G.0166.98) and Ares Serono International for supporting this research project. Ms Sandra De Schaepdryver is thanked for secretarial assistance.

REFERENCES 1 Vanderhyden BC, Cohen JN, Morley P. Mouse oocytes regulate granulosa cell steroidogenesis. Endocrinology (1993); 133:423–6. 2 Eppig JJ, Wigglesworth K, Pendola F, Hirao Y. Murine oocytes suppress expression of luteinizing hormone receptor messenger ribonucleic acid by granulosa cells. Biol Reprod (1997); 56:976–84. 3 Elvin JA, Clark AT, Wang P, Wolf man NM, Matzuk MM. Paracrine actions of growth differentiation factor-9 in the mammalian ovary. Mol Endo (1999); 13:1035–47. 4 Joyce IM, Pendola FL, Wigglesworth K, Eppig JJ. Oocyte regulation of Kit Ligand expression in mouse ovarian follicles. Dev Biol (1999); 214:342–53. 5 Wickramasinge D, Albertini DF. Centrosome phosphorylation and the developmental expression of meiotic competence in mouse oocytes. Dev Biol (1993); 152:62–74. 6 Fair T, Hyttel P, Greve T. Bovine oocyte diameter in relation to maturational competence and transcriptional activity. Mol Reprod Dev (1995); 42:437–45. 7 Gougeon A. Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev (1996); 17:121–55.

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8 Whitaker M. Control of meiotic arrest. Rev Reprod (1996); 1:127–35. 9 Downs SM, Daniel SAJ, Bornslaeger EA, Hoppe PC, Eppig JJ. Maintenance of meiotic arrest in mouse oocytes by purines: modulation of cAMP levels and cAMP phosphodiesterase activity. Gamete Res (1989); 23:323–34. 10 Richard F, Sirard MA. The effect of co-culture of follicular components with bovine oocytes on meiotic resumption, Biol Reprod (1993); 48:123. 11 Ismail RS, Dubé M, Vanderhyden BC. Hormonally regulated expression and alternative splicing of kit ligand may regulate kitinduced inhibition of meiosis in rat oocytes. Dev Biol (1997); 184:333– 42. 12 Pincus G, Enzman EV. The comparative behavior of mammalian eggs in vivo and in vitro. J Exp Med (1935); 62:665. 13 Eppig JJ, Schroeder AC, O’Brien MJ. Developmental capacity of mouse oocytes matured in vitro: effects of gonadotrophic stimulation, follicular origin and oocyte size. J Reprod Fertil (1992); 95:119–27. 14 De Smedt V, Crozet N, Gall L. Morphological and functional changes accompanying the acquisition of meiotic competence in ovarian goat oocyte. J Exp Zool (1994); 269:128. 15 Lonergan P, Monaghan P, Rizos D, et al. Effect of follicle size on bovine oocyte quality and developmental competence following maturation, fertilisation and culture in vitro. Mol Reprod Dev (1994); 37:48–53. 16 Crozet N, Ahmed-Ali M, Dubos MP. Developmental competence of goat oocytes from follicles of different size categories following maturation, fertilization and culture in-vitro. J Reprod Fertil (1995); 103:293–8. 17 Tsuji K, Sowa M, Nakano R. Relationship between human oocyte maturation and different follicular sizes. Biol Reprod (1985); 32:413–7. 18 Durinzi KL, Saniga EM, Lanzendorf SE. The relationship between size and maturation in vitro in the unstimulated human oocyte. Fertil Steril (1995); 63:404–6. 19 Dubey AK, Wang HA, Duffy P, et al. The correlation between follicular measurements, oocyte morphology, and fertilisation rates in an in vitro fertilization program. Fertil Steril (1995); 64:787–90. 20 Thibault C, Szollosi D, Gérard M. Mammalian oocyte maturation. Reprod Nutr Dev (1987); 27:865–96. 21 Taieb F, Thibier C, Jessus C .On cyclins, oocytes, and eggs. Mol Reprod Dev (1997); 48:397–411. 22 Crozet N, Dahirel M, Gall L .Meiotic competence of in vitro grown goat oocytes. J Reprod Fertil (2000); 118:367–73. 23 Lévesque JT, Sirard MA. Resumption of meiosis is initiated by the accumulation of Cyclin B in bovine oocytes. Biol Reprod (1996); 55:1427–36.

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182 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. 183 Adashi EY, Resnick CE, D’Ercole J, Svoboda ME, Van Wyk JJ. Insulin-like growth factor as intraovarian regulators of granulosa cells. Endocrinol Rev (1985); 6:400–20. 184 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. 185 May JV, Schomberg DW. Granulosa cell differentiation in vitro: effect of insulin on growth and functional integrity. Biol Reprod (1981); 25:421–31. 186 Armstrong DT, Xia P, Gannes G, Teckpetey FR, Khamsi F. Differential effect of insulin-like growth factor I and folliclestimulating hormone on proliferation and differentiation of bovine cumulus cells and granulosa cells. Biol Reprod (1996); 54:331–8. 187 Dekel N, Sherizly I. Epidermal growth factor induces maturation of rat follicle enclosed oocytes. Endocrinology (1985); 116:406–9. 188 Ding J, Foxcroft GF. Epidermal growth factor enhances oocyte maturation in pigs. Mol Reprod Dev (1994); 39:30–40. 189 Das K, Stout LE, Hensleigh HC, Tagatz GE, Phipps WR, Leung BS. Direct positive effect of epidermal growth factor on the cytoplasmic maturation of mouse and human oocytes. Fertil Steril (1991); 55:1000.4. 190 Kobayashi K, Yamashita S, Hoshi H. Influence of epidermal growth factor and transforming growth factor α on in vitro maturation of cumulus cell-enclosed bovine oocytes in a defined medium. J Reprod Fertil (1994); 100:439–46. 191 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. 192 McGaughey RW, Van Blerkom J. Patterns of polypeptide synthesis of porcine oocytes during maturation in vitro. Dev Biol (1977); 56:241–54. 193 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. 194 Apa R, Lanzone A, Miceli F, et al. Growth hormone induces in vitro maturation of follicle- and cumulusenclosed rat oocytes. Mol Cell Endocrinol (1994); 106:207–12. 195 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.

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196 Saito S, Nakamura T, Titani K, Sugino H. Production of activinbinding protein by rat granulosa cells in vitro. Biochem Biophys Res Commun (1991); 176:413–22. 197 Krummen EA, 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. 198 Ueno N, Ling N, Ying SY, Esch F, Shimasaki S, Guillemin R. 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. 199 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. 200 Shimonaka M, Inouye S, Shimasaki S, Ling N. Follistatin binds to both activin and inhibin through the common beta-subunit. Endocrinology (1991); 128:3313–5. 201 Ronghao LI, Philips DM, Mather JP. Activin promotes ovarian follicle development in vitro. Endocrinology (1995); 136:849–56. 202 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. 203 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. 204 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. 205 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. 206 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. 207 Hulshof SCJ. Bovine preantral follicles and their development in vitro. Thesis 1995, Utrecht University, The Netherlands. 208 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. 209 Stock AE, Woodruff TK, Smith LC. Effects of inhibin A and activin A during in vitro maturation of bovine oocytes in hormone- and serumfree medium. Biol Reprod (1997); 56:1559–64. 210 Silva CC, Knight PG. Modulatory actions of activin-A and follistatin on the developmental competence of invitro matured bovine oocyte. Biol Reprod (1998); 58:558–65.

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211 Alak BM, Coskun S, Friedman CI, Kennard EA, Kim MH, Seifer DB. Activin A stimulates meiotic maturation of human oocytes and modulates granulosa cell steroidogenesis in vitro. Fertil Steril (1998); 70:1126–30. 212 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. 213 Gwatkin RBL, Haidri AA. Oxygen requirements for the maturation of hamster oocytes. J Reprod Fertil (1974); 37:127–9. 214 Eppig JJ, Wigglesworth K. Factors affecting the developmental competence of mouse oocytes grown in vitro: oxygen concentration. Mol Reprod Dev (1995); 42:447–56. 215 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), accepted. 216 Eppig JJ, O’Brien MJ, Pendola FL, Watanabe S. Factors affecting the developmental competence of mouse oocytes grown in vitro: folliclestimulating hormone and insulin. Biol Reprod (1998); 59:1445–53. 217 Gosden RG, Byatt-Smith JG. Oxygen concentration gradient across the ovarian follicular epithelium: model, predictions and implications. Hum Reprod (1986); 1:65–8. 218 Hyttel P, Fair T, Callesen H, Greve T. Oocyte growth, capacitation and final maturation in cattle. Theriogenology (1997); 47:23–32. 219 Wassarman PM. Oogenesis. In: Adashi EY, Rock JA, Rosenwacks Z, eds. Reproductive endocrinology, surgery and technology. Philadelphia: Lippincott-Raven (1996); 341–57. 220 Decker C, Parker R. Mechanisms of mRNA degradation in eukaryotes. Trends Biochem Sci (1994); 19:336–40. 221 Bachvarova R. A maternal tail of poly(A): the long and short of it. Cell (1992); 69:895–7. 222 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. 223 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. 224 Moor RM, Gandolfi F. Molecular and cellular changes associated with maturation and early development of sheep eggs, J Reprod Fertil (1987); 34:55–69. 225 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. 226 Bilodeau S, Fortier MA, Sirard MA. Effect of adenylate cyclase on meiotic resumption and cyclic AMP content of zona-free and cumulusenclosed bovine oocytes in vitro. J Reprod Fertil (1993); 97:5–11.

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227 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. 228 Mermillod P, Lonergan P, Carolan C, Khatir H, Poulin N, Cognie Y. Maturation ovocytaire in vitro chez les ruminants domestiques. Contracept Fertil Sex (1996); 24:552–8. 229 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. 230 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. 231 Fulka A, Leibfried-Rutledge ML, First NL. Effect of 6dimethylaminopurine on germinal vesicle breakdown of bovine oocytes. Mol Reprod Dev (1991); 29:379–84. 232 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. 233 Alak BM, Smith GD, Woodruff TK, et al. Enhancement of primate oocyte maturation and fertilization in-vitro by inhibin A and activin A. Fertil Steril (1996); 66:646–53. 234 Cohen J, Scott R, Alikani M, et al. Ooplasmic transfer in mature human oocytes. Mol Hum Reprod (1998); 4:269–80. 235 Hashimoto N, Kishimoto K. Regulation of meiotic metaphase by a cytoplasmic maturation-promoting factor during mouse oocyte maturation. Develop Biol (1988); 126:242–52.

10 Equipment and general technical aspects of micromanipulation of gametes and embryos Frank L Barnes

INTRODUCTION Over the past 20 years micromanipulation has increased in importance in the livestock and human assisted reproductive technologies (ART) laboratory. Applications of micromanipulation include embryo bisection for embryo twining,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 and aneuploidy,5 intracytoplasmic sperm injection (ICSI) for the treatment of male 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 course 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 clearly visualize 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 stereo microscope to prepare eggs and embryos for manipulation and appropriate environmental control to maintain temperature and atmosphere as may be required.

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HANDLING CONDITIONS AIR QUALITY AND TEMPERATURE As with any experimental or clinical procedure, day to 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 30–40% 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 setup may have a similar impact on subsequent embryo development. Thirty millimeter plates with 2–3ml of media without oil overlay will remain 3–5 degrees cooler than the controlling heat source owing to evaporation. When there is no heat source a plate will cool precipitously below room temperature within five minutes. Alternatively, micro drops 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 (four to eight) 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, that use optical quality plastic and heat capability to eliminate the stage opening and cooling effect of the objective. Time will tell if this observation leads to improved ICSI outcomes. 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 like Hams F10 or TCM 199 into

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Dulbecco’s PBS, a common manipulation medium, can elicit calcium movement within the cell and ultimately effect development.12 It is always preferable to manipulate eggs and embryos in a media that maintains a similar salt balance while keeping temperature constant from incubation to manipulation.

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 specimens to room conditions that may effect development. There are two basic types of manipulation systems today, motorized and mechanical. I have not used 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 Eppendorf system and some of its clones 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 setups have a blend of motorized course movement with joystick and hydraulic fine movement translated through a separate joystick (Fig 10.1). 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 10.2). Whether performing microinjection or blastomere biopsy the hydraulic joystick offers good range of motion across a 300–400×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 10.2). Hand position on the bench top is very comfortable when using the “drop down” joysticks. The disadvantage of this system is the hydraulic lines (Fig 10.1). If pinched in some way it may be impossible to fix on location. Additionally, these systems are not very portable and require a considerable amount of time to assemble and disassemble. Research Instruments (RI) produces completely mechanical systems. The RI system attaches entirely to the microscope; there are no lines or cords or plugs to deal with, and it provides a very clean and neat workstation. The course alignment of the manipulator is adjusted with small joysticks that protrude upright from the suspended arms off each side of the microscope (Fig 10.3). The fine movement joysticks hang down from the suspended arms. They have good three-dimensional movement across a 300–400×field; the joysticks are well oriented to the

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focus knobs of an inverted microscope. The RI system, once set, requires almost no course adjustment. There are levers on each side of the manipulator above the microscope stage that allow the micro tools to be raised and lowered within a fraction of an inch of the bottom of the manipulation plate (Fig 10.3). 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 clearly visualize 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 setup 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 the 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 moved back again, to reduce the time held at room atmosphere. A stereomicroscope with a magnification range of 10 to 100× can be valuable for placing specimens into the micromanipulation plate. The “setup” station should be close to the micromanipulation workstation to avoid unnecessary chair movements (Fig 10.4).

Fig 10.1 Narishige micromanipulator (left arm), combines motorized course adjustment (background) with hydraulic fine adjustment (foreground).

Fig 10.2 Narishige course and fine adjustment joysticks are separate from the microscope and inhibit operator induced vibration of the specimen on the microscope stage. Note the position of the SAS syringe and the joysticks, which allows even horizontal movement of the left hand to effect the positioning of the specimen and manipulation. The SAS syringe tool chuck is under the control of the right handed joystick and allows positioning of the glass micro tool while the left hand performs the aspiration or injection.

Fig 10.3 Mechanical micromanipulator from Research Instruments (RI) suspends from the objective pillar of the microscope. Suspension of the joysticks from the mounting arms provide a clean workstation but allow 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 micro tools to be raised and lowered easily when changing out manipulation plates.

Fig 10.4

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Setup 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. 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 setup stereomicroscope. Beware 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 36–38°C. 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: (1) 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 out and thus reduce the time required to perform a micromanipulation procedure, and (2) a mixed gas atmosphere with high humidity allows culture in bicarbonate buffered media and limits the exposure to HEPES or phosphate buffered media to the time 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 acknowledge the fact that one cannot maintain gas atmosphere without humidity. Also, humidity helps to prevent evaporation of media when working without an oil overlay. Evaporation can decrease temperature and increase solution osmolarity. There are a variety of bench top containment systems available that control temperature, humidity, and mixed gas atmosphere (Fig 10.5). 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,

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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, like sperm, or remove something, such as blastomeres. A micro syringe 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

Fig 10.5 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.

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Fig 10.6 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. 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 micro tool to the microsyringe and those that simply contain air within that tubing (Figs 10.6 and 10.7). Both types have their advocates, but I prefer the air filled systems because I feel I have better control and 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 micro tool, leading to disastrous results. Possibly more important is what to do when you cannot afford an expensive microsyringe or what to do when the micro syringe is not working properly. One workable “homemade” system is a combination of a three-way valve, 10 cc rubber plunger syringe and 1 cc rubber plunger syringe (Fig 10.8). Fill the 10 cc syringe with oil and connect it to the three-way valve (Baxter, Three-Way Stopcock, Cat. No. K75) to which the 1 cc syringe and manipulation tubing are connected. By moving the valve closure sequentially between the 1 cc syringe and the tubing, deliberately inject the oil into the system removing all air bubbles. The 1 cc syringe should contain at least 0.5ml of oil, and the tubing should contain oil all the way into the glass micro tool; there can be an air space

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between the oil interface and the media within the glass micro tool. Close off the valve at the 10 cc syringe and control the manipulation by gently moving the plunger in and out of the 1 cc 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 cc rubber plunger syringe

Fig 10.7 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 media within the glass micro tool during manipulation.

Fig 10.8 Homemade micro syringe to be used with oil filled tubing. This device can yield extremely sensitive control and is very useful as a backup system. Shown with polyethylene tubing and tool chuck.

Fig 10.9 Air filled 20 cc syringe requires additional skill by the operator but it may be useful as a backup system. Shown with polyethylene tubing and tool chuck.

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filled with air connected to the manipulation line. There may be some benefit to back loading a small amount of oil into the glass micro tool to improve control (Fig 10.9). The holding pipette also needs some degree of control and a simple air filled syringe appears to be 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). The type of tubing used to connect the microsyringe and the glass micro tools can be important. Soft tubing allows for too much expansion and ultimately a loss of control. Select a hard polyethylene tubing with little expansion capability. Finally, tool chucks or holders make the connection between the line and the glass micro tool. These are usually acquired through the micromanipulator distributor. Some tool chucks require a small silicon gasket to form a tight seal between the glass micro tool and the manipulation tubing while others simply attach with a type of lock-nut 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 MICRO TOOLS 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 micro tool 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 micro tools 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 10.10). VIBRATION 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 the

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micromanipulation plate with small drops (5µl) overlaid with oil. Place the microscope on a rubber pad; a typewriter pad works well.

Fig 10.10 Parallel orientation of angled micro tools to the bottom of the manipulation plate provides a straight-on approach to the egg or embryo with a clear focus of the tool throughout the observation field, (200×). The setup of the pictured glass micro tools 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 (micro syringe) 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 micro syringe is on the right side of the microscope. An example of the convenience of this orientation is the acid drilling pipette may be moved around inside a secured embryo to easily allow fragment removal without hand cross over at he level of the manipulator.

PROCEDURE STEPWISE MICROMANIPULATOR SETUP 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. The respective controls (syringes) are on the opposing side to avoid hand

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crossover during the procedure. The holding pipette is attached to a 10 ml syringe via approximately 1 meter of polyethylene tubing. The manipulation pipette is attached to a microsyringe via 1 meter of polythene tubing obtained from the microsyringe distributor. The micro tools should be oriented such that they are perpendicular to the micro drop interface at the 9 o’clock and 3 o’clock position and parallel to the bottom of the manipulation plate (Fig 10.10). MANIPULATION PLATE SETUP 1. Label a Falcon 1006 plate with the identity and/or ownership of the specimen to be manipulated. 2. Place small micro drops in the center of the plate of sufficient size to contain the specimens (5–10µl). The drops are overlaid with 4–5ml of mineral oil and placed into a GE-80 or other suitable incubators to temperature equilibrate. 3. The micromanipulation plate setup should be performed at least 1 hour prior to manipulation so that temperature is equilibrated at 37°C. It is important that the drops be close together so they easily fit within the objective opening of the microscope stage. 4. Prepare enough manipulation plates such that any given plate is only used once for a given patient or procedure. Care should be taken to keep plates warm. The time micro tools are exposed to air should be minimized when changing plates. Micro tools become sticky when exposed to air. MICROMANIPULATION TECHNIQUE At 200× magnification, focus on the zona pellucida of 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 this is performed exactly in the order described, the micro tools will be aligned with the equator of the oocyte or embryo. 5. Change magnification to 400× and focus on the area to be manipulated. 6. Bring the manipulation pipette into focus. 7. Adjust 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.

1. 2. 3. 4.

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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 (1984); 307: 634–6. 3 Willadsen SM. Nuclear transplantation in sheep embryos. Nature (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. Prod Natl Acad Sci (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); 340:17–8. 7 Muggelton-Harris A, Whittingham DG, Wilson L. Cytoplasmic control of preimplantation development in-vitro in the mouse. Nature (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. 9 Powell R, Barnes FL. The kinetics of oocyte activation and polar body formation in bovine embryo clones. Mol Reprod Devel (1992); 33:53– 8. 10 Barnes FL, Collas P, Powell R, King WA, Westhusin M, Shepherd D. Influence of recipient oocyte cell cycle stage on DNA synthesis, nuclear envelope breakdown, chromosome constitution and development in nuclear transplant bovine embryos. Mol Reprod Devel (1993); 36:33–41. 11 Keefe D. New morphologic criteria for imaging viable eggs and embryos. In: 13th Annual in vitro fertilization and embryo transfer, a comprehensive update—2000. (Santa Barbara, CA: UCLA School of Medicine. Mini-symposium on the IVF laboratory (2000): 83–3. 12 Collas P, Fissore R, Robl JM, Sullivan EJ, Barnes FL. Electricallyinduced calcium elevation, activation and parthenogenetic development of bovine oocytes. Mol Reprod Devel (1992); 34:212–23.

11 ICSI: technical aspects Gianpiero D Palermo, Ricciarda Raffaelli, June J Hariprashad, Queeni V Neri, Takumi Takeuchi, Lucida Veeck, Zev Rosenwaks

INTRODUCTION Spermatozoa sometimes fail to fertilize even when they are artificially placed in close proximity of 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

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need to be analyzed for gene defects, the avoidance of contaminating spermatozoa on the zona pellucida reduces the chance of false positivity with PCR.

MATERIAL 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 a high viscosity, this can be reduced within three to five minutes by usually adding it to 2–3mL of HEPESbuffered human tubal fluid (HTF-HEPES) containing 200 to 300IU 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 absence of the vas deferens (CABVD) 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 non-obstructive situations. Variable volumes of fluid (1–500µl) 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 are 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 100–200X, 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. The sperm suspension (adjusted to a concentration of ~

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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 1ml aliquots of the final solution are placed in 1ml 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 (3.5mM 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 by using the strict criteria of Kruger et al. Classification is usually made after spreading 5µL 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 <20×10ml, 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 500×g for five minutes in HTF medium supplemented with 30mg/ml BSA fraction V (A-9647; Sigma Chemical Co). Semen samples with <5×106/ml spermatozoa or <20% motile spermatozoa are washed in HTF medium by a single centrifugation at 1,800×g for five minutes. The resuspended pellet is layered on a discontinuous Percoll gradient (Pharmacia, Upsala, Sweden) on three layers (90%, 70%, and 50%), and centrifuged at 300×g for 20 minutes. A Percoll gradient in two layers (95% and 47.5%) is used when samples have a sperm density <5×10/ml spermatozoa and <20% motile spermatozoa. The sperm rich Percoll fraction is washed twice by adding 4ml of HTF medium and centrifuged at 1,800×g for five minutes to remove the silica gel particles. For spermatozoa with poor kinetic characteristics, the sperm suspension is exposed to a 3mM solution of pentoxifylline and is washed again. The concentration of the assessed sperm suspension is adjusted to 1 to 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.

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COLLECTION AND PREPARATION OF THE OOCYTES Oocyte retrieval is performed after pituitary desensitization with a gonadotropinreleasing hormone agonist, with ovulation induction carried out by administering a combination of human menopausal gonadotropins (hMG) (Pergonal®, Serono, Waltham, MA, USA; Humegon®, Organon Inc, West Orange, NJ, USA), and follicular stimulating hormone (FSH) (Gonal-F®, 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 100X, the cumulus corona cell complexes are scored as mature, slightly immature, completely immature, or slightly hypermature. Thereafter, the oocytes are incubated for more than four hours. Immediately prior to micromanipulation, the cumulus corona cells are removed by exposure to HTF-HEPESbuffered medium containing 80IU/ml of hyaluronidase (Type VIII, Sigma Chemical Co). 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 −200µm. 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 pipette 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 78mm 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 60µm and an inner one of 20µm. The injection pipette is prepared by opening and sharpening the pulled capillary on a grinder; the bevel angle is 30°, the outer and inner diameters are approximately 7µm and 5µm, respectively. On the microforge, a spike is made on the injection pipette and both pipettes are bent to an angle of approximately 35° at 1mm from the tip, to be able to perform the injection procedure with the tip of the tools horizontally positioned in a plastic Petri dish (Model 1006, Falcon, Becton & Dickinson, Lincoln Park, NJ, USA). Immediately before injection, 1µl of the sperm suspension is diluted with 4µl of a 10% polyvinyl pyrrolidone solution (approximately

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290mOsm) (PVP-K 90, MW 360 000, INC Biochemicals, Cleveland, OH, USA) 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 cells from sticking to the injection pipette. When there are <500000 spermatozoa per sample, the sperm suspension is concentrated in approximately 3µl and transferred directly to the injection dish. Each oocyte is placed in a 5µl droplet of medium surrounding the central drop containing the sperm suspension. HTF-HEPES medium supplemented with 6% Plasmanate® is used in the injection dish. The droplets are covered with lightweight paraffin oil (BDH Limited, Poole, England). Spermatozoa are aspirated from the central droplet or the concentrated 3µl 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 400X using Hoffman Modulation Contrast optics. This microscope is equipped with two motor driven coarse control manipulators and two hydraulic micromanipulators (MM188 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 400X, it is not easy to select spermatozoa according to morphological 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, a 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 demonstrated to improve fertilization rates.17–19 Owing to physiological 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

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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 midpiece. This induces a permanent crimp in the tail section, making it kinked, looped, or convoluted (Fig 11.1). 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 ejaculated spermatozoa, the difference was less remarkable. A possible explanation of the variation in the 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 a 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 11.2). 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 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 11.3). 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

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Fig 11.1 Aggressive immobilization of the spermatozoon for ICSI. The correctly immobilized spermatozoon has its tail permanently kinked (a), convoluted (b), or looped (c). 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 reaspirated 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, the order of the opening should maintain a funnel shape with a vertex into the egg (Fig 11.4). 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

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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. When the patient is ≤30 years old, 2 or 3 embryos are usually transferred; while in the 31–34, 35–41, and ≥42 years age groups the number of embryos increase to 3, 4, and ≥5, 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, thus permitting the transfer of a lower number of them. The embryonic endometrial synchronization as well as the possibility to assess the viability of the blastocyst, since genomic activation most likely occurs at day 4 after fertilization, may explain a higher chance of implantation.

Fig 11.2 ICSI procedure. Prior to penetrating the oolemma, the spermatozoon is brought in proximity to bevel opening of the injection pipette.

Fig 11.3 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.

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Fig 11.4 ICSI procedure. After withdrawal of the needle from the oocyte, the breach in the oolemma should be observed as a coneshaped opening with its vertex toward the center of the oocyte. Sequential culture media that meet changing physiological 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 twopronuclear 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 more hours.27–29 Thereafter, blastocyst formation is assessed and blastocysts selected according to the established criteria for subsequent transfer.30

RESULTS From September 1993 through December 1999, ICSI was performed in 3731 cycles with ejaculated spermatozoa, and in 537 cycles with surgically retrieved sperm. The mean maternal age was 36.2±5 years for the ejaculated group, and 34.5±5 years in the couples undergoing surgical retrieval of spermatozoa. 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 3731 ICSI cycles were performed with ejaculated spermatozoa, consisting of 405 with normal and 3326 with abnormal semen parameters. A total of 31 401 MII oocytes were obtained from 3560 oocyte retrievals. After ICSI, 94.3% (29612/31401) of these oocytes survived, and 23699 were fertilized and displayed two pronuclei (2PN). The final fertilization rate of 75.5% appeared to be influenced by the condition (fresh or cryopreserved) and the collection method (masturbation, electroejaculation, or bladder catheterization) of the spermatozoa used (P=0.0001) (Table 11.1). The same factors had no influence on the clinical pregnancy rate. However, both the fertilization and pregnancy rates achieved with ejaculated spermatozoa were significantly different than those obtained with surgically retrieved spermatozoa (P=0.0001) (Table 11.2).

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ICSI WITH SURGICALLY RETRIEVED SPERMATOZOA A total of 341 cycles were performed with epididymal spermatozoa and 196 cycles with testicular samples. When the fertilization and the pregnancy characteristics were analyzed according to the origin of the spermatozoa, it was observed that cryopreservation clearly impaired motility parameters (P<0.0001) and pregnancy outcome (P=0.0001) though without affecting the fertilization rate. On the other hand, when testicular samples were used for ICSI, the lower fertilization and pregnancy rates were not different regardless of the spermatozoa being fresh or cryopreserved (Table 11.3). BLASTOCYST TRANSFER AFTER ICSI From July through December 1999, blastocyst transfer was performed in a total of 23 ICSI cycles, 22 with ejaculated spermatozoa, and 1 cycle with an epididymal sample. Out of 257 injected oocytes at MII stage, 210 were successfully fertilized and showed 2PN, thus giving a fertilization rate of 81.7%. The cleaving embryos observed at day 3 were 185 (88.1%). On day 5, a total of 27 morulas and 58 blastocysts were obtained. Of these, 17 (63.0%) morulas and 28 (48.3%) blastocysts were replaced into the uterine cavity. Additional good quality blastocysts (n=30) were cryopreserved at this stage for later use. A total of 14 patients presented with a positive βhCG (60.9%). Of these, five pregnancies were biochemical (21.7%), while the remaining nine cases (39.1%) were clinical pregnancies with a positive fetal heartbeat detected by ultrasound, achieving an implantation rate per embryo of over 30% (Table 11.4). Among these pregnancies, 56%

Table 11.1. Fertilization rates according to semen origin and specimen condition. Semen origin Cycles Oocyte fertilized/oocyte Clinical inseminated (%) pregnancies (%) Fresh ejaculate 3509 22173/29338(75.6)* 1525(43–5) Frozen ejaculate 177 1181/1627 (72.6)* 86 (48.6) Electroejaculate 31 250/312 (80.1)* 15 (48.4) Frozen 8 40/65 (61.5)* 4 (50.0) electroejaculate Retrograde ejaculate 6 55/59 (93.2)* 3 (50.0) *χ2, 5×2, 4 df, Effect of etiology and collection method of spermatozoa on fertilization rate, P=0.0001.

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Table 11.2. Fertilization and pregnancy rates according to semen origin. Spermatozoa No of Ejaculated Surgically retrieved Cycles 3731 537 Fertilization (%) 23699/31401(75.5)* 3 575/5 123 (69.8)* Clinical pregnancies (%) 1633 (43.8)† 283 (52.7)† *χ2, 2×2, 1 df, Effect of spermatozoal source on fertilization rate, P=0.0001 †χ2, 2x2, 1 df, Effect of spermatozoal source on clinical pregnancy rate, P=0.0001 Table 11.3. Spermatozoal parameters and ICSI outcome according to retrieval sites and specimen condition. Spermatozoa Epididymal Testicular Fresh Frozen/thawed Fresh Frozen/thawed Cycles 162 179 164 32 Density 27.1±41 22.4±28 0.4±1 0.2±0.4 (106/ml±SD) Motility (%±SD) 18.6±17* 2.9±7* 7.1±13 1.8±5 Morphology 2.6±3 1.9±2 0 0 (%±SD) Fertilization (%) 1286/1740 1157/1600 966/1517 166/266(62.4) (72.9) (72.3) (63.7) Clinical 109 (67.3)† 82 (45.8)† 80 (48.8) 12 (37.5) pregnancies (%) *Student’s t-test, two independent samples; Effect of cryopreservation on sperm motility, P<0.0001 †χ2, 2×2, 1 df, Effect of cryopreservation on clinical pregnancy rate, P=0.0001 Table 11.4. Implantation rate according to embryo culture. Embryo replacement No of (%) Day 3 Day 5 Embryos replaced No. 13480 45 Sacs implanted 3408 (25.3) 16 (35.6) Positive fetal heartbeats 3018 (22.4) 14 (31.1) were singleton pregnancies, 33% twin, and 11% triplet (Table 11.5). However, when the rate of higher order gestations was compared between

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blastocyst transfer (day 5) and embryo replacement (day 3), no significant differences were found. PREGNANCY AND DELIVERY CHARACTERISTICS The pregnancy outcome of 4268 ICSI cycles is described in Table 11.6. Of a total of 2399 patients presenting with a positive βhCG (56.2%), 339 were biochemical (8.0%), and 144

Table 11.5. Influence of embryonic stage on pregnancy outcome and gestational order. Embryo replacement No of (%) Day 3 Day 5 ICSI 4245 23 Replacements 4049 (95.4) 23 (100) Embryos replaced (mean) 3.3 2.0 Pregnancies (+FHB*) 1907 (44.9) 9 (39.1) Singletons 1170 5 Twins 610 3 Triplets 123 1 Quadruplets 4 0 *FHB=fetal heartbeat Table 11.6. Evolution of ICSI pregnancies in 4268 cycles. No of Positive outcomes ICSI cycles 4268 Embryo replacements 4072 Positive hCGs 2399 Pregnancy 56.2% (2399/4268) Biochemical pregnancies 339 Blighted ova 144 Positive fetal heartbeats 1916 Clinical pregnancy 44.9% (1916/4268) Miscarriages/therapeutic abortions 196 Ectopic pregnancies 18 Deliveries 1395 Ongoing pregnancy 39.9% (1702/4268) Ongoing gestations 307

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Table 11.7. Occurrence of congenital abnormalities with assisted reproductive techniques. No of (%) ICSI IVF Cycles 4268 3713 Offspring delivered 2053 1560 Newborns with major malformations 22 (1.1) 32 (2.0) Newborns with minor malformations 14 (0.7) 24 (1.5) Total malformations 36 (1.8)* 56 (3.5)* *χ2, 2×2. 1 df, Difference in congenital malformations between ICSI and IVF, P<0.001 were blighted ova (3.4 %). Among 1916 patients in which a viable fetal heartbeat was observed, 196 had a miscarriage or were therapeutically aborted, and 18 had an ectopic pregnancy. The final ongoing pregnancy rate was 39.9% per retrieval (1702/4268), and 41.8% per replacement (1702/4072). A total of 2053 babies were born from 1395 deliveries, included 1009 female babies and 1044 male and, with an overall frequency of multiple deliveries of 41.4% (578/1395): 489 twin (35.0%), 88 triplet (6.3%), and one quadruplet (0.1%). Only 36 (1.8%) of the 2053 newborns exhibited congenital abnormalities at birth: 22 (1.1%) were major and 14 (0.7%) were minor. Compared with the frequency of malformations in offspring born after standard IVF, ICSI newborns experienced a lower rate of congenital malformation (Table 11.7). In the present study, we encountered only one sex chromosomal abnormality (Klinefelter’s syndrome; 47, XXY). Thus our figures fail to show that any specific genetic aberration can be directly linked to the use of ICSI.

DISCUSSION AND CONCLUSIONS ICSI is the newest and, to date, most successful technique used to overcome fertilization failure. In addition, it has helped us to better understand some important key steps of the fertilization process. The results demonstrate that the injection of mechanically immobilized spermatozoa achieved fertilization at a higher rate than the injection of motile spermatozoa. This is the result of a 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 physiological changes induced onto the sperm membrane by its

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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 sperm surgically retrieved 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 concerns focused on the ICSI procedure itself has been shifted to the subfertile men who may transmit his genetic defects to the 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. However, data available from 23 ICSI cycles with blastocyst transfer did not show any significant difference in terms of occurrence of multiple gestations compared with the cleavage stage embryo replacement. On the other hand, the implantation and pregnancy rates achieved with this procedure appeared promising but not yet significantly superior. The reason for this improvement in implantation may lie in a more physiological environment for the conceptus at this stage, while the earlier embryo finds its environment in the fallopian tube. However, more data are necessary to draw any definitive conclusions about the extended embryo culture. 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

REFERENCES 1 Gordon JW, Grunfeld L, Garrisi GJ, Talansky BE, Richards C, Laufer N. Fertilization of human oocytes by sperm from infertile males after zona pellucida drilling. Fertil Steril (1988); 50:68–73.

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2 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. 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, Adler A, Talansky BE, Rosenwaks Z. 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. Intracytoplasmic 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. 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, Boulos A, Foster C, Craft IL. 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, Soffer Y, Komarovsky D, Ron-El R. Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy in men with nonobstructive azoospermia. Hum Reprod (1997);12:1488– 93. 14 Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, Tournaye H, Devroy P. High fertilization and pregnancy rates after intracytoplasmic sperm injection with spermatozoa obtained from testicular biopsy. Hum Reprod (1995); 10:148–52.

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15 Verheyen G, Pletinck I, Van Steirteghem AC. Effect of freezing method, thawing temperature and post-thaw 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, Camus M, Devroey P, Van Steirteghem A. 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 oocytepenetrating capacity of spermatozoa in the human epididymis. Int J Androl (1983); 6:310–8. 23 Palermo GD, Schlegel PN, Colombero LT, Zaninovic N, Moy F, Rosenwaks Z. 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 Reproductive and Technology. Assisted reproductive technology in the United States and Canada. Results generated from the American Society for Reproductive Technology Registry. Fertil Steril (1998); 69:389–98. 26 Gardner DK, Schoolcraft WB. Elimination of high order multiple gestations by blastocyst culture and transfer. In Female infertility therapy: current practice. In: Shoham Z, Howles C, Jacobs H, eds. London: Martin Dunitz, (1998); 267–74. 27 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.

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28 Gardner DK, Lane M. Culture and selection of viable blastocysts: a feasible proposition for human IVF? Hum Reprod Update (1997); 3:367–82. 29 Gardner DK, Vella P, Lane M, Wagley L, Shlenker T, Schoolcraft WB. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril (1998); 69:84–8. 30 Schoolcraft WB, Gardner DK, Lane M, Schlenker MA, Hamilton F, Meldrum DR. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril (1999); 72:604–9. 31 Takeuchi T, Tsai MC, Hariprashad JJ, Rosenwaks Z, Palermo GD. Ultrastructure of immobilized spermatozoa used for ICSI. Fertil Steril (1999); 72:S118–S119 (abstract). 32 Wolny YM, Fissore RA, Wu H, et al. Human glucosamine-6phosphate isomerase, a homologue of hamster oscillin, does not appear to be involved in Ca+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.

12 Assisted hatching AnnaVeiga, Irene Boiso

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 and 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 the protection of the embryo and the maintenance of its integrity.4 It has been postulated that the blastomeres may be weakly connected and that the ZP is needed during the migration of the 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 the 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 was described by Trounson and Moore5 that could be ascribed to an immune response. HATCHING Once in the uterus, the blastocysts must get out from the ZP (hatching) so that the trophectoderm cells interact with endometrial cells and implantation occurs. The loss of ZP in utero is the result of embryonic and uterine functions. Zona hardening after zona reaction subsequent to fertilization occurs 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.

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It has been postulated that additional zona pellucida 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 thick or hardened ZP.8 Schiewe et al performed a study to characterize ZP hardening on unfertilized and abnormal embryos and to correlate it with culture duration, patient’s age, and ZP thickness.9 The dispersion of ZP glycoproteins and the time needed for complete digestion after achymotrypsin treatment were assessed. The results obtained proved that zona hardening of fertilized eggs was increased compared with inseminated unfertilized eggs. Wide patient to patient variations in zona hardness were observed, but no correlation between zona hardness or thickness and patient’s 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. Contractionexpansion cycles as well as cytoplasmic extensions of trophectoderm (trophectoderm projections, TEP) have been documented in time lapse video recording10 in human blastocysts. TEP could be a component of zona escape in cultured embryos. It is not clear whether TEP are needed in vivo for ZP hatching, but they seem to have a role in attachment, implantation and possibly in 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 cells are responsible for secreting the zona lysins required for hatching. Recent data on mouse blastocysts indicate that hatching in vitro depends on a sufficiently high number of cells. Hatching in vivo must be different to that in vitro, the difference involving uterine and/or uterine induced trophectoderm lytic factors.14

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ASSISTED HATCHING The first report on the use of assisted hatching (AH) in human embryos was published by Cohen et al in 1990.7 These authors reported an important increase of implantation rates with mechanical AH in embryos from unselected IVF patients. WHY PERFORM ASSISTED HATCHING? The ratio of lysin production to ZP thickness could determine whether the embryo will lyse the zona and perform the 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 lysins action could also be impaired in some patients or cycles.16 Khalifa et al have shown that ZP thinning significantly increases the complete hatching of mouse embryos.17 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 after the transfer of assisted hatched embryos that had cell numbers reduced in pseudopregnant female mice. In a randomized study Liu et al18 demonstrated that implantation occurred significantly earlier in patients whose embryos were submitted to AH when compared to the control group, possibly by allowing earlier embryo endometrium contact.

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 the embryos for AH after the adherence between blastomeres has increased, just before compaction.19 Embryos at the 6 to 8 cell stage, at day three 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. Micro pipettes are mounted on micromanipulators. It is very important to minimize the time

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the embryo is out from the incubator and to optimize the 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 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 permits blastomere loss.20,21,22,23 Monozygotic twinning has been described as a consequence of AH.24 The adequate size of the hole seems to be 30–40µm. Different protocols have been described, but a minimum of 30 minutes culture period seems to be sufficient before transfer of the manipulated embryos. Embryo transfer to the uterus has to be performed as atraumatically as possible to avoid damage of ZP manipulated embryos. Treatment during four days starting on the day of oocyte retrieval with broad spectrum antibiotics and corticosteroids (methylprednisolone, 16mg 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 is similar to the one described for oocytes to assist oocyte zona pellucida penetration by spermatozoa25 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 before, 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 1 o’clock to 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 two times in fresh culture medium and placed in the transfer dish. Three-dimensional partial zona dissection (PZD) in the shape of a cross has been described recently.23 The procedure starts as conventional PZD, and a second cut is made in the ZP under the first slit. A cross shaped cut can be seen on the surface of the ZP. This method allows the creation of larger openings while permitting the protection of the embryo by the ZP flaps during embryo transfer.

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ACID TYRODE’S ASSISTED HATCHING It has been described that zona hardening and the increase in the 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 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 al26 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.7,22,27 The embryo is held with a holding pipette in such a way that the micropipette containing acid tyrode’s (internal diameter 3–5µm) 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 using mouth control suction or 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 in the perivitelline space. If the inner region of the ZP is difficult to breach, the creation of the hole can be facilitated by pushing the AT micropipette against the ZP.28 It is necessary to rinse the embryo several times in fresh culture medium. ZONA PELLUCIDA THINNING Zona pellucida thinning with AT has been described in mice and in humans.17,29 It involves bidirectional thinning of the ZP of a cross shaped area over about one quarter of the embryo circumference. Care has to be taken not to rupture the ZP completely. Embryos are washed in fresh droplets of medium and cultured before transfer. The methodology has proved useful for hatching enhancement in mice but not in humans, probably because of differences observed in both the morphological and the biophysical characteristics of the ZP between the two species. The mouse ZP has a monolayer structure whereas the human

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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.29 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.30,31 For fast and efficient clinical use of lasers 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 laser on the ZP for AH results in photoablation of the zona pellucida. The first use of laser for ZP drilling was reported by Palanker et al with an ArF excimer laser (UV region, 193nm wavelength).32 This laser system makes necessary to touch the ZP with the laser delivering pipette (contact mode laser). The erbium:YAG (Er:YAG) (2940nm radiation) also working in contact mode have been used for ZP assisted hatching and thinning, and its safety and efficacy have been demonstrated in clinical practice.33,34 Obruca et al performed a study to evaluate the ultrastuctural effects of the Er:YAG laser on the ZP and membrane of oocytes and embryos.35 No degenerative alterations were observed using light and scanning electron microscopy after ZP drilling with such system. 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. Antinori et al36 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 in a length of 20µm. The necessity of sterile micropipettes and optical fibers to deliver the laser beam to the target are the main disadvantages of contact mode lasers.37 NON-CONTACT LASERS Non-contact 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.

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Blanchet et al first reported the use of a non-contact laser system (248nm KrF excimer) for mouse ZP drilling.38 Neev et al described the use of a non-contact laser Ho:YSGG (2.1µm wavelength) for AH in mice.39 The study shows the lack of embryo toxic effects as well as improved blastocyst hatching. Similar results were reported by Schiewe et al.40 Rink et al recently designed and introduced a non-contact infrared diode laser (1.48µm) that delivers laser light through the microscope objective.41 The drilling mechanism is explained by a thermal effect induced at the focal point by the absorption of the laser energy by water and/or ZP macromolecules, leading to a thermolisis of the ZP matrix.42 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 (10–40 minutes) can be minimized. The safety and usefulness of the system was demonstrated in mice and humans.43–45 Its use for PB as well as blastomere and blastocyst biopsy has also been reported.46–48 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 and easy to use. Two pulses of 15–20 minutes are usually needed to drill a 30–403µm hole in 15–17µm thick zonae. Fig 12.1 shows the holes created with such a system in a 8 cell embryo. Antinori et al have reported on the use of a compact non-contact ultraviolet (337nm wavelength) laser microbeam system to create holes in the zona pellucida of human embryos.49,50 This equipment requires the manipulation of the oocytes and embryos in Petri dishes with membrane bottom. Depending on the equipments, different methods are used, varying in the energy, time, and number of pulses needed to open the zona pellucida. 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. A monozygotic twin pregnancy was achieved after transfer of a frozen thawed human blastocyst, on which zona pellucida rubbing with a microneedle was applied.51 Fong et al recently described a method for enzymatic treatment of the zona pellucida of blastocysts.52 The culture to the blastocyst stage was achieved with the use of sequential media and late cavitating embryos; early and expanding blastocysts were treated with 10IU/ml pronase for approximately 1 minute at 37°C for assisted hatching. 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.

Fig 12.1 Day 3 embryo in which the ZP has been driled with 2 laser shots (Fertilase, MTM, Montreaux, Switzerland).

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Fig 12.2 Laser assisted hatching (Fertilase, MTM, Montreaux, Switzerland) in an expanded blastocyst. A trophectoderm cell is protruding through the thin ZP, Park et al recently reported the use of 1.48µm non-contact diode laser for assisted hatching of in vitro matured/in vitro fertilized/in vitro cultured (IVM/IVF/IVC) blastocysts.53 Short irradiation exposure times (3–5 minutes) were applied and a significant increase in the hatching rate was observed. We have described the use of a 1.48µm diode laser for AH in human blastocysts.54 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 non-drilled blastocysts (44.4% v. 23.8% and 30.6% v. 11.6%). Fig 12.2 shows the effect of laser AH in an expanded human blastocyst.

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RESULTS AND CONCLUSIONS Several studies have been performed into the usefulness and efficacy of AH in different groups of patients of AH using the different methods described. Most of the studies have been done in patients with poor prognosis, including advanced age patients, patients with elevated concentrations of follicle stimulating hormone (FSH), patients with previous implantation failures, or with embryos with thick ZP. Assisted hatching has also been applied to cocultured and frozen thawed embryos. Table 12.1 shows the results reported by different authors. From the published results and taking into account the variability in the methods and study designs, the conclusions concerning AH benefits are: — 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. — It is not clear whether AH is beneficial for patients of an advanced age, for patients with thick ZP, or for frozen thawed embryos.

Table 12.1. Assisted hatching results reported by different authors. Author(s) Method Population Randomized Pregnancy rate study increase 8 Cohen 92 AT Normal FSH Yes Yes NS ≥153µm ZP <5 cell Yes Yes sig day 3 ≥20% frag Tucker 9329 AT ZP All IVF Yes No thinning Impl. failure No (no control) — Olivennes PZD 9455 Day 3 FSH>15 Yes Yes sig 34 Obruca 94 Er:YAG Impl. failures No Yes sig laser Age ≥38 years Impl. Yes (control: Yes sig Tucker 9456 AT CC failures AT) Schoolcraft AT Elevated FSH ≥39 No Yes sig 57 94 years impl. failures Schoolcraft AT ≥40 years No retrosp. Yes sig 9557 PZD ≥3 impl failures Yes Yes Sig for Stein 9558

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Hellebaut59 PZD Antinori 9636 UV laser Check 9660 AT Antinori 9649 Er:YAG laser Tucker 9661 AT Bider 9762 AT Chao 9763 PZD Hurst 9864 Magli 9865

AT AT

Lanzendorf AT 9866 Meldrum AT 9867 Antinori 9950 Er:YAG laser Edirisinghe PZD 9968

Baruffi 9945 Diode laser ZP thinning Veiga 9954 Diode laser

258

>38 years No Yes sig Yes NS Yes sig

1st cycle Impl. failures Frozen ET 1st cycle

Yes No No Yes

Impl. failures ICSI≥35 years ≥38 years Impl. failures

Yes No No Yes

1st cycle ≥38 years ≥3 impl. failures both ≥36 years

Yes No

Yes

Yes sig Yes sig No Yes IVF No TET No Yes sig Yes sig No No

≥35 years

No

Yes NS

1st cycle

Yes

Yes?

≥6 impl.failures ≥38 years

Yes No

Yes? No

ZP≥15 ≥1 impl.fail. <37

Yes

No

Yes

Yes NS

1st cycle Impl.fail. CC

Yes No (unpubl.) Yes (control: Yes sig conv.PZD) AT: acid Tyrode’s; PZD: partial zona dissection; CC: coculture; NS: not significant; sig: significant.

Cieslak 9923 3D PZD

1st cycle All IVF

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REFERENCES 1 Dean J. Biology of mammalian fertilization: the role of the zona pellucida. J Clin Investig (1992); 89:1055–9. 2 Shabanowitz RB, O’Rand MG. Characterization of the human zona pellucida from fertilized and unfertilized eggs. J Reprod Fert (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 Fertil and Embryo Transfer (1991); 8:179–90. 5 Trounson AO, Moore NW. The survival and development of sheep eggs following complete or partial removal of the zona pellucida. J Reprod Fert (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 Ass 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.

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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 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. 18 Liu HC, Cohen J, Alikani M, Noyes N, Rosenwaks Z. Assisted hatching facilitates earlier implantation. Fertil Steril (1993); 60:871–5. 19 Dale B, Talevi R, Gualtieri R, Tosti E, Santella L, Elder K. Intercellular communication in the early human embryo. Mol Reprod Dev (1991); 29:22–8. 20 Talansky BE, Gordon JW. Cleavage characteristics of mouse embryos inseminated and cultures after zona pellucida drilling. Gamete Res (1998); 21:277–8. 21 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. 22 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. 23 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. 24 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. 25 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. 26 Hogan B, Constantini F, Lacy E. (1986). Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbour Laboratory Press. New York: Cold Spring Harbour, 1986. 27 Malter H, Cohen J. Blastocyst formation and hatching in vitro following zona drilling of mouse and human embryos. Gamete Res (1989); 24:67–80. 28 Schoolcraft W, Schenker 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. 29 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 Ass Reprod Genet (1993); 10:187–91. 30 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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31 Tadir Y, Wright WH, Vafa O, Liaw LH, Asch R, Berns MW. Micromanipulation of gametes using laser microbeams. Hum Reprod (1991); 6:1011–6. 32 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. 33 Strohmer H, Feichtinger W. Successful clinical application of laser for micromanipulation in an in vitro fertilization program. Fertil Steril (1992); 58:212–4. 34 Obruca A, Strohmer H, Sakkas D, et al. Use of lasers in assisted fertilization and hatching. Hum Reprod (1994); 9:1723–6. 35 Obruca A, Strohmer H, Blaschitz A, Schönickle E, Dohr G, Feichtinger W. Ultrastuctural observations in human oocytes and preimplantation embryos after zona opening using an Er:YAG laser. Hum Reprod (1997); 12:2242–5. 36 Antinori S, Panci C, Selman HA, Caffa B, Dani G, Versaci C. Zona thinning with the use of laser: a new approach to assisted hatching in humans . Hum Reprod (1996); 11:590–4. 37 Neev J, Tadir Y, Ho P, Berns MW, Asch R, Ort T. Microscopedelivered ultraviolet laser zona dissection: principles and practices. J Assist Reprod Genet (1992); 9:513–23. 38 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. 39 Neev J, Schiewe M, Sung, et al. Assisted hatching in mouse embryos using a noncontact Ho:YSGG laser system. J Ass Reprod Genet (1995); 12:288–93. 40 Schiewe M, Neev J, Hazeleger NL, Balmaceda JP, Berns M, Tadir Y. Developmental competence of mouse embryos following zona drilling using a non-contact Ho:YSGG laser system. Hum Reprod (1995); 10:1821–4. 41 Rink K, Delacretaz G, Salathe RP, et al. 1.48µm diode laser microdissection of the zona pellucida of mouse zygotes. SPIE (1994); 21134 a:412–22. 42 Rink K, Delacretaz G, Salathe RP, et al. Non-contact microdrilling of mouse zona pellucida with an objectivedelivered 1.48µm diode laser. Laser Surg Med (1996); 18:52–62. 43 Germond M, Nocera D, Senn A, et al. Microdissection of mouse and human zona pellucida using a 1.48µm diode laser beam: efficacy and safety of the procedure. Fertil Steril (1995); 64:604–11. 44 Germond M, Nocera D, Senn A, et al. Improved fertilization and implantation rates after non touch zona pellucida microdrilling of mouse oocytes with a 1.48µm diode laser beam. Hum Reprod (1996); 11:1043–8. 45 Baruffi R, Mauri Al, Petersen C, et al. Assisted hatching with a laser diode in patients <37 years old with no previous failure of

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implantation: a prospective randomized study. Hum Reprod (1999); 14 (Abstract book 1) Abstracts of the 15th Annual meeting of the ESHRE, Tours, France. 46 Montag M, Van der Ven K, Delacretaz G, et al. Laser asssisted microdissection of the zona pellucida facilitates polar body biopsy. Fertil Steril (1998); 69:539–42. 47 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. 48 Veiga A, Sandalinas M, Benkhalifa M, et al. Laser blastocyst biopsy for preimplantation diagnosis in the human. Zygote (1997); 5:351–4. 49 Antinori S, Selman HA, Caffa B, Panci C, Dani GL, Versaci C. 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. 50 Antinori S, Versaci C, Dani L, et al. Laser assisted hatching at the extremes of the IVF spectrum: first cycle and after 6 cycle: a randomized prospective trial. Hum Reprod (1999); 14 (Abstract book 1) Abstracts of the 15th Annual meeting of the ESHRE, Tours, France. 51 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. 52 Fong CY, Bongso A, Ng SC, Kumar J, Trounson A, Ratnam S. Blastocyst transfer after enzymatic treatment of the zona pellucida: improving in-vitro fertilization and understanding implantation. Hum Reprod (1998); 13:2926–32. 53 Park S, Kim EY, Yoon SH, Chung KS, Lim JH. Enhanced hatching rate of bovine IVM/IVF/IVC blastocysts using a 1.48µm diode laser beam. J Assist Reprod Genet (1999); 16:97–101. 54 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. 55 Olivennes F, Bergere M, Fanchin R, Vialle MN, Frydman R, Selva J. L’éclosion embryonnaire assistée. Contracept Fertil Sex (1994); 22:493–7. 56 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. 57 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. 58 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.

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59 Hellebaut S, De Sutter P, Dozortsev D, Onghena A, Qian C, Dhont M. 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. 60 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. 61 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. 62 Bider D, Livshits A, Yonish M, Yemini Z, Mashiach S, Dor J. Assisted hatching by zona drilling of human embryos in women of advanced age. Hum Reprod (1997); 12:317–20. 63 Chao KH, Chen SU, Chen HF, Wu MY, Yang YS, Ho HN. 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. 64 Hurst BS, Tucker KE, Awoniyi CA, Schlaff WD. Assisted hatching does not enhance IVF success in goodprognosis patients. J Assist Reprod Genet (1998); 15:62–4. 65 Magli MC, Gianaroli L, Ferraretti AP, Fortini D, Aicardi G, Montanaro N. Rescue of implantation potential in embryos with poor prognosis by assisted zona hatching. Hum Reprod (1998); 13:1331–5. 66 Lanzendorf SE, Nehchiri F, Mayer JF, Oehninger S, Muasher SJ. A prospective, randomized, double-blind study for the evaluation of assisted hatching in patients with advanced maternal age. Hum Reprod (1998); 13:409–13. 67 Meldrum DR, Wisot A, Yee B, Garzo G, Yeo L, Hamilton F. Assisted hatching reduces the age-related decline in IVF outcome in women younger than age 43 without increasing miscarriage or monozigotic twinning. J Assist Reprod Genet (1998); 15:418–21. 68 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.

13 Cytoplasmic fragmentation in human embryos in vitro: implications and the relevance of fragment removal Mina Alikani

OVERVIEW FRAGMENTATION IN THE HUMAN EMBRYO IN VIVO AND IN VITRO During the early days of human embryo culture in vitro, Edwards1 observed a phenomenon he described as “cell division without nuclear division.” This was noted in both developing and arrested embryos and, reminiscent of a similar occurrence in somatic cells, was later termed fragmentation. That observation still holds true: partial or total loss of cellular integrity can affect a large percentage (34521/51 599) of cleavage stage embryos in vitro (EggCyte™ database, Art Institute of New York and New Jersey, 1995–2000). Fragments are independent, membrane bound structures that form when segments of the cytoplasm protrude from the cell and then separate. While cytoplasmic blebbing may be reversible, once the bridge between a bleb and a cell has been severed, reincorporation of the fragment in the cell is unlikely. The exact timing of this in the cell cycle is not clear, but it is likely that most fragments form during mitosis rather than in interphase, precompaction, and particularly at the first and second cleavage divisions. Fragmentation before syngamy also occurs, albeit very rarely, and in many cases leads to zygotic/embryonic arrest. In one study by Schmiady and Kentenich,2 42/111 or 38% of arrested zygotes exhibited fragmentation. Our own database reflects the rarity of this type of fragmentation (less than 2% of all zygotes) and shows that 65% (51/78) of pronucleate embryos with fragmentation either arrested on day 1 (8/51 or 16%), arrested on day 2, or became excessively fragmented by day 3 (43/51 or 84%) and were suitable for neither transfer nor cryopreservation. Some pronucleate eggs do not show fragmentation until syngamy; this may lead to lower than normal cell numbers and higher than normal fragmentation on day 3.3 Antczak and Van Blerkom4 reported complete arrest at either pronuclear or syngamic stage, if fragmentation at

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the 1 cell stage was pervasive and affected a significant portion of the oolema. Fragmentation is not exclusive to embryos developing in vitro. In vivoproduced cleavage stage embryos collected from a number of species, including the human, apparently exhibit various degrees of cellular fragmentation. Embryos recovered after natural or induced ovulation in the baboon5 and rhesus monkey6–8 contained fragmenting blastomeres. Killeen and Moore9 reported a high incidence of “anucleate particles” in sheep embryos collected from fallopian tubes and portions of the uterine horn 60 hours following uterine insemination or natural mating. In humans, it is difficult to say whether fragmentation is as common in vivo as it is in vitro. Information on morphological features of in vivo fertilized and developed human embryos is understandably limited. Three important studies, by Hertig et al,10 Ortiz and Croxatto,11 and Buster et al12 provide some clues, however. Hertig et al10 examined four ova that they judged to be abnormal. Apart from multinucleated blastomeres, the authors referred to “cellular degeneration or necrobiosis” in these ova, which may vaguely imply fragmentation, but this is not obvious from the thick sections through the embryos. Ortiz and Croxatto11 associated fragmentation with aging of the unfertilized oocyte: 42% of unfertilized ova recovered 96 or more hours after the luteinizing hormone (LH) surge from women practicing sexual abstinence showed fragmentation. They also identified 25 abnormal ova recovered from the genital tract of women who had intercourse both outside and within the fertile period. Nine of the 25 ova had not cleaved and were presumed to be unfertilized. Thirteen ova exhibited no sign of cleavage or heterogeneous cytoplasmic fragments. Whether fragmentation resulted from aging of unfertilized ova or it occurred subsequent to fertilization could not be determined. In the study of Buster et al,12 the four 1 cell ova recovered by uterine lavage at 100 or more hours from fertile donors were found to be fully intact. These investigators collected 15 other ova in which development was arrested between 2 and 16 cell stages. One 6 cell embryo had fully intact but uneven cells with severely contracted cytoplasm, while the intact cells of an unevenly divided 2 cell embryo were in close proximity to necrotic debris (lysed cell?) in the perivitelline space (PVS). Another embryo evaluated as a 14 cell with “non-uniform” cells may have actually contained some fragments, but this is difficult to ascertain. Overall, it may be safe to say that although fragmentation seems to be a feature of both in vitro and in vivo conceived embryos, it is more prevalent among the former. Perhaps in vitro conditions and/or ovarian hyper-stimulation are more conducive to fragmentation.

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CAUSES OF FRAGMENTATION The causes of fragmentation remain speculative since an appropriate model in which these may be tested has not yet been found. However, a number of factors may play a part. Apart from media composition, the conditions under which embryos are maintained can impact the pattern of cytokinesis. Extra-cellular pH, temperature, as well as oxygen tension, for instance, can impose their effects by altering the subcellular architecture. A decrease in the pH of the culture medium may lead to destabilization of cell membranes through inhibition of actin polymerization.13 This may in turn lead to blastomere fragmentation in the embryo. Similarly, high oxygen tension has been shown to lead to accumulation of reactive oxygen species in blastomeres, jeopardizing the integrity of their membranes, triggering apoptosis, and leading to fragmentation.14 A lowering of temperature, on the other hand, may lead to anomalies in spindle assembly during mitosis, causing atypical patterns of cytokinesis. Ovarian hyperstimulation and oocyte maturation under adverse follicular conditions may contribute to abnormal embryonic behavior as well. Poorly vascularized and hypoxic follicles have been reported to produce eggs with increased cytoplasmic and chromosomal abnormalities and embryos with reduced development potential.15 However, fragmentation is clearly not limited to embryos generated following exogenous gonadotrophin administration; embryos from non-stimulated cycles also display fragmentation in mild to severe forms.16 Numerical and structural chromosome abnormalities inherent to male and female gametes17–18 may not preclude fertilization but may produce embryos that subsequently fragment and fail to develop. Indeed, embryos with excessive fragmentation have been shown to be chromosomally abnormal. Pellestor et al19 examined the karyotypes of 118 embryos with irregular blastomeres and extensive fragmentation. A normal diploid chromosome complement was found in only 12% of the embryos examined, the remainder showing a variety of abnormalities such as aneuploidy and mosaicism. Munné et al20 applied fluorescent in situ hybridization (FISH) with probes for chromosomes X, Y, 18, and 13/21 to 154 slow and/or fragmented embryos. Chromosomal anomalies found in this group included extensive diploid mosaicism, aneuploidy, and polyploidy. In further studies, 66% of embryos exhibiting more than 35% fragmentation were shown to be chromosomally abnormal (see Chapter 25). This was significantly higher than the 47% abnormality rate among embryos with less than 35% fragmentation. The latter findings imply that a fair number of highly fragmented embryos may in fact be chromosomally intact, and that severely reduced development/implantation potential among such embryos should not be presumed to reflect chromosomal incompetence.

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THE MECHANISMS OF FRAGMENTATION The mechanisms of fragmentation are largely unknown, but the most prevalent theory is apoptosis, or programmed cell death (PCD).21 Apoptosis is a genetically programmed process in somatic cells that includes chromatin condensation, nuclear DNA fragmentation, cellular shrinkage, and fragmentation of a whole cell into membrane bound vesicles that are ingested by neighboring cells. This is different from necrosis, the other form of cell death, which usually results from injury and affects entire groups of cells.22 The apoptotic process is rapid and culminates in the degradation and disposal of damaged cells during the course of normal development in many organisms.23 The occurrence of nuclear fragmentation in dying cells of the human blastocyst24 as well as the morphological similarities between apoptotic somatic cells and fragmented blastomeres in cleavage stage embryos prompted the investigation of apoptosis in these embryos.25 Application of transferase-mediated dUTP nick-end labeling (TUNEL) to arrested fragmented embryos identified extensive condensation and degradation of chromatin, leading some investigators to conclude that embryo fragmentation is the result of activated PCD in some blastomeres.25 Levy et al26 argued that TUNEL alone may fail to distinguish between apoptosis and necrosis; they used TUNEL in combination with annexin V labeling in order to detect cells in early stages of apoptosis. Annexin V binds phosphatidylserine, a molecule that translocates from the inner to the outer cell membrane during apoptosis.27 All arrested and/or fragmented human embryos stained positively for annexin V, while cryopreserved embryos that continued to develop normally after thaw did not show any staining.26 But similar experiments by Antczak and Van Blerkom4 dispute the direct correlation between apoptosis and fragmentation. Intact and fragmented blastomeres, as well as membrane intact propidium iodide negative fragments associated with fragmented blastomeres were virtually all TUNEL and annexin V negative, suggesting that apoptosis may be more an effect than a cause of fragmentation. After TUNEL labeling of 31 fragmented, non-fragmented, and arrested day 3 embryos,28 we noted three patterns of DNA staining: intact DNA with no incidence of apoptotic nuclei (n=16), intact DNA with small, scattered apoptotic bodies in the surrounding cytoplasm (n=8) and apoptotic nuclei (n=6). In further studies, over 200 embryos were subjected to nuclear staining (Fig 13.1a-f). Approximately half displayed some DNA degradation represented by small intensely fluorescing bodies (Fig 13.1c, d, e and f) (Stachecki JJ et al, unpublished observations). Nuclear material was rarely located within cytoplasmic fragments but they could occasionally be visualized in blastomeres associated with the fragments (Fig 13.1e). Also, some patient specificity was observed in that, regardless of the degree of fragmentation, embryos from the same patient

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tended to stain similarly, either exhibiting or lacking areas of DNA degradation. More experiments of this nature are currently under way in our laboratory. Considering the tremendous diversity among human embryos, and the contradictory findings of the few studies in this area, it is our opinion that firm conclusions regarding the relationship between fragmentation and apoptosis should be withheld until more experimental data are available.

Fig 13.1 Fragmented and non-fragmented day 3 human embryos subjected to nuclear staining. Different staining patterns were observed. Intact nuclei (panels A and B), intact nuclei with small, scattered intensely staining nuclear fragments in the surrounding cytoplasm (panels C and D), and degrading nuclei (panels E and F).

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Embryos in panels A, E and F are all fragmented but show different patterns of staining. DEGREE AND PATTERNS OF FRAGMENTATION DEFINITIONS Along with development rate, fragmentation has been widely accepted as the most important parameter in assessment of embryo viability during cleavage stages.29 Despite this, the early descriptions of fragmentation were rather vague and not quantified. Embryos were graded based on the presence or absence of fragments30 or alternatively, minor or major fragmentation.31 An exception to this is a publication by Puissant et al,32 in which embryos with fragments covering less than one third of the embryonic surface were described as fair while those with more extensive fragmentation were classified as poor. We have described fragmentation both with respect to degree and pattern. The degree of fragmentation, or the close approximation of the embryonic volume occupied by anucleate cytoplasmic fragments, is expressed as a percentage. This may vary from 0 to 100%. The proportion of day 2 (n=52624) and day 3 (n=51 599) embryos with various degrees of fragmentation is shown in Fig 13.2. While the proportion of 0–15% fragmented embryos is reduced between day 2 and day 3, that of embryos with more than 15% fragmentation is increased, demonstrating the progressive nature of fragmentation at least during those days. We further proposed that human embryos not only fragment to various degrees, but they do so in five spatially and morphometrically distinct patterns.33–34 Representative embryos with these patterns are depicted in Fig 13.3. Fig 13.4 shows the frequency of their occurrence among 34598 fragmented embryos. Fragmentation of type I is minimal in volume and the fragments are typically, but not always, associated with only one blastomere. The number and position of these fragments in some instances suggest they may be remnants of the first and/or second polar bodies. Type II fragments are strictly localized and often form continuous columns occupying the PVS. Evidence presented in the next segment suggests that such a pattern may emerge as a result of complete fragmentation of one or more cells into small fragments. The most common pattern of fragmentation is type III fragmentation; fragments of this type are small and scattered and may be positioned between blastomeres, peripherally, or both. Pattern IV is represented by large size fragments that sometimes resemble whole blastomeres. These fragments are often randomly distributed, and are associated with uneven cells. As in type II fragmentation, type IV fragmentation may result from complete fragmentation of one or more blastomeres. Type V fragments appear

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necrotic, with a characteristic granularity and cytoplasmic contraction within the intact blastomeres. This pattern does not occur commonly. IMPLICATIONS FOR PREGNANCY AND IMPLANTATION AFTER SHORT-TERM CULTURE That fragmentation limits the potential of human embryos for development became evident from early clinical data such as those presented by Puissant et al.32 Many studies have been published on this topic since, but the lack of sufficient data, low overall

Fig 13.2 The extent of fragmentation in day 2 (white bars) (n=52 624) and day 3 (grey bars) (n=51 599) embryos. Twenty-eight per cent of day 2 embryos had more than 15% fragmentation; this proportion increased to 37% on day 3, indicating the progressive nature of fragmentation during those days.

Fig 13.3 Five distinct patterns of fragmentation that emerge by day 3 of development in vitro. Each pattern is depicted before (left panel) and after fragment removal (right panel; designated “a”). Fragmentation of type I (I) is minimal in volume, in this case about 5% and associated mostly with one blastomere. Note that the removal of these fragments has not altered the appearance of the remaining blastomeres (Ia). Type II fragments are strictly localized (II); here they form a continuous column (about 25%

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of the total volume) occupying the perivitelline space. After fragment removal, the blastomeres appear to be disorganized, with reduced cell-cell contact (IIa). This pattern emerges as a result of complete fragmentation of one or more cells into small fragments. The most common pattern of fragmentation is III (III); fragments are small and scattered (here about 20% of the total volume) positioned between blastomeres and peripherally. The blastomeres are well organized but seem to have reduced contact after fragments are removed (IIIa). In pattern IV, large size fragments (here about 30% of the total volume) are associated with uneven cells (IV). This is very apparent after fragments are completely removed (IVa). Type V fragments (here about 40% of the total volume) appear necrotic, with a characteristic granularity and cytoplasmic contraction within the intact blastomeres apparent before (V) and after fragment removal (Va). implantation rates, and ambiguous categorization of embryos have made it difficult to draw useful conclusions. Three large and more recent studies on the impact of the degree of fragmentation on the outcome of IVF are those of Staessen et al,35 Giorgetti et al,36 and Ziebe et al.37 These investigators reported an implantation rate of approximately 5% after transfer of embryos with 10–50% fragmentation on day 2 of development. But these reports did not address the differences among embryos within this wide range, nor did they consider the size and distribution of the fragments.

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Fig 13.4 The frequency of five distinct patterns of fragmentation in fragmented day 3 embryos (n=37918). NP=no distinct pattern. Fragmentation pattern III, small scattered fragments, was the most common pattern, followed by pattern IV, predominantly large fragments associated with numerous uneven cells. Pattern V occurred least frequently. Our preliminary data showed that the developmental potential of fragmented embryos was partly dependent on the patterns of fragmentation; patterns IV and V were detrimental to the embryo.33 In an extensive follow up study,38 we retrospectively analyzed 1727 transfers homogeneous with respect to the degree of fragmentation, and 570 transfers homogeneous with respect to the pattern of fragmentation. A transfer was considered homogeneous when more than one half of the embryos replaced were in the same morphological category. This was done in order to eliminate or minimize correlative uncertainties when multiple embryos were transferred. All transferred embryos in the study were subjected to selective assisted hatching (AHA)39 and fragment removal. Patients/embryos were selected for hatching based on maternal age and embryonic morphology criteria, a detailed list of which appears in Table 13.1. None of the transfers in the study included embryos with more than 25% fragmentation after the fragments were removed. With increasing fragmentation, more fragments were removed. In extensively fragmented embryos, virtually all fragments were removed (Fig 13.5). The analyses

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showed that pregnancy and implantation decreased significantly with increasing fragmentation. The proportion of fetal hearts developing over the total number of embryos replaced was 30.1% (1681/5496) when the majority of the replaced embryos were ≤15% fragmented. Implantation decreased significantly to 20.2% (85/420), when embryos with >15% were replaced. However, under the conditions of our study, that is, with the application of assisted hatching and fragment removal, the dramatic decrease in implantation occurred only when fragmentation exceeded 35%. In total, 6% of such embryos implanted (all delivered). Embryos with 16–35% fragmentation still showed a good ability to implant. The pattern of fragmentation also had a profound impact on developmental competence of embryos. Transfers involving embryos with type IV fragmentation on day 3 resulted in a low implantation rate of 18.2% (58/318), significantly lower than types I, II, or III (implantation rate of 37.9%, 33.8%, and 30.5%, respectively). Embryos with localized fragments (type II) had an average of 3.22±1.14 cells on day two. This was significantly lower in comparison to all other fragment types, implying that this type of fragment arose after complete disintegration of one or more blastomeres. On day 3, both types II (5.98±1.99 cells) and IV (5.58±1.97 cells) embryos had fewer cells compared with others, providing evidence, for the first time, that total loss of one or more blastomeres led to the appearance of these types. The average degree of fragmentation on day 3 among type IV fragmented embryos was 25%, higher than that in other types (Fig 13.6), hence the possibility that the apparent loss of development potential among these embryos was solely due to the high degree of fragmentation. We therefore investigated transfers homogeneous for both degree and pattern of fragmentation. Transfer of type IV fragmented embryos, with 6–35% fragmentation, led to implantation rates lower than their type II or III counterparts, or the

Table 13.1. Criteria for selection of patients and embryos for assisted hatching and fragment removal. Age Fragmentation Fragmentation Zona Attempt Assisted Fragment (years) Degree (%) Pattern (Type) Thickness Number Hatching Removal (micrometers) <38 0–15 I,II,III <17 1 No n/a <38* 0–15 IV,V,NP >17 >1 Yes minimal <38 >15 all all all Yes extensive ≥38 0–15 all all all Yes minimal *Assisted hatching is applied if one or more of the conditions listed for fragmentation type, zona pellucida thickness, and/or number of attempts occurs.

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implantation rate of fragmented embryos regardless of the pattern of fragmentation. Although this was considered to be a very meaningful trend, the reduction in implantation did not reach statistical significance. Electron microscopic evidence suggests that large fragments originate from cells of 2 or 4 cell embryos, as these fragments have mitochondria that are more electron-dense than in later stage cells.40 The release of large fragments at an early stage may deplete the embryo of essential organelles such as mitochondria, or structures such as pinocytotic caveolae that are involved in uptake of exogenous protein.41 There is also some evidence suggesting the loss of cortically positioned regulatory proteins such as leptin and STAT3 through the process of fragmentation.4 Collectively or each of these factors alone can contribute to reduced viability among type IV fragmented embryos. RELATION TO MATERNAL AGE With increasing maternal age, the proportion of embryos with 0–5% fragmentation appears to increase and that of embryos with more than 35% fragmentation appears to decrease (Fig 13.7). This would imply an overall decrease in fragmentation rate with aging of (at least) the female gamete. If one assumes that fragmentation is integral to normal development, and an active, energy driven process, it is the absence of it in this case that is abnormal. And perhaps it should not be entirely surprising in view of the accumulation of mitochondrial mutations (J Barrett and C Brenner, unpublished data) and the associated reduction in ATP levels among aged oocytes.42

Fig 13.5 The relation between the degree of fragmentation on the morning of day 3 of development and the extent of fragment

removal. The mean volume of fragments removed increased with increasing fragmentation. Most fragments were removed from embryos with more than 35% fragmentation.

Fig 13.6 Embryos with patterns II and IV fragmentation before (II and IV) and after (IIa and IVa) fragment removal. Arrows indicate the site of zona pellucida breach. Average degree of fragmentation was higher among type IV embryos than all other patterns, including type II. Note the uneven appearance of remaining blastomeres after fragment removal from Type IV (IVa).

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Fig 13.7 The relation between maternal age and degree of fragmentation. With increasing maternal age, the proportion of embryos with 0–5% fragmentation (Black bars and trendline) seem to increase, while that of embryos with more than 35% fragmentation (white bars and trendline) seems to decrease.

Fig 13.8

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The relation between the degree of fragmentation on day 3 and the incidence of normal compaction (◊), cavitation (□), and blastocyst formation ( ) in extended culture. Fragmentation has a significant negative impact on all three processes. On the other hand, there is no evidence that age is correlated with the pattern of fragmentation. All patterns of fragmentation occurred with the same frequency among different age groups, indicating that the mechanisms giving rise to various types of fragments are not unique to any specific age group.

BLASTOCYST FORMATION AFTER PROLONGED CULTURE We evaluated the relation between common cleavage anomalies and apparently normal blastocyst formation after five days in culture.43 Normal blastocyst formation rate decreased significantly with increasing fragmentation: 33.3% (311/935) among embryos with 0–15% fragmentation was significantly higher than

Fig 13.9 The relation between the pattern of fragmentation on day 3 and the incidence of normal compaction (◊), cavitation (□), and blastocyst formation ( ) in extended culture. Fragmentation type IV has a

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significant negative impact on all three processes, while types I, II, and III do not. 16.5% (46/279) among embryos with more than 15% fragmentation (P<0.001). The difference was apparent at compaction (51.1% v 24.6%, respectively) and cavitation (47.9% v 29.3%, respectively). When fragmentation exceeded 35%, all processes were severely compromised, and normal compaction (5/50; 10%), cavitation (5/47; 10.6%) and blastocyst formation (6/51; 11.8%) were lowest (Fig 13.8). The pattern of fragmentation also correlated with blastulation. Blastocyst formation rates were not different among embryos with fragmentation type I (54/140, 38.6%), type II (27/82, 32.9%), or type III (143/442, 32.4%). However, type IV fragmentation led to a significant reduction in normal blastocyst formation (25/170, 14.7%) (P<0.001). The same trend was apparent at compaction and cavitation (Fig 13.9). METHODS FOR MICROSURGICAL FRAGMENT REMOVAL This technique is applied immediately after assisted hatching. Once the gap in the zona pellucida is made and the acidified solution is cleared from the vicinity of the embryo, fragments may be removed by aspiration with the hatching needle. Great caution must be exercised, since even the slightest touch of the hatching needle on the membrane of a blastomere can result in the loss of membrane integrity and cell lysis. Fragment removal is attempted at the highest magnification and using a state-of-theart interference microscope. Continuous refocusing and repositioning of the embryo are essential to this technique. A needle, manufactured in house, with an outer diameter of 12µm, is preferred for this technique (a regular AHA needle is approximately 10µm in diameter). Suction is mouth controlled and is instantly ceased if it appears that the membrane of a blastomere is reacting to the suction in any way. Fragments in clefts between blastomeres and those opposite the zona opening are usually removed last. While the procedure is performed carefully and patiently, time spent on each procedure is closely monitored, and must not exceed 10 minutes per embryo. Micromanipulation of more than one embryo at a time is avoided. EVIDENCE FOR AND AGAINST MECHANICAL REMOVAL OF FRAGMENTS Fragment removal has been applied routinely in our clinical program. This was initially based on the following observation:44 when assisted hatching and fragment removal were applied simultaneously to embryos with >=15% fragmentation, 14.7% (120/819) implanted, significantly higher than a 6.5% (8/124) implantation rate achieved after transfer of similar

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embryos with zona drilling only. Subsequent retrospective analyses demonstrated a high implantation rate among embryos with 6–35% fragmentation.38 Admittedly, the efficacy of fragment removal has not been tested in large prospective randomized clinical trials, hence the controversy and criticism surrounding this work. Moreover, it is not entirely clear how the removal of fragments from certain embryos helps their survival, but several possibilities have been explored. First, fragments and apoptotic cells may undergo secondary necrosis. Since blastomeres of early human embryos do not appear to have any phagocytic activity, the necrotic fragments and cell corpses are not effectively removed from the developing embryo. The close proximity of these components to the surrounding vital blastomeres may cause them to deteriorate, and eventually arrest. In fact, intact blastomeres adjacent to groups of fragments have been observed to show signs of degeneration, such as lysosome accumulation and vaculation.40 In support of this, there is some firm indication that cleavage between day 2 and 3 is promoted if fragments are microsurgically removed on day 2 of development.45 Embryos displaying on average 30% fragmentation on day 2 were subjected to extensive fragment removal on day 2; control embryos (from the same patient) were subjected to assisted hatching only. Both groups were cultured to day 3. Mean cell number on day 3 for embryos with and without fragment removal were compared, with the former showing significantly higher cell numbers and more rapid development to >=6 cells on day 3. Secondly, fragments may be positioned in an orientation that hinders normal cell-cell contact, possibly interfering with the proper establishment of cell division axes,46–48 compaction, and, ultimately, normal blastocyst formation. Experimental evidence suggests that fragment removal on day 3 promotes compaction between day 3 and day 4 (Fig 13.10). In 19 experiments, at least two embryos (one experimental and one control) with comparable morphology (cell number, degree of fragmentation, pattern of fragmentation, and multinucleation) were selected from each of 19 patients. Mean fragmentation degree among these embryos was 29%; fragmentation patterns included type III and IV. On day 3 of development, after embryo evaluation, assisted hatching was applied to both control and experimental embryos. All fragments were removed from experimental embryos, while the fragments were left intact in the control embryos. Embryos were placed on bovine epithelial cells for extended culture until day 5 or 6. On days 4 and 5, the morphology of both groups was evaluated. Examples of control and experimental embryos from three different patients are shown in Figs 13.11–13. Compaction on days 4 and 5 was defined as complete, incomplete, or regional. Complete or incomplete compaction involved all cells within the embryo (Figs 13.11 and 12, E4).

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Regional compaction (Figs 13.11 and 12, C4) marked exclusion of one or more cells from the compacted morula. A single defined cavity was expected to appear on day 5 (Figs 13.11, E5), but the persistence of large vacuoles, small and multiple cavities on day 5 was more usual (Figs 13.11 and 12, C4, C5) and was considered abnormal as it led to development arrest. Compaction rate was significantly higher among experimental embryos than the control (9/19 or 47.4% v2/19 or 10.5%, respectively; P<0.05) (Fig 13.10). Cavitation rate as well as normal blastocyst formation rate were, however, low in both experimental and control groups, but this was expected as these were severely compromised embryos to begin with, and the culture conditions were suboptimal at best. Embryos that displayed 40% or more fragmentation rarely showed any improvement after removal of fragments (Fig 13.13, E5), and not all embryos with less than 40% fragmentation and fragment removal showed clear morphological superiority to their counterparts with intact fragments, especially on day 5 (Fig 13.12, C5 compared with E5). The results of this study are nevertheless interesting as they show that (1) an advantage for fragmented embryos with fragment removal may exist with respect to division and compaction, (2) the in vitro culture system is unable to fully support the development of fragmented embryos with or without fragment removal for extended periods, and (3) embryos with more than 40% fragmentation may suffer from inherent abnormalities, not reversible by simple removal of fragments. So it is within such a context that arguments against the application of microsurgical fragment removal should be discussed. Using a mouse model, Dozortsev et al49 attempted to determine the impact of experimentally induced fragmentation on hatching rate and total cell number at the blastocyst stage. At the late pronuclear stage, five to six cytoplasts were removed, amounting to about one quarter of the total zygote volume. These were then returned to the same zygote, to serve as fragments. Neither hatching nor blastocyst cell number was affected when compared to a control

Fig 13.10 The impact of day 3 fragment removal on day 4 and day 5 compaction and cavitation. The incidence of compaction increased significantly (P<0.05) among embryos with fragment removal (grey bars) compared to embryos with assisted hatching only and no fragment removal (white bars). The difference in cavitation was not statistically significant. Both experimental and control embryos were from the same patient in the 19 experiments represented here.

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Fig 13.11 Control (C) and experimental (E) embryos from patient 1. The control embryo was a 6-cell with 25% type IV fragmentation and was zona drilled on day 3 (C3); fragments were not removed. On day 4 (C4), this embryo had developed several intracellular vacuoles and was showing signs of regional compaction. On day 5 (C5), two large cavities were visible. Development arrested at this abnormal state. The experimental embryo was a 7 cell, also with 25% type IV fragmentation. Assisted hatching was performed, and all fragments were subsequently removed on day 3 (E3a). On day 4 (E4), several of the cells had divided further and had begun to compact normally. By day 5 (E5) a blastocyst developed with reasonably cohesive trophectoderm, but irregularly arranged inner cell mass. Hatching had started at the point of zona breach. group with reduced cytoplasmic volume. The second experimental group consisted of 8 cell embryos in which two blastomeres were subjected to karyoplast removal. All blastomeres in such embryos subsequently participated in compaction. Moreover, blastocyst cell count and hatching ability were not compromised. The authors then concluded that fragments formed around the time of the first mitotic division or before compaction,

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in an otherwise healthy embryo, did not interfere with development, and that fragment removal provides no benefit for embryo development. The general conclusion of these authors regarding the benign nature of some fragments is actually in agreement with our own observations in the human. Fragmentation type I, for example, appearing early in development but remaining limited in degree to about 5% of the embryonic volume, appears inconsequential to the developing embryo. However, this conclusion could not be extended to all other types of fragments. The basic (and major) flaw in the study by Dozortsev et al49 is the experimental model itself. Fragmentation is normally rarely seen in the mouse, and when it occurs, it is invariably associated with embryonic demise. At the zygote stage, what Dozortsev et al introduced in their experimental embryos were homologous cytoplasts, rather than fragments. Similarly, removal of the nucleus or a karyoplast from a blastomere of an 8 cell embryo is not equivalent to fragmentation at that stage. The mechanisms that give rise to fragmentation, and consequently the fragments

Fig 13.12 Control (C) and experimental (E) embryos of patient 2. The control embryo was a 5 cell with 30% type III fragmentation and

was zona drilled on day 3 (C3); fragments were not removed. On day 4 (C4), this embryo had developed several intracellular vacuoles and was showing signs of regional compaction. On day 5 (C5), one small cavity was visible, and the embryo appeared to have begun to differentiate. However, several cells and fragments had been excluded and cells allocated to the trophectoderm were large and irregular. A distinct inner cell mass was not seen. The experimental embryo was also a 5 cell with 30% type III fragmentation before fragment removal (E3b). Assisted hatching was performed and all fragments were subsequently removed on day 3 (E3a). On day 4 (E4), normal compaction occurred. By day 5 (E5) a single cavity developed, but few cells seemed to have been allocated to the trophectoderm, and the inner cell mass cells were loose and disorganized.

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Fig 13.13 Control (C) and experimental (E) embryos from patient 3. The control embryo was an 8 cell with 45% type V fragmentation and was zona drilled on day 3 (C3); fragments were not removed. On day 4 (C4), this embryo had excluded several cells and fragments and was regionally compacted. On day 5 (C5), several intracellular vacules as well as the beginnings of a cavity were visible. Development arrested at this abnormal state. The experimental embryo was a 6 cell, also with 45% type V fragmentation before fragment removal (E3b). Assisted hatching was performed, and all fragments were subsequently removed on day 3 (E3a). The embryo did not make any further progress during the next two days in culture (E4 and E5) and subsequently arrested with severe contraction of the cytoplasm. themselves, are undoubtedly very different in humans. Moreover, in the same animal model, we have demonstrated the detrimental effects of deliberate partial degeneration,50 which may more closely represent some types of fragmentation in the human. Microsurgical removal of degenerate cells from such embryos promoted hatching and restored their viability. Antczak and Van Blerkom4 have also argued against fragment removal. Referring mainly to the loss of cortically positioned regulatory proteins, they state that whatever effects fragmentation may have on embryo viability are likely to have already occurred, so the beneficial effects on outcome after the mechanical removal of fragments may be “more apparent than real.” But this conclusion seems to be somewhat contradictory to their own findings that (1) the developmental potential of fragmented embryos varies and may be dependent on the specific patterns of fragmentation, and (2) localization of protein domains as well as the extent of domain loss may vary from blastomere to blastomere and from embryo to embryo, even within the same pattern of fragmentation. Moreover, the significance of these protein domains (and their loss) to early development clearly needs further investigation, as only a few embryos have been examined so far. But even if one presumes that the protein loss is universal and irreversible in every fragmented embryo, it is still possible that the removal of fragments from such embryos stops the degenerative processes that are secondary to fragmentation.

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CONCLUSIONS Human embryos seem to be particularly prone to cytoplasmic fragmentation while developing in vitro, with profound implications for development following intrauterine transfer or in extended culture. The underlying mechanisms of fragmentation are still far from clear, however, the discovery of the different patterns of fragmentation and their correlation to implantation certainly sheds some light on this and disputes the existence of a single mechanism such as apoptosis. The relevance of fragment removal in maintaining or restoring the viability of fragmented embryos is justifiably debated. But clinical and experimental data are very suggestive that the removal of fragments promotes division and compaction and does in fact contribute to better implantation rates among fragmented embryos. Undoubtedly, large randomized prospective trials can clarify this further. But ultimately, the relevance of this technique is in the understanding of fragmentation itself, as well as enhancing implantation in patients whose embryos are in the difficult predicament of excessive fragmentation. As with any other microsurgical technique in embryology, fragment removal should be applied cautiously and conservatively by those with expertise in the area of embryo micromanipulation.

REFERENCES 1 Edwards RG, Steptoe PC, Purdy JM. Fertilization and cleavage in vitro of preovulatory human oocytes. Nature (1970); 227:1307–9. 2 Schmiady H, Kentenich H. Cytological studies of human zygotes exhibiting developmental arrest. Hum Reprod (1993); 8:744–51. 3 Zaninivic N, Papale MN, Xu KP, Menendez S, Palermo GD, Veeck LL, Rosenwaks Z. The impact of early fragmentation during subsequent pre-embryo development. Hum Reprod (supplement) (1996); 11:211 (abstract). 4 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. 5 Hendrickx AG, Kraemer DC. Preimplantation stages of baboon embryos. Anat Rec (1968); 162:111–20. 6 Batta SX, Stark RA, Brackett BG. Ovulation induction by gonadotropin and prostaglandin treatments of rhesus monkeys and observations of the ova. Biol Reprod (1978); 18:264–78. 7 Hurst PR, Wheeler AG, Eckstein P. A study of uterine embryos recovered from rhesus monkeys fitted with intrauterine devices. Fertil Steril (1980); 33:69–79.

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8 Enders AC, Hendrickx AG, Binkerd PE. Abnormal development of blastocysts and blastomeres in the Rhesus Monkey. Biol Reprod (1982); 26:353–66. 9 Killeen ID, Moore NW. The morphological appearance and development of sheep ova fertilized by surgical insemination. J Reprod Fert (1971); 24:63–70. 10 Hertig AT, Rock J, Adams EC, Menkin MC. On the preimplantation stages of the human ovum: a description of four normal and four abnormal specimens ranging from the second to the fifth day of development. Contrib Embryol (1954); 35:199–220. 11 Ortiz ME, Croxatto HB. Observations on the transport, aging, and development of ova in the human genital tract. In: Talwar GP, ed. Recent advances in reproduction and regulation of fertility. New York: Elsevier/North-Holland Biomedical Press, 1979:307–17. 12 Buster JE, Bustillo M, Rodi IA, et al. Biologic and morphologic development of donated human ova recovered by nonsurgical uterine lavage. Am J Ostet Gynecol (1985); 15:211–7. 13 Begg DA, Rebhun LI. PH regulates the polymerization of actin in the sea urchin egg cortex. J Cell Biol (1979); 83:241–8. 14 Yang HW, Hwang KJ, Kwon HC, Kim HS, Choi KW, Oh KS. Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos. Hum Reprod (1998); 13:998–1002. 15 Van Blerkom J, Antczak M, Schrader R. The developmental potential of the human oocyte 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. 16 Monks NJ, Turner K, Hooper MAK, Kumar A, Verma S, Lenton EA. Development of embryos from natural cycle in vitro fertilization: impact of medium type and female infertility factors. Hum Reprod (1993); 8:266–71. 17 Pellestor F. Frequency and distribution of aneuploidy in human female gametes. Hum Genet (1991); 86:283–8. 18 Martin RH, Radenmaker AW, Hildebrand K. Variation in the frequency and type of sperm chromosomal abnormalities among normal men. Hum Genet (1987); 77:108–14. 19 Pellestor F, Dufour MC, Arnal F, Humeau C. Direct assessment of the rate of chromosomal abnormalities in grade IV human embryos produced by in-vitro fertilization procedure. Hum Reprod (1994); 9:293–302. 20 Munné S, Alikani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmental rates and maternal age are correlated with chromosome abnormalities. Fertil Steril (1995); 64:382–91. 21 Hardy K. Apoptosis in the human embryo. Rev Reprod (1999); 4:125– 34.

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22 Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytology (1980); 68:251–306. 23 Fraser A, Evan G. A license to kill. Cell (1996); 85:781–4. 24 Hardy K, Handyside AH, Winston RML. The human blastocyst: cell number, death and allocation during late preimplantation development in vitro. Development (1989); 107:597–604. 25 Jurisicova A, Varmuza S, Caspar RE Programmed cell death and human embryo fragmentation. Mol Hum Reprod (1996); 2:93–8. 26 Levy R, Benchaib M, Cordonier H, Couchier C, Guerin JF. Annexin V labelling and terminal transferasemediated DNA end labelling (TUNEL) assay in human arrested embryos. Mol Hum Reprod (1998); 4:775–83. 27 Martin SJ, Reutelingsperger CPM, McGahon AJ, Rader JA, van Schie RCAA, LaFace DM, Green DR. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J Exp Med (1995); 182:1545–56. 28 Stachecki JJ, Alikani M, Brenner C. Incidence of apoptosis in human cleaved embryos. Annual meeting program supplement, ASRM, Cincinnati, USA 1998; S122 (abstract). 29 Mohr LR. Assessment of human embryos. In: Trounson A, Wood C, eds. In vitro fertilization and embryo transfer. New York: Churchill Livingston, 1984:159–71. 30 Plachot M, Mandelbaum J. Oocyte maturation, fertilization and embryonic growth in vitro. Brit Med Bull (1990); 46:675–94. 31 Veeck LL. Oocyte assessment and biological performance. Ann NY Acad Sci (1988); 541:259–74. 32 Puissant F, Van Rysselberge M, Barlow P, Deweze J, Leroy F. Embryo scoring as a prognostic tool in IVF treatment. Hum Reprod (1987); 2:705–8. 33 Alikani M, Cohen J. Patterns of cell fragmentation in the human embryo. J Asst Reprod Genetics (1995); 12:28S (abstract). 34 Warner CM, Cao W, Exley GE, et al. Genetic regulation of egg and embryo survival. Hum Reprod (1998); 13 (supplement): 178–90. 35 Staessen C, Janssenwillen C, Van Den Abbeel E, Devroey P, Van Steirteghem AC. Avoidance of triplet pregnancies by elective transfer of two good quality embryos. Hum Reprod (1993); 8:1650–3. 36 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); 10:2427–31. 37 Ziebe S, Petersen K, Lindenberg S, Andersen AG, Gabrielsen A, Andersen AN. Embryo morphology or cleavage stage: how to select the best embryos for transfer after in vitro fertilization. Hum Reprod (1997); 12:1545–49.

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38 Alikani M, Cohen J, Tomkin G, Garrisi GJ, Mack C, Scott R. Human embryo fragmentation in vitro and its implications for pregnancy and implantation. Fertil Steril (1999); 71:836–42. 39 Cohen J, Alikani M, Trowbridge J, Rosenwaks Z. Implantation enhancement by selective assisted hatching using zona drilling of embryos with poor prognosis. Hum Reprod (1992); 7:685–91. 40 Sathananthan AH, Wood C, Leeton J. Ultrastructural evaluation of 8– 16 cell human embryos developed in vitro. Micron (1982); 13:193– 203. 41 Sathananthan H, Bongso A, Ng SC, Ho J, Mok H, Rtnam S. Ultrastructure of preimplantation human embryos co-cultured with human ampullary cells. Hum Reprod (1990); 5:309–18. 42 Van Blerkom J, Davis P, Lee J, ATP content of human oocytes and developmental potential and outcome after in vitro fertilization and embryo transfer. Hum Reprod (1995); 10:415–54. 43 Alikani M, Tomkin G, Calderone G, Garrisi GJ, Kokot M, Cohen J. Cleavage anomalies in early human embryos and survival after prolonged culture. Hum Reprod (2000) in press. 44 Cohen J, Alikani M, Ferrara T, et al. Rescuing abnormally developing embryos by assisted hatching. In: Mori T, Aono T, Tominaga T, Hiroi M, eds. Frontiers in endocrinology, perspectives on assisted reproduction. Rome: Ares Serono Symposia, (1994); 4:536–44. 45 Zaninovic N, Veeck L, Xu K, Rosenwaks Z. Microsurgical fragment removal on day two enhances preembryo quality and increases pregnancy rates in poor prognostic patients. Annual meeting program supplement, ASRM, Toronto, Canada. (1999) 72: S13 (abstract). 46 Goldstein B. Cell contacts orient some cell division axes in the Caenorhabditis elegans embryo. J Cell Biol (1995); 129:1071–80. 47 Suzuki H, Togashi M, Adachi J, Toyoda Y. Developmental ability of zona-free mouse embryos is influenced by cell association at the 4-cell stage. Biol Reprod (1995); 53:78–83. 48 Edwards RG, Beard HK. Oocyte polarity and cell determination in early mammalian embryos. Mol Hum Reprod (1997); 3:863–906. 49 Dozortsev D, Ermilov A, El-Mowafi DM, Diamond M. The impact of cellular fragmentation induced experimentally at different stages of mouse preimplantation development. Hum Reprod (1998); 13:1307– 11. 50 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.

14 Human embryo biopsy for preimplantation genetic diagnosis Alan H Handyside

INTRODUCTION In the mid eighties, the development of PCR strategies for amplification of specific fragments of DNA from single cells1,2,3 paved the way for preimplantation genetic diagnosis (PGD) of inherited disease from one or more cells biopsied from embryos at preimplantation stages after IVF.4 As the human oocyte and embryo up to the expanded blastocyst stage are enclosed within the zona pellucida, any sampling procedure requires micromanipulation to penetrate this protective glycoprotein layer. The second challenge is to remove the target cells with minimal damage to the embryo again requiring micromanipulation. Various approaches have been advocated from biopsy of polar bodies at the zygote stage to removal of some of the outer trophectoderm (TE) cells from blastocysts and each has particular advantages and disadvantages (Table 14.1).5

Table 14.1. Biopsy of genomic DNA from human preimplantation embryos in vitro. Features Applications Periconception Both polar bodies biopsied separately soon Chromosomal First and second polar after fertilization, generally intracytoplasmic aneuploidy in maternal meiosis Single gene body biopsy sperm injection (ICSI). Analysis of both required for either chromosomal aneuploidy defects involving maternal mutations or single gene analysis. For single gene defects, limited to analysis of maternal mutations. Preimplantation stages Cleavage Removal of one or two blastomeres at late Chromosomal stage biopsy cleavage stages (6 to 10 cell stage) does not aneuploidy (from affect cleavage rate or development to either parent) Single blastocyst. Single cell genetic analysis gene defects required. Removal of blastomeres reduces (involving mutations

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the implantation and developmental potential of the embryo. Accuracy compromised by chromosomal mosaicism. Excision of outer trophectoderm cells is possible without affecting the inner cell mass from which the fetus is derived. Multiple cells available for analysis. Although affected by chromosomal mosaicism, likely to be more accurate than cleavage stage analysis. Loss of trophectoderm cells may adversely affect implantation.

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in either parent)

All of the above with increased scope for multiple analysis e.g. combined chromosomal and single gene analysis

PENETRATION OF THE ZONA PELLUCIDA Until the advent of non-contact lasers for use in micromanipulation (see below), two basic methods have been used for penetrating the zona. Both of these were pursued initially as a means to enhance fertilization rates with oligospermic 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 round the circumference.6 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 holes 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 has been put forward as a possible approach for embryo biopsy, but it is difficult to control, does not allow precise selection of blastomeres, and the risk of lysis is high. A modification developed by Verlinsky et al is to make two slits to create a “flap” of zona that can be flipped open, allowing more flexibility in the size of the opening created.7 This group has shown the effectiveness of this approach for both polar body and blastomere biopsy. Mechanical methods for zona penetration are more time consuming and require skilful micromanipulation. As an alternative, zona drilling using acidified Tyrode’s solution (pH 2.2 to 2.4) to dissolve the zona glycoproteins has been extensively used. Again, this method was developed by using the mouse embryo as a model, as a possible means to improve fertilization rates with low sperm densities.8 However, its use with human oocytes while increasing the incidence of fertilization arrested the further development of the zygote presumably consequent to changes

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in intracellular pH.9 With zona drilling, the effect of the acid Tyrode’s is localized to a small area of the zona using a fine micropipette, typically 5– 10um. The micropipette filled with acid Tyrode’s 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. Global pH is normally maintained by using HEPES buffered modifications of the culture medium and when the drilling is complete the micropipette is immediately withdrawn.

POLAR BODY BIOPSY Originally, it was suggested that biopsy and genetic analysis of the first polar body would allow PGD of maternal defects prior to conception.10 Apart from some arguable practical advantages (see below), this concept was also attractive as it only involves manipulation of the human egg and not the fertilized embryo and would therefore be more acceptable to those with moral or ethical objections to screening embryos. For single gene defects, however, identification of the maternal allele remaining in the oocyte is not possible in many cases because of recombination in meiosis I which results in a heterozygous first polar body and oocyte. Additional analysis of the second polar body is therefore essential to infer which allele has been retained in the zygote. Furthermore, although molecular genetic analysis of the products of conception and affected individuals indicates that most aneuploidies arise in maternal meiosis, particularly meiosis I, analysis of polar bodies and oocytes has now shown a significant incidence in both meiotic divisions. For these reasons, therefore, both first and second polar bodies are now biopsied from zygotes following IVF or ICSI for either chromosomal or single gene defect analysis. Verlinsky et al in Chicago pioneered and continue to advocate this approach for PGD and have used it effectively both for aneuploidy screening in advanced maternal age and for single gene defects involving maternal mutations.11,12,13,14 After mechanical zona dissection to form a flap a narrow micropipette is introduced into the perivitelline space, and the two polar bodies are separately biopsied.7 The polar bodies are distinguished on the basis of morphology; the first polar body tends to have crinkled surface and may fragment, the second polar body is generally smooth and may have a visible interphase nucleus under interference contrast. In addition to the obvious advantages of not damaging the embryo (the polar bodies do not contribute structurally to the developing embryo), and allowing a maximum time for genetic analysis, at a technical level, analysis of both polar bodies allows detection of allele dropout (ADO). ADO is the random amplification failure of one parental allele after PCR from single cells and is therefore

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one source of errors in PGD. However, as polar body biopsy is limited to analysis of maternal defects, the Chicago group advocates a combination of this and/or later blastomere biopsy depending on the type of defect to be diagnosed.

CLEAVAGE STAGE BIOPSY The first PGD cycles were carried out in late 1989 in a series of couples at risk of X-linked disease.15 The sex of each embryo was identified by biopsying single cells from cleavage stage embryos by PCR amplification of a Y-linked sequence and transferring female embryos which could carry the defect but should not be affected. In these cases, acid Tyrode’s was used to drill relatively large holes (20–30um) in the zona and a second micropipette, filled with normal medium and held in a double holder alongside the acid Tyrode’s pipette, used to aspirate single cells.16,17,18 Chen et al19,20 have shown that it is possible to use a single micropipette for both drilling and aspiration, but care is needed to prevent overexposure to acid. The pregnancy rate in this first series of predominantly fertile patients was impressive (Table 14.2) and stimulated research into assisted hatching using the same approach (Cohen, personal communication). Predictably, these rates have not been sustained. Pregnancy rates in the largest series analysed in detail to date, mostly following cleavage stage biopsy, are only 18% per oocyte retrieval and 22% per embryo transfer.21 The reasons are many fold but are not so surprising considering a proportion of embryos cannot be transferred because they are diagnosed as affected, and in most cases the number of embryo transferred is limited to a maximum of two. Biopsy at cleavage stages is based on the principle that at these stages cells, or blastomeres, remain totipotent and that the mammalian embryo, in contrast to those of lower vertebrates and invertebrates, shows a remarkable ability to regulate for the loss of some blastomeres. Even mouse embryos, for example, in which one 2 cell blastomere is removed or damaged, can develop normally into normal sized offspring.22 However, as the cell mass is reduced, first implantation and then fetal development is reduced.23 It is important therefore to minimize the cellular mass removed at biopsy. 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. For this reason, cleavage stage embryos are biopsied early in the morning on day 3 after insemination at about the 6 to 10 cell stage and cells identified as having completed the third cleavage division (on the basis of their size) selected for biopsy. Theoretically, therefore, each blastomere removes only 1/8 of the cellular mass of the embryo. As zona drilling for assisted hatching may be

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beneficial, it is also possible that this offsets to some extent the adverse effects of lowering the cell mass of the embryo.

Table 14.2. Outcome of preimplantation genetic diagnosis (PGD) in the first series of couples at risk of X-linked disease.15 • 5/8 (62.5%) women treated became pregnant • 10/22 (45%) embryos transferred implanted • 7/22 (32%) developed to the fetal heart stage • 6/7 confirmed to be female by CVS • One misdiagnosed male terminated at 11 weeks • Six females born (one stillborn) CVS=Chorionic villus sampling To show that cleavage stage biopsy did not compromise the preimplantation development of the biopsied embryos prior to clinical application, one or two blastomeres were biopsied from human embryos on day 3 and cultured to the blastocyst stage 16. Although there was some evidence of depressed metabolism in the 24 hours after biopsy, the same proportion of embryos developed to the blastocyst stage and cell numbers in the trophectoderm (TE) and inner cell mass (ICM) of blastocysts were in proportion to the cellular mass removed at biopsy, i.e. the cleavage rate of the biopsied embryos was not affected by the micromanipulative procedure. This is exactly what would be expected from similar work in the mouse in which regulation for removal of cells at cleavage stages only occurs after implantation.24 It is also important to consider any effects on postimplantation development. Clearly, any increase in fetal malformations or congenital abnormalities would be unacceptable. However, studies of pregnancies and children born after in vitro fertilization (IVF) in infertile couples had not identified a significant increase in perinatal defects, despite the transfer in many cases of partially fragmented or degenerate embryos. Furthermore, cryopreservation of embryos, which often damages a proportion of cells, had been widely practised clinically with no reports of problems. Recently, the European Society of Human Reproduction and Embryology (ESHRE) has formed a PGD consortium with the aim of monitoring PGD in clinical practise and, importantly, following up pregnancies and children. Relative to mainstream IVF, the number of PGD cycles and pregnancies is low. However, the data collected so far does not suggest there is a significant increase in perinatal abnormalities and almost all of the cycles involved cleavage stage biopsy.21 Cleavage stage biopsy remains the most widely practised form of embryo biopsy although there have been a number of modifications and improvements (see appendix for typical clinical protocol). When PDG started, culture media were not as optimal as the newer generation of

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media that 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 calcium and magnesium free medium to reverse the initial calcium dependent adhesion.25 Another development is the use of non-contact lasers for zona drilling (see below). Finally, some groups advocate altering 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 development).26 This then allows more time for genetic analysis of the biopsied cells. As an alternative, many other groups are using newer sequential media and delaying transfer until day 4 or 5, which similarly allows additional time for analysis and may also improve pregnancy rates because developing embryos that have undergone further cleavage divisions can be selected for transfer. The consistency of cleavage stage biopsy has now been established in many centres and in the latest ESHRE PGD consortium report the efficiency of embryo biopsy ranged between 94.5% and 97.5% depending on the diagnostic class with an average of 96.5% success rate in over 5000 cleavage stage embryos in PGD cycles for chromosomal and single gene defect analysis excluding aneuploidy screening and structural chromosomal abnormalities.21 This is also shown in results from a small series of consecutive cases of PGD for X-linked disease (Table 14.3). In these cases, the aim was to remove a single blastomere with a single visible interphase nucleus since this is all that is required for accurate identification of sex by multicolour fluorescence in situ hybridization (FISH) analysis with chromosome specific probes for X, Y, and 18.27 In 80% of embryos, biopsy of a single nucleated blastomere was successful, but in the remainder one or two further blastomeres had to be biopsied to ensure that at least one would provide a normal nucleus for analysis. Only in two out of 50 embryos did biopsy fail completely because the embryo was too damaged to be considered for transfer. 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

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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. it to remove specific blastomeres is essential to attain the efficiencies required for clinical effectiveness. The reasons for this are that, firstly, 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 would not be visible and are likely to get lost.28 Secondly, postzygotic chromosomal mosaicism arising during cleavage is known to be associated with nuclear abnormalities.29 The exception is binucleate blastomeres, in which there are two normal sized nuclei. In most cases, these are generated through failure of cytokinesis and both nuclei contain the normal diploid chromosomal complement for that embryo.30 However, even with careful selection, diagnostic efficiency is not 100% and aneuploid results are not uncommon (Table 14.3b). A full discussion of the impact of chromosomal mosaicism on the accuracy of PGD is beyond the scope of this review but some groups advocate biopsy of two blastomeres even for identification of sex for this reason. Biopsy

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Table 14.3. Efficiency of cleavage stage biopsy in clinical practise: Summary of biopsy outcome in a series of seven cycles in couples at risk of X-linked disease, in which the aim was to biopsy a single blastomere with a single visible interphase nucleus.

(a) No of blastomeres biopsied 1 2 3 Failed (b) Normal FISH* result XX1818 XY1818

No of embryos biopsied n=so 40 (80%) 6 (12%) 2 (4%) 2 (4%)

No Overall diagnostic diagnosed efficiency 37 (92.5%) 5 (83%) 43/50 (86%) 1 (50%)

No of embryos Abnormal FISH result 17 (40%) X1818 12 (28%) Tetraploidy Trisomy 18 Other aneuploidies *Fluorescent in situ hybridization.

No of embryos 3 (7%) 3 (7%) 2 (5%) 6 (13%)

Table 14.4. Efficiency of cleavage stage biopsy in clinical practise: Summary of biopsy outcome in a series of 25 cycles in couples at risk of cystic fibrosis (deltaF508) in which the aim was to biopsy two blastomeres to avoid errors resulting from a low level of contamination.17 No of blastomeres biopsied No of embryos biopsied n=214 1 80 (37%) 2 115 (54%) 3 15 (7%) Failed 4 (2%) 20% cell lysis of two nucleated blastomeres is only possible in good quality embryos at a sufficiently advanced stage, and the efficiency is lower so that the end result is a mixture of embryos with one or two blastomeres for analysis (Table 14.4).

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TROPHECTODERM BIOPSY The primary advantage of biopsy of TE cells, which form the spherical outer epithelial monolayer of the blastocyst, is that theoretically, multiple cells can be biopsied without affecting the ICM from which the fetus is derived. In the mouse, TE biopsy is easily achieved by PZD followed by a period in culture during which the expansion of the blastocoel cavity forces the TE to herniate out of the slit.31 The herniating TE vesicle can then be excised on a bed of agarose by using a needle (which can be handheld) 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 the incubation. A similar approach has been used successfully to biopsy human blastocysts.32,33 More recently, non-contact lasers have been used not only to create an opening to assist hatching but also to excise the herniating trophectoderm.34 Furthermore, pregnancy rates after culture in sequential media and blastocyst transfer can be high.35 The main obstacles to clinical application remain heterogeneity among embryos in the rate of development and quality of blastocysts in vitro, both of which contribute to comparatively low biopsy efficiencies, secondly, concern that implantation will be affected by reducing TE cell numbers and thirdly, uncertainty about whether embryos that reach the blastocyst stage include all those that would have developed successfully after transfer at cleavage stages. The latter is an important principle. If this is the case and because only about 50% of normally fertilized embryos reach the blastocyst stage, even under the best available conditions in vitro, delaying biopsy and analysis until this stage should not affect overall pregnancy rates although the chances of finding unaffected embryos are halved. However, it is unlikely that prolonged culture could improve the implantation and developmental potential of an embryo (unless through altered synchronization with the uterine cycle of receptivity) and it remains more likely that implantation and development rates are reduced per embryo. Nevertheless, 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 would be one approach.36

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NEW DEVELOPMENTS AND ALTERNATIVE STRATEGIES A major new development is the use of non-contact infrared lasers for zona drilling.37 With this approach, localized heating causes denaturation of the zona proteins in a cylindrical spot where the laser beam is focussed, and the size of the hole created is controlled by adjusting the length of the laser pulse. The advantages of the laser are that it is quick, controlled, and consistent. Many clinics are now using this equipment for assisted hatching as well as PGD,38 and there have been several studies showing that there is no effect on development to the blastocyst stage or pregnancy rates in animal and human studies.39,40,41 However, our recent studies on the immediate effects at the blastomere level in which we used the mouse as a model have shown that it can cause damage if used inappropriately.42 Certainly, if the laser beam is fired in an area in direct contact with a blastomere its viability is always compromised. As expected from basic physical principles, 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 as far away as possible and also to use minimum pulse lengths to restrict any damaging effects. Blastomere viability was also affected in a minority of cases by acid Tyrode’s, which may explain the high frequency with which blastomeres biopsied using this approach are lysed (Table 14.4). As an alternative to blastocyst biopsy, it is possible to coculture biopsied blastomeres with the biopsied embryo.43 Over a period of three days, division and development of the biopsy significantly mirrors the behaviour 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 (Fig 14.2). 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 8 cell stage blastomeres and 9.1±1.1 (n=11) cells where two blastomeres were biopsied and encouraged to form a single morula. The advantages of this approach are that the behaviour of the biopsy in vitro could predict the potential for the biopsied embryo,44 and avoid the difficulties and damage of biopsy at the blastocyst stage itself. Towards this end, there is now preliminary evidence, using sequential media, that cleavage stage biopsy and culture of the biopsied embryo to the blastocyst stage does not seriously affect implantation rates.45 In a series of five cases, about 48% of embryos developed to the blastocyst stage following biopsy and screening for aneuploidy and six out of nine (66%) blastocysts implanted. This compares favourably with an implantation rate of 48% per embryo in >300 routine IVF cases.

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Another challenge for the future will be to develop effective methods for cryopreservation of biopsied embryos. To date, 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 the protection to ice crystals in the medium provided by an intact zona pellucida.46,47 With the high rate of multiple pregnancies reported after PGD, it is imperative to develop effective methods of cryopreservation that would allow storage of unaffected embryos for later transfer so that the numbers transferred could be limited to two or even single embryo transfers.

Fig 14.2 Human embryo biopsied on day 3 and the biopsied embryo and isolated blastomere co-cultured for 3 days. Note that the biopsied embryo has hatched from the zona pellucida on the left which still contains some degenerating cellular debris and on the right a small trophectoderm vesicle which developed from the biopsied blastomere (from Geber et al., 1995 with permission).43

REFERENCES 1 Li A, Gyllenstein UB, Cui X, Saiki RK, Erlich HA, Arnheim N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature (1988); 335:414–9.

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2 Coutelle C, Williams C, Handyside A, Hardy K, Winston R, Williamson R. Genetic analysis of DNA from single human oocytes: a model for preimplantation diagnosis of cystic fibrosis. BMJ (1989); 299, 22–4. 3 Holding C, Monk M. Diagnosis of beta-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 Cohen J, Malter H, Wright G, Kort H, Massey J, Mitchell D. Partial zona dissection of human oocytes when failure of zona pellucida penetration is anticipated. Hum Reprod (1989); 4:435–42. 7 Cieslak J, Ivakhnenko V, Wolf G, Sheleg S, Verlinsky Y. Threedimensional partial zona dissection for preimplantation genetic diagnosis and assisted hatching. Fertil Steril (1999); 71:308–13. 8 Gordon JW, Talansky BE. Assisted fertilization by zona drilling: a mouse model for correction of oligospermia. J Exp Zool (1986); 239:347–54. 9 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. 10 Verlinsky Y, Ginsberg N, Lifchez A, Valle, Moise J, Strom CM. Analysis of the first polar body: preconception genetic diagnosis. Hum Reprod (1990); 5:826–9. 11 Verlinsky Y, Cieslak J, Freidine M, et al. Pregnancies following preconception diagnosis of common aneuploidies by fluorescent in-situ hybridization. Hum Reprod (1995); 10:1923–7. 12 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. 13 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. 14 Rechitsky S, Strom C, Verlinsky O, et al. Accuracy of preimplantation diagnosis of single-gene disorders by polar body analysis of oocytes. J Assist Reprod Genet (1999); 16:192–8. 15 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. 16 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. J Reprod Fert Abstr Series (1990); 5 (abst). 17 Ao A, Handyside AH. Cleavage stage human embryo biopsy. Hum Reprod Update (1995); 1:3.

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18 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. 19 Chen SU, Ho HN. Chen HF, et al. A simplified technique for embryo biopsy: use of the same micropipette for zona drilling and blastomere aspiration. J Assist Reprod Genet (1997); 14:157–61. 20 Chen SU, Chao KH, Wu MY, Chen CD, Ho HN, Yang YS. The simplified two-pipette technique is more efficient than the conventional three-pipette method for blastomere biopsy in human embryos. Fertil Steril (1998); 69:569–75. 21 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); 14:3138–48. 22 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. 23 Rossant J. Postimplantation development of blastomeres isolated from 4- and 8-cell mouse eggs. J Embryol Exp Morphol (1976); 36:283–90. 24 Lewis NE, Rossant J. Mechanism of size regulation in mouse embryo aggregates. J Embryol Exp Morphol (1982); 72:169–81. 25 Dumoulin JC, Bras M, Coonen E, Dreesen J, Geraedts JP, Evers JL. 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. 26 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. 27 Kuo HC, Ogilvie CM, Handyside AH. Chromosomal mosaicism in cleavage-stage human embryos and the accuracy of single-cell genetic analysis. J Assist Reprod Genet (1998); 15:276–80. 28 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. 29 Munne S, Cohen J. Unsuitability of multinucleated human blastomeres for preimplantation genetic diagnosis. Hum Reprod (1993); 8:1120–5. 30 Hardy K, Winston RML, Handyside AH. Binucleate cells in human preimplantation embryos in vitro: failure of cytokinesis during early cleavage . J Reprod Fert Abstr Series (1990); 6:24–20 (abst). 31 Nijs M, Van Steirteghem A. Developmental potential of biopsied mouse blastocysts. J Exp Zool (1990); 256:232–6.

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32 Dokras A, Sargent IL, Ross C, Gardner RL, Barlow DH. Trophectoderm biopsy in human blastocysts. Hum Reprod (1990); 5:821–5. 33 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. 34 Veiga A, Sandalinas M, Benkhalifa M, et al. Laser blastocyst biopsy for preimplantation diagnosis in the human. Zygote (1997); 5:351–4. 35 Schoolcraft WB, Gardner DK, Lane M, Schlenker T, Hamilton F, Meldrum DR. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril (1999); 72:604–9. 36 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. 37 Handyside AH, Chatzimeletiou K, Picton H. Unpublished observations. 38 Boada M, Carrera M, De La Iglesia C, Sandalinas M, Barri PN, Veiga A. 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. 39 Montag M, Van der Yen H. Laser-assisted hatching in assisted reproduction. Croat Med J (1999); 40:398–403. 40 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. 41 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. 42 Chatzimeletiou K, Picton H, Handyside AH. Unpublished observations. 43 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. 44 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. 45 Stevens J, Schoolcraft WB, Schlenker T, Wagley L, Munne S, Gardner DK. Day 3 blastomere biopsy does not affect subsequent blastocyst development or implantation rate. Fertil Steril (2000); 74: Suppl. 1:4246. 46 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.

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47 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.

APPENDIX

TYPICAL CLINICAL PROTOCOL FOR CLEAVAGE STAGE EMBRYO BIOPSY DAY 1 (1 DAY AFTER EGG COLLECTION) 1 Set up culture dishes (one for each normally fertilized embryo): Label a 4 well dish (Nunclon) with the patient’s name and embryo number on the base of the dish and on the front panel. Put 0.5 ml of blastocyst culture medium in each well and cover with a monolayer of washed Squibb oil. Place the dishes in the incubator to equilibrate overnight.

DAY 2 (2 DAYS AFTER EGG COLLECTION) 2 After scoring the embryos, transfer each embryo into the blastocyst culture medium in well 1 of the appropriately labelled 4 well dish, wash and transfer to well 2 and transfer to the incubator for overnight culture. (The timing of the switch between cleavage stage and blastocyst culture media may differ depending on the medium used.) 3 In the warming oven, place 10ml of HEPES buffered biopsy medium (Ca2+/Mg2+-free) and Falcon dishes (1006) for the biopsy. In the incubator, place enough washed oil for the biopsy procedure (allow 4ml per embryo). DAY 3 (DAY OF EMBRYO BIOPSY)

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4 Half an hour before the biopsy: (1) set up a biopsy dish (1006 Falcon) for each embryo and label it with the patient’s name and embryo number. Take a Gilson pipette set at 10µµl and a sterile yellow tip and flush the tip (×10) with the HEPES buffered biopsy medium. Pipette three drops of HEPES buffered biopsy medium and one drop of AT as shown in the diagram—it is important that the dish is oriented as shown in relation to the “bumps” on the outside of the dish.

Immediately cover the dish with 4ml of washed and pre-equilibriated Squibb oil to avoid evaporation and put the prepared dishes in the warming oven until required; (2) Set up a 4 well dish for transferring the embryos into biopsy medium with 0.5ml of HEPES buffered biopsy medium in each of the wells and cover with oil and place in the warming oven. 5 After 15 minutes before each biopsy, take the appropriately labelled biopsy dish and the 4 well transfer dish from the warming oven, and carefully wash successive embryos through each well of HEPES buffered medium transferring minimal medium between wells. Leave the embryo in each well for at least a minute. (It is essential to completely remove the divalent cations from the culture medium to promote the reversal of any compaction). Then place the embryo into the middle of the three droplets in the biopsy dish (the two other droplets are spare in case of difficulties during biopsy) and take the dish through to the downflow and hand the dish over to the person performing embryo biopsy. 6 At the end of the biopsy, transfer the embryo into well 3 of the 4 well culture dish. (This is a washing stage to remove the HEPES buffered biopsy medium). Finally, transfer the embryo with minimal medium to well 4 and return to culture. 7 Return the biopsy dish with the isolated blastomeres to the downflow for sample preparation.

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8 Repeat until all the embryos have been biopsied. 9 When the PGD is complete assess the morphology of each of the embryos and count the number of cells as accurately as possible to get an indication of division post biopsy. 10 In consultation with the other members of the PGD team and finally with the couple themselves, select a maximum of two unaffected embryos with the best morphology for transfer.

15 Analysis of fertilization Lynette Scott

INTRODUCTION The efficiency of in vitro fertilization (IVF) and embryo transfer (ET) in the human is low, with fewer than 30% of embryos that are transferred ever realizing full developmental potential.1 Since the implantation rates have remained relatively low there has been a practice of replacing multiple embryos in order to increase the likelihood of a pregnancy.2–6 This has lead to an unacceptable level of high order multiple pregnancies. To overcome this problem some countries have mandated the number of embryos that can be replaced, in some instances limiting this number to two in certain age groups. Although this will reduce the level of multiple pregnancies it can also reduce pregnancy rate since there are data showing that the number of embryos replaced affects pregnancy rates. 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 patients. Initially, most 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 al demonstrated an increased implantation rate.2 This system has been widely adopted, with a concomitant increase in implantation rates. For both day 2 and day 3 embryo transfers, embryo selection is based on key morphological features of cleaving embryos that have been previously correlated with increased implantation.3–5 With the introduction of extended culture and blastocyst transfer the pregnancy and implantation rates have increased,6 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 zygotes placed in extended culture reach the blastocyst stage and of these 30–50% implant. Further, in some countries, allowing development in vitro as a means of choosing embryos with the most potential is not feasible, since their state and government policies preclude the destruction of any fertilized embryos.7 In order to reduce the numbers of embryos replaced with constraints such

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as these, embryos at the first stages of development, or even oocytes, would need to be selected for transfer. The morphology of early human zygotes, at the 1 cell stage, has been used as a means of embryo selection, prospectively for day 1 pronuclear transfers8 and retrospectively for day 3 transfers.9 Scott and Smith8 based their grading on empirical data correlated with pregnancy and on previously published observations of zygote morphology.10,11 The zygote grading system was a combination of pronuclear size, nucleoli number and distribution and cytoplasmic appearance. Additionally, the changes in the zygote cytoplasm and the progression to first cleavage division were considered. Testarik et al reported a single observation grading system in which the nucleoli size, number and distribution were utilized.9 Embryos were replaced on the third day of culture, at which point embryo morphology was used as primary selection criteria. 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. The advantages of the Testarik zygote grading system compared with the Scott system are the single-observation and fewer parameters for consideration. Ludwig et al7 used a pronuclear grading system based on a combination of the Scott and Testarik systems and demonstrated no reduction in pregnancy rate using only two embryos rather than three 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. Recently, it has been shown that there is a correlation between the zygote morphology and ability to grow to the blastocyst stage in vitro (Scott et al, unpublished data; submitted). Since day 5 blastocyst transfers result in higher implantation rates and zygote morphology is positively correlated with blastocyst formation, scoring of zygotes 16–18 hours after insemination could be used proactively for selecting candidates for extended culture and as an initial screen for blastocyst selection. This would further reduce the incidence of high order multiple pregnancies without a reduction of pregnancy rates and allow early embryo selection in countries where “in vitro dropout” is not allowed.

FERTILIZATION 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

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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 the resumption of the first meiotic division and progression to metaphase II. Those of cytoplasmic maturation include all the 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 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 feeding will not occur correctly. 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.12–14 The ovulated or retrieved oocyte is activated when the sperm enters, either by normal fertilization or artificially with intracytoplasmic sperm injection (ICSI). Activation is a complex series of events that result in the release of the cortical granules; activation of membrane bound ATPase, resumption of meiosis, and, finally, the formation of the male and female pronuclei with the 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. In human fertilization the centriole, which is the microtubule organizing center, is derived from the sperm.15,16 These structures are responsible for bringing the male and female pronuclei together. Within the nuclei are structures known as the nucleoli which form at areas known as the “nucleolus organizing regions.” The nucleolus organizing regions are located on the chromosomes in the nucleus where the genes coding for ribosomal RNA are located. They are the sites where pre-rRNA is synthesized. These sites are very active on the chromosomes of the oocyte and sperm pronuclei. Nucleoli are comprised of a chromatin and a ribosomal nuclear protein portion. The number and location of nucleolus organizing regions are species specific but nucleoli differ according to the cell type, the activity of the cell and the stage of development of the cell in any one species.17,18 Nucleoli are first seen in oocytes in antral follicles where they are well defined and synthesize rRNA. This synthesis is essential for meiotic competence.19,20 During oocyte maturation leading to ovulation RNA synthesis decreases and the nucleoli become small and scattered.21 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.22,23 Ovulated oocytes and early embryos rely exclusively on maternal RNA that was synthesized preovulation. However, as the nucleoli in the decondensing sperm nucleus and the female nucleus form, new embryonic rRNA can be synthesized and can take over, which in the human occurs at about the 4 cell stage.24

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Nucleoli tend to fuse, which is cell cycle related, with more nucleoli being present at the beginning of the GI phase.12,22,23 This fusion continues until, at the S phase, only one or two nucleoli per nucleus are left. During a mitotic cycle, daughter cells display synchrony in their nuclei content and fusion. There is also a correlation between ploidy and nucleoli content. Asynchrony in daughter cells is a result of aberrant chromosomal function. Zygote scoring involves the careful analysis of the pronuclei and the nucleoli within the nuclei in a single observation 16–18 hours after fertilization. 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 lab, a single observation can aid in embryo selection for transfer and decisions of day of embryo transfer.

ZYGOTE SCORING Pronuclei: 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 post fertilization (Fig 15.1b) could indicate some disruption in aster and microtubule formation, which will lead to abnormal development.15,16 These embryos rarely progress well or form blastocysts. The pronuclei should be approximately the same size (Fig 15.1a). Zygotes that have pronuclei of very different sizes (Fig 15.1c) have an 87% incidence of chromosomal abnormalities.25,26 Likewise, zygotes that have very small nuclei are generally abnormal, displaying poor and retarded development (Fig 15.1d). The position of the pronuclei within the zygote 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 placed centrally in the oocyte or just into the hemisphere containing the first polar body.

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.

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Fig 15.2 Cartoon representation of Z-scores. The Zscore describes the number, size and position of the nucleoli and the equality between the nuclei for these characteristics. Z-1 zygotes have equal numbers of nucleoli between 3–7 that are aligned at the pronuclear junctions. Z-2 zygotes have equality in size and number between the nuclei, but the nucleoli have not yet aligned at the pronuclear junction. Z-3 zygotes are characterized by inequality between the nuclei; unequal sized nucleoli, unequal numbers of nucleoli, or unequal alignment at the pronuclear junction. Z-4 zygotes are grossly abnormal and present with unequal sized nuclei, nuclei that have not aligned, small, and misplaced nuclei. Therefore, zygotes with non-aligned pronuclei, pronuclei in the hemisphere without the second polar body and pronuclei of distinctly different sizes or very small pronuclei are probably abnormal. They

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should not be considered for transfer. Thus, only zygotes with normal sized pronuclei, aligned and in the hemisphere of the second polar body are used for the second phase of zygote scoring. Nucleoli: The nucleoli can be visualized within the nuclei on any inverted microscope with contrast optics (Hoffman or Nomaski). They can also be seen on high power, good binocular microscopes that have the ability to tilt the mirror and throw shadows through the zygote. This technique can be used for initial sorting but contrast optics is recommended for final scoring. The size, number and distribution of the nucleoli form the central aspect of zygote scoring in which zygotes are described as Z-1, Z-2, Z-3 or Z-4 depending on the size, number, and distribution of nucleoli within the nuclei (Fig 15.2). The number of nucleoli should ideally be between 3 and 7 per nucleus (Fig 15.3a,b). Zygotes with many small pinpoint nucleoli are probably delayed in nuclear events and the formation of the nucleolar organizing centers (Fig 15.4a). These zygotes are slow in development and result in suboptimal embryos with only 10–15% blastocyst formation (unpublished data). Zygotes 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. The alignment of the nucleoli at the pronuclear junction is a desired feature (Fig 15.1a; 15.2, and 15.3a–c). This is related to the metabolic status of the embryo and for the ability of the nuclei to fuse and form the unique embryonic genome.10,27,28 There is a pH gradient between the two sets of nucleoli which is lower than elsewhere in the zygote and which is an important factor in the event of nuclei fusion. This alignment is also seen in mouse zygotes, where it is speculated that this feature is indicative of intact DNA, normal metabolic characteristics and appropriate assembly of microtubules.29 Failure to align or

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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-f) Z2: equal numbers of equal sized nucleoli still scattered in the nuclei. the inequality of nuclei could alter this pH gradient leading to abnormal development when the male and female genomes combine. Therefore, the desired zygote is one that has equal numbers of even sized nucleoli with between three and about seven per pronucleus that are beginning to or have aligned at the pronuclear junction (Fig 15.1a; 15.3a,b,c). These have been shown to give optimal development and implantation at the 1 cell,8 cleaving day 3 cell stage,7,9 and blastocysts stage (Scott, unpublished data; submitted). Those in which there is

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alignment at the pronuclear junction are designated Z1 (Fig 15.2), and those whose nucleoli are still scattered are designated Z2 (Fig 15.2; Fig 15.3d,e,f). Zygotes with unequal numbers and or unequal sizes of nucleoli are designated Z3 (Fig 15.4b-f). These zygotes have been shown to result in lower grade day 3 embryos, lower in vitro blastocyst formation and lower implantation rates when using day 5 transfers (Scott et al, unpublished data). Inequality in the state of coalesces of nucleoli in the two nuclei and the progression to the S phase will lead to gross abnormalities in development. This is directly reflected in the decreased development recorded for these zygotes. A second level of assessment of the zygotes that can be used is the appearance of the cytoplasm. The presence of a “halo”8 is associated with the development of high quality embryos on day 3 and day 5 (Fig 15.1a; 15.3a,b,c). The halo has not shown to be associated with a specific zygote morphology but has been consistently linked to cohorts of zygotes that developed to good quality blastocysts (unpublished data). The clearing of the cytoplasm on the zygote periphery has been termed cytoplasmic streaming.30 Mouse31 and hamster32 1 cell fertilized embryos have differential mitochondrial distribution, which

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Fig 15.4 Z3 Zygotes: a) many small pin-point nucleoli. b-e) Inequality in size or number or alignment of nucleoli between the 2 nuclei. f) Many small scattered pin-point nucleoli. is related to the cell cycle. The mouse zygote mitochondria migrate to the periphery of the cell, whereas in hamster zygotes 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 zygotes seems to mimic that seen in hamster zygotes with an aggregation in the center of the cell.8,30 It seems reasonable to assume that the mitochondria are aggregating at the site of highest metabolic activity, the pronuclei. Zygotes in which this movement is not so pronounced or it does not occur

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could be metabolically compromised, leading to delayed and poor development. 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 zygote and embryo.33,34 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 only occur after the events of fertilization. An increase in fertilization rates, embryo morphology34 and implantation rates33 of embryos resulting from oocytes with polar bodies that were round or oval with no fragmentation or rough cell membranes was shown. It is assumed that oocytes that display polar body abnormalities have some form of cytoplasmic or chromosomal abnormalities. Polar body abnormalities include fragmentation (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. How the first and second polar body morphology at 16–18 hours after insemination affects development is not known. Further, there are no data correlating the polar body morphology with zygote score. If there were, this could be yet another level of screening at a very early stage of development that again is directly related to the oocyte that could help select embryos for transfer and limit the number used. It has been observed that the small acellular debris or inclusions in the perivitaline space, attached to the zona pellucida are correlated with high estrogen levels. However, this was not related to any decrease in pregnancy. There have been no studies in which this phenomenon is correlated with either polar body morphology or with zygote score.

APPLICATION OF ZYGOTE SCORING Zygote scoring involves grading zygotes at 16–18 hours postinsemination, in a one-time scoring, and designating them as Z1–4 (Scott et al, unpublished data; as depicted in Fig 15.2. Zygotes of a like score can be cultured in groups or separately, as per laboratory protocol. The spread of Z-scores is not affected by age, infertility type, route of sperm entry, 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 of Z1 and 2 whereas others will have predominately Z3. Overall, less than 10% of zygotes are Z4. The Z4s are generally grossly abnormal but can develop into embryos with good morphology and into good grade blastocysts. However, since Z-4 zygotes are considered abnormal they should not be used for transfer or cryopreservation. Fig 15.5 shows the spread of Z scores for a large cohort of zygotes over an 18 month period in a group of patients ranging in age

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from 22 to 43 years, with varying infertility presentations. Overall, no one type is predominant. Once zygotes are scored and sorted they can be followed to day 2, 3, or 5 where a secondary scoring system, based on key morphological features, can be used. In this way, the embryos most likely to implant can be used for transfer. Fig 15.6 shows the day 3 embryo grading of a large cohort of Z-scored embryos. This demonstrates that, although most high grade embryos, with little fragmentation and adequate cell number, are from Z1 and Z2 zygotes, there is a wide spread of morphologies for all Z-scores. This underscores how important it is to screen embryos as early as possible, and repeatedly as they develop, in order to select only those that have the highest potential of implanting. Fig 15.7 shows the relation between zygote morphology and development to the blastocyst stage on day 5 of culture and for total blastocyst development (day 5+6). Again, although the majority of good grade blastocysts on day 5 originate from Z1 zygotes, there are still a number of blastocysts that are morphologically high grade arising from lower scoring zygotes. When these blastocysts are transferred they do not give the same results that the Z1 or Z2 blastocysts do (Scott, unpublished data). When a continuous grading system is used for embryo selection, based initially and primarily on zygote score the implantation and pregnancy rates for both day 3 and day 5 embryo transfers can be significantly increased (Scott, unpublished data). This is very important for limiting the number of embryos used to achieve high pregnancy rates. By applying zygote scoring in conjunction with day 3 or 5 morphology only one or two

Fig 15.5 Distribution of Z-scores for 4318 zygotes.

Fig 15.6 The distribution of day 3 embryo morphologies of the 4 Z-scores for a large cohort of embryos over a 12 month period.

Fig 15.7

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The percent of blastocyst development after 96 hours of culture (day 5) and 120 hours of culture (day 5+6) for each of the Zscores over a two year time period for all infertility and age presentations. embryos per transfer need be used, thus limiting the potential of high order multiple pregnancies, a highly desired outcome in human IVP-ET. Thus, the application of scoring fertilized embryos at the one 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 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 zygotes with a Z3 score should not be frozen since they have very little implantation potential. Zygote scoring can also be used to select patients who would benefit from a day 3 versus day 5 transfer based on how many good grade zygotes they have. For countries where there are strict laws regarding the culture and freezing of cleaving embryos, the application of zygote scoring could also reduce the numbers of embryos they both freeze and that they use for embryo transfer.

REFERENCES 1 Edwards RG, Beard HK. 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 post-insemination, to select non-arrested embryos, increases development to the fetal heart stage. Hum Reprod (1995); 10:177–82. 3 Pruissant F, Van Rysselberg M, Barlow P, Deweze J, Levoy F. Embryo scoring as a prognostic tool in IVF treatment. Hum Reprod (1987); 2:705–80. 4 Steer CV, 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) 7:117–9. 5 Tan SL, Royston P, Campbell S. Cumulative conception and live birth rates after in-vitro fertilization. Lancet (1992); 339:1390–4. 6 Gardner DK, Vella P, Lane M. Culture and transfer of blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril (1998); 69:84–8. 7 Ludwig M, Schopper B, Katalinic A, Strum R, Al-Hasani S, Deidrich K. Clinical use of a pronuclear stage score following intracytoplasmic

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sperm injection: impact on pregnancy rates under the conditions of the German embryo protection law. Hum Reprod (2000); 15:325–9. 8 Scott LA, Smith S. The successful use of pronuclear embryo transfers the day after oocyte retrieval. Hum Reprod (1998); 13:1003–13. 9 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. 10 Van Blerkom J. Occurrence and developmental consequences of aberrant cellular organization in meiotically mature human oocytes after exogenous ovarian hyperstimulation. J Electron Microscopy Technique (1990); 16:324–46. 11 Wright G, Wiker S, Elsner C, Kort H, Massey J, Mitchell D, Toledo A, Cohen J. Observations on the morphology of pronuclei and nucleoli in human zygotes and implications for cyropreservation. Hum Reprod (1990); 5:109–15. 12 Anderson LD, Hirshfield AN. An overview of follicular development in the ovary: From embryo to the fertilized ovum in vitro. Maryland Med J (1992); 41:614–20. 13 Eppig JJ, Schultz RM, O’Brien M, Chesnal F, Smith A. Relationship between the developmental programs controlling nuclear and cytoplasmic maturation of mouse oocytes. Develop Biol (1994); 164:1– 9. 14 Eppig JJ, O’Brien M, Wigglesworth K. Mammalian oocyte growth and development in vitro. Mol Reprod Devel (1996); 44:260–73. 15 Schatten G. The centrosome and its mode of inheritance: the reduction of the centrosome during gametogenesis and its restoration during fertilization. Develop Biol (1984); 165:299–335. 16 Sathananthan AH, Kola I, Trounson A, Ng SC, Bongso A. Centrioles in the beginning of human development. Proc Natl Acad Sci USA (1991); 88:4806–10. 17 Goessens G. Nucleolar structure. Int Rev Cytol (1984); 87:107–58. 18 King WA, Nair I, Chartrain KJ, Betteridge KJ, Guay P. Nucleolus organizer regions and nucleoli in preattachment bovine embryos . J Reprod Fert (1988); 82:87–95. 19 Motlik J, Kopecny V, Pivko J. RNA synthesis in pig follicular oocytes. Autoradiographic and cytochemical study. Biol Cell (1984); 50:229– 36. 20 Motlik J, Crozet N, Fulka J. Meiotic competence in vitro of pig oocytes isolated from early antral follicles. J Reprod Fertil (1984); 72:323–8. 21 Crozet N, Kanka J, Motlik J, Fulka J. Nucleolar fine structure and RNA synthesis in bovine oocytes from antral follicles. Gamete Res (1986); 14:65–73. 22 Tesarik J, Kopecny V. Development of human male pronucleus: Ultrastructure and Timing. Gamete Res (1989); 24:135–49.

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23 Tesarik J, Kopecny V. Assembly of the nuclear precursors bodies in human male pronuclei is correlated with an early RNA synthetic activity. Exper Cell Res (1990); 191:153–6. 24 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. 25 Munne S, Cohen J. Chromosome abnormalities in human embryos. Hum Reprod Updates (1998); 4:842–55. 26 Sadowy S, Tomkin G, Munne S. Impaired development of zygotes with uneven pronuclear size. Zygote (1988); 63:137–41. 27 Van Blerkom J, Runner MN. Mitochondrial reorganisation during resumption of arrested meiosis in the mouse oocyte. Am J Anatomy (1984); 171:335–55. 28 Van Blerkom J, Henry G. Oocyte dysmorphism and aneuploidy in meioticall-mature human oocytes after ovulation stimulation. Hum Reprod (1992); 7:379–90. 29 Dozortsev D, Coleman A, Nagy P, Diamond M, Ermilov A, Weier U, et al. Nucleoli in pronuclei-stage mouse embryo are represented by major satellite DNA of interconnecting chromosomes. Fertil Steril (2000); 73:366–71. 30 Payne D, Flaherty SP, Barry MF, Mathews CD. Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography. Hum Reprod (1997); 12:532–41. 31 Muggleton-Harris AL, Brown JJG. Cytoplasmic factors influence mitochondrial reorganization and resumption of cleavage during culture of early mouse embryos. Hum Reprod (1988); 3:1020–8. 32 Barnett DK, Kimura J, Bavister DD. Translocation of active mitochondria during hamster preimplantation embryo development studied by confocal laser scanning microscopy. Developmental Dynamics (1996); 205:64–72. 33 Ebner T, Moser M, Yaman C, Feichtinger O, Hartl O, Tews G. 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. 34 Ebner T, Yaman C, Moser M, Sommergruber M, Feichtinger O, Tews G. Prognostic value of first polar body morphology on fertilization rate and embryo quality in intracytoplasmic sperm injection. Hum Reprod (2000); 15:427–30.

16 Embryo culture David K Gardner, Michellen Lane

INTRODUCTION Through considerable research efforts by numerous laboratories around the world, there have been great improvements in embryo culture conditions. However, it would be complacent to propose that culture conditions were now optimized. When discussing embryo culture it is important to consider embryo culture systems rather than embryo culture media in isolation, as all aspects of the culture system need to be optimized in order for culture media to achieve their best results. This concept highlights the interactions that exist between the embryo and its physical surroundings. It is also important to acknowledge that it is not possible to make a good embryo from poor quality gametes. Rather, it is the role of the laboratory to maintain the inherent viability of the oocyte and sperm from which the embryo is derived. This means that the in vitro fertilization (IVF) laboratory is ultimately dependent on the quality of the stimulation by the physician and emphasizes the importance of the team in IVF. So to successfully culture embryos one has to take a more holistic look at patient management and the laboratory.

THE EMBRYO IN CULTURE Serendipitously the human embryo exhibits a considerable degree of plasticity enabling it to develop under a wide variety of culture conditions. However, this should be perceived as a testament of the resilience of the embryo and not our ability to culture it, as undoubtedly having to adapt to suboptimal culture conditions comes at the cost of lost viability.1 It is important that one discusses the generation of viable embryos, as it is evident that embryo development in culture per se does not necessarily equate to the development of a viable embryo. Viability is best defined as the ability of the embryo to implant successfully and give rise to a normal healthy baby. Subsequently, implantation rates (fetal heart rates, as opposed to fetal sacs) should always be reported and considered, as they best represent the efficacy of the IVF system. Today clinics are not only faced with a multitude of embryo culture media to choose from, but also with the relatively new decision of

Fig 16.1 Effect of sequential culture media on the development of F1 (C57/BL6×CBA/Ca) mouse zygotes in vitro. Zygotes were collected at 20 hours post human chorionic gonadotropin (hCG). All media were supplemented with BSA (2mg/ml). All embryos were transferred to fresh medium after 48 h of culture, with the exception of embryos in medium G1, where the embryos were transferred to either medium G1 or G2. To compensate for this, twice the number of embryos were originally cultured in medium G1, although only a designated 50% of these embryos were

used in the statistical analysis of the 44 to 52 h data set. (a) Embryo cell number after 44, 48 and 52 h of culture. Values are mean±sem. n=200 embryos/medium. Media: G1 (solid bar); HTF (open bar); Ham’s F-10 (hatched bar). Significantly different from other media; **, P<0.01. (b) Embryo development after 72 h of culture. n=150 embryos/medium. G1/G2; embryos cultured for 48 h in medium G1 and then transferred to medium G2. Blastocyst (solid bar), hatching blastocysts (as a percentage of total blastocysts; open bar). Like pairs are significantly different; a, c, d, P<0.05; b, P <0.01. (c) Embryo development after 92 h of culture. n=150 embryos/medium. G1/G2; embryos cultured for 48 h in medium G1 and then transferred to medium G2. Blastocyst (solid bar), hatching blastocysts (as a percentage of total blastocysts; open bar). Like pairs are significantly different; a, b, c, P<0.05. Significantly different from medium G1 and G1/G2; **, P<0.01. (d) Cell allocation in the blastocyst after 92 h of culture. n=150 embryos/medium. G1/G2; embryos cultured for 48 h in medium G1 and then transferred to medium G2. Trophectoderm (solid bars), inner cell mass (open bars). Significantly different from other media; *, P<0.05; **, P<0.01. (e) Viability of cultured blastocysts

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n=at least 60 blastocysts transferred per treatment. G1/G2; embryos cultured for 48 h in medium G1 and then transferred to medium G2. Implantation (solid bar), fetal development per implantation (open bar). Like pairs are significantly different; a, d, P<0.05; b, c, P<0.01. From Gardner and Lane with permission.10 whether to transfer at the cleavage stage or the blastocyst. For the most part there seems to be little consensus as to what media/day of transfer laboratories should choose. It is therefore the purpose of this chapter to discuss the types of media and culture systems currently available and to describe how they can be implemented in a clinical setting. The potential benefits of extended culture are subsequently discussed and the move to single blastocyst transfer proposed for a significant number of patients.

A. TYPES OF CULTURE MEDIA 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 therefore poses an intriguing problem in that it undergoes important changes in physiology during the preimplantation period. Subsequently, nutrient requirements by the embryo change with successive stages of development. Furthermore, the environment to which the embryo is exposed in the female reproductive tract differs as the embryo progresses through the oviduct to the uterus. The preimplantation human embryo is a highly dynamic entity. The pronucleate embryo, like the 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.2–4 Glucose consumption is limited by the early embryo, but it is still consumed.5 As development proceeds, and energy demands increase with cell multiplication and an increase in protein synthesis, there is a concomitant increase 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. The nutrients available within the human female reproductive tract mirror the changing 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 (0.32mM) and lactate (10.5mM), and a comparatively low

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concentration of glucose (0.5mM).6 In contrast, uterine fluid is characterized by comparatively low levels of pyruvate (0.1mM) and lactate (5.87mM), and a higher concentration of glucose (3.15mM). So it is for these reasons that the formulation of the culture media is changed during extended culture (should one decide to grow embryos past day 3). If one does not address the changing requirements of the embryo but enforces a single culture environment on the entire preimplantation period, then any resultant blastocysts can be significantly compromised. Optimal conditions for the early embryo do not support good blastocyst development and differentiation. Conversely, those conditions, that do support blastocyst development and differentiation are detrimental to the early embryo. This helps to explain the paradoxical findings of Bolton et al,7 who obtained a 40% blastocyst development of human embryos using Earle’s medium supplemented with pyruvate and serum, and yet only obtained a 7% implantation rate. Such a culture system may support the first couple of cleavage divisions, but is not able to support the development and differentiation of a viable blastocyst. More recently Huisman et al8 used a more complex culture medium for extended culture, a mixture of Earle’s balanced salts with Ham’s F-10. This approach produced blastocysts with an implantation potential of 26% in patients on their first IVF cycle. Although this figure is higher than that reported by Bolton et al,7 it is below that obtained when using sequential media, 32.4%, used for a non-selected group of patients.9 The effects of sequential media on mouse embryo development, differentiation, and viability are shown in Fig 16.1. Although the culture of embryos in medium G1 for the entire preimplantation period results in high levels of blastocyst formation and hatching, the development of the inner cell mass and subsequent viability are reduced. This is because the needs of the trophectoderm alone were addressed by medium G1 but those of the inner cell mass were not met. As a result high implantation rates were attained, but with low levels of fetal development. In contrast, when embryos were transferred to medium G2 after 48 hours, blastocyst formation and hatching were equivalent to that observed in medium G1, but significantly more inner cell mass cells developed culminating in high levels of fetal development.10 COMPOSITION OF CULTURE MEDIA There are several extensive treatise on the composition of embryo culture media,3,4,11–13 and it is beyond the scope of this chapter to discuss in detail the role of individual medium components. However, four specific components; glucose, amino acids, ethylenediaminetetraacetic acid (EDTA), and macromolecules need to be discussed as there is considerable confusion regarding their role in embryo culture media. Understanding their effects on embryo physiology should help clinics make a more informed decision regarding their choice of culture media. It

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is also important to consider that sequential media were specifically developed to work together and that mixing different pairs of media from different sequential systems is not advisable. GLUCOSE: TO BE OR NOT TO BE? There is a considerable literature on the effects of glucose in embryo culture, from which glucose has received the unfair tag of being the “villain of embryo culture.” Studies on the mouse,14 hamster,15 sheep,16 cattle,17–19 and human20–21 have all demonstrated that comparatively high levels of glucose (>1mM) in culture medium containing phosphate but lacking amino acids is responsible for the retardation or developmental arrest of cleavage stage embryos in culture. Significantly, this apparent toxicity of glucose is manifest only in the presence of phosphate. However, this inhibition of glucose in the presence of phosphate can be alleviated by the presence of amino acids,22–25 EDTA, and vitamins26 highlighting the interactions between medium components and the potential hazards of using simple salt solutions for embryo culture (see section on amino acids). In light of the potential toxicity of glucose in such media as Human Tubal Fluid (HTF),27 there has been a trend to remove it from embryo culture media.21,28–29 Such a course of action may work for the culture of the cleavage stage embryo, but the removal of glucose from medium used for blastocyst culture results in a significant reduction in subsequent fetal development, highlighting its intrinsic role in the development of a viable embryo.24,30 Indeed, the removal of glucose from embryo culture medium is really the alleviation of a culture-induced artifact, by the introduction of a second artifact as glucose is present in oviduct and uterine fluids,6 and oocytes and embryos do possess a specific carrier for it.31 Reasons for the inclusion of glucose in embryo culture medium are that not only is glucose required for energy production, but it is also essential for lipid/membrane biosynthesis, and nucleic acid and triacylglycerol biosynthesis.32–33 Glucose therefore becomes increasingly important once the embryonic genome is activated and biosynthetic levels increase. Furthermore, at the time of implantation the environment around the blastocyst is relatively anoxic.34–35 This means that glycolysis may well be the only means of generating energy before angiogenesis in the endometrium is complete.2 A source of this glucose for glycolysis could be the embryo’s own glycogen stores. Should the embryo have prematurely used such endogenous glucose stores during development because there was no glucose present in the culture medium, then these embryos will have a reduced ability to implant. Indeed, mouse blastocysts in culture which exhibit excessive lactate production from their endogenous energy reserves have a significantly reduced developmental potential after transfer. However, in all likelihood the oocyte and early zygote are not likely to be exposed to high glucose levels in vivo as the surrounding cumulus cells readily metabolize it to pyruvate and lactate.6

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In summary, glucose toxicity to the early embryo when using simple culture media lacking amino acids such as HTF and T6, can be alleviated by its removal, or the removal of phosphate. Alternatively, one can keep glucose and phosphate in the culture medium, but prevent any toxic effects by including amino acids and EDTA. Both approaches have worked clinically. However, when culturing from the 8-cell stage onwards glucose should be present in the medium. AMINO ACIDS It is certainly the case that human embryos can grow in the absence of amino acids. The real question, however, is how well do they grow 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,37–41 while both oocytes and embryos possess specific transport systems for amino acids42 to maintain an endogenous pool.43 As amino acids are readily taken up and metabolized by the embryo,43,44 the above information supports the notion that amino acids have a physiological role in the preand 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.38–40 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 non-essential amino acids.45 Studies on the embryos of several mammalian species, such as mouse,24,46–48 hamster,49–50 sheep,16,23 cow,17,51 and human,52 have all demonstrated that the inclusion of amino acids in the culture medium enhances embryo development to the blastocyst stage. Even a transient exposure (less than five minutes) of mouse zygotes to medium lacking amino acids impairs subsequent developmental potential.24 During this five minute period in a simple medium the embryo 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). It has been demonstrated that the preimplantation embryo exhibits a switch in amino acid requirements. Up to the 8 cell stage non-essential amino acids and glutamine increase cleavage rates.51,53 However, after compaction, nonessential amino acids and glutamine increase blastocoel formation and hatching, while essential amino acids stimulate cleavage rates and increase development of the inner cell mass in the blastocyst.54 Most importantly, amino acids have been reported to increase viability of cultured embryos from several species after transfer to recipients23,53,55,56

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as well as increasing embryo development in culture. In the mouse equivalent implantation rates to in vivo developed blastocysts were achieved when pronucleate embryos were cultured with non-essential amino acids to the 8 cell stage followed by culture with all 20 amino acids from the 8 cell stage to the blastocyst. The inclusion of amino acids in embryo culture medium warrants further comment, as amino acids are important regulators of several cellular function. Intracellular roles for amino acids include; chelators, osmolytes, pHi buffers, antioxidants, regulators of energy metabolism, as well as serving as biosynthetic precursors and energy substrates (reviewed by Gardner and Lane4,57). It is proposed that by filling such niches in the embryo’s physiology, amino acids facilitate normal cellular function. The work of Ho et al58 on gene expression in mouse embryos goes some way to confirm this 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—in a medium based on a simple salt solution— exhibited delayed gene expression. In light of this, it is advisable that media based on simple salt solutions, such as HTF27 and Earles’ salts,59 should either be supplemented with amino acids or their use discontinued. Certainly the use of such simple culture media supplemented with any form of serum can no longer be condoned in clinical IVF (see below). CHELATORS: EDTA The beneficial effects of the divalent cation EDTA in embryo culture media was first reported by Ambruzak et al,60 over 20 years ago. Subsequently Mehta and Kiessling61 demonstrated that EDTA at a concentration between 10µM and 150µM stimulated development of mouse zygotes through the 2 cell block to the blastocyst stage. In light of these studies, many media designed to support embryo development in culture contain EDTA, such as CZB,14 KSOM,62 G1,56,57 x-basal HTF,21 DM2,24 and SOF.63 However, the beneficial effects of EDTA are confined to the cleavage stage embryo.24,63,64 Culture of post-compacted embryos with EDTA significantly reduces both inner cell mass development and fetal development after transfer compared to culture without EDTA.24,63 EDTA was found to have its beneficial effects on the cleavage stage mouse embryo by preventing the abnormal activation of glycolysis, by inhibiting cytosolic kinases such as 3-phosphoglycerate kinase. However, in contrast to the cleavage stage embryo the inner cell mass uses glycolysis as its main energy producing pathway65 and therefore the presence of EDTA in the medium for development of the post-compaction stage embryo inhibits the development of the inner cell mass. Therefore media designed specifically for the development of the post-compaction stage embryo

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omit EDTA from their formulations such as G2,56,57 DM324 and Ham’s F10 (blastocyst medium66). MACROMOLECULES A commonly used protein source in human IVF and embryo culture is patient’s serum, added to the culture medium at a concentration of 5–20%. In some programs fetal cord serum is used in preference. The use of serum in embryo culture medium has several inherent drawbacks; the considerable expense and time required for its collection and processing (and screening of the fetal cord serum), and the risk of infection to both the embryo and laboratory staff. Furthermore, serum contains many components that are poorly characterized. Proteins in serum have macromolecules attached, such as hormones, vitamins, and fatty acids, as well as chelated metal ions and pyrogens.67 As the concentration of such macromolecules and other serum components varies between patients and even within the menstrual cycle, it makes any comparison between batches of medium which contain serum almost impossible. There are several reasons for the elimination of serum from mammalian embryo culture systems. From a physiological perspective the mammalian embryo is never exposed to serum in vivo. The fluids of the female reproductive tract are not simple serum transudates,68 but rather specialized environments for the development of the embryo.6 Serum can best be considered a pathological fluid. More significant, however, is the growing evidence that serum is detrimental to the developing mammalian preimplantation embryo in culture. Studies using defined serum free embryo culture systems have shown that.serum induces premature blastulation,69,70 changes in embryo morphology,23,70 perturbations in ultrastructure70,71 and in energy metabolism.23 Perhaps more disturbing are the reports that the presence of serum in culture medium used on domestic animal embryos is associated with high rates of fetal loss as well as abnormally elevated fetal weights.70 Therefore, should any abnormalities potentially arise after extended culture in clinical assisted reproductive technologies (ART), they should have arisen after the use of coculture and serum, like that observed in sheep and cattle. However, Menezo et al,72 have not reported any difference in gestation lengths, nor have they reported any birth abnormalities after using coculture and 15% serum. It is therefore plausible that the large offspring phenomenon is peculiar to ruminants. However, it is strongly recommended that human embryos are not exposed to whole serum at any time. 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.73 Not only are there significant differences between sources of serum albumin74,75 but also between batches from the same source.74,76 Therefore when using serum albumin or

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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. Importantly, recombinant human albumin has recently become available, which should eliminate the problems inherent with using blood derived products, and lead to the standardization of medium formulations.77

B. CULTURE SYSTEMS Several key components of the culture system are reviewed here, none of which should be considered in isolation as all directly affect 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 disturbances to avoid are temperature and pH changes. This means that ideally the environment in which the embryo is placed is not disturbed during the culture period. Practically, this is very difficult to achieve in a busy clinical laboratory. Two approaches can be considered: first, the employment of a single use incubation chamber, such as a modular incubator chamber or glass desiccator, which can be purged with the appropriate gas mix. 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. Second, an alternative to the use of modular chambers is the use of inner doors within an incubator. Although this does not work as effectively in maintaining the environment around embryos during culture, it does significantly reduce fluctuations in the gaseous environment upon opening the incubator door. Several incubator manufacturers make incubators with inner doors. What is evident is that it is imperative to have sufficient numbers of incubators to match the caseload. This is especially true when performing extended culture. For around 20 retrievals per week we employ 14 incubator chambers (present in the laboratory as seven double stacks). The top chamber of each stack is for media equilibration, while the bottom chamber is 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 two to three retrievals per week.

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pH AND GAS PHASE 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; 7.278–80). It is the specific components of the culture media, such as lactic and amino acids that 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.80 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,80 amino acids increase the intracellular buffering capacity and help maintain the pHi at around 7.2. 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 Sorensons’s phosphate buffer standards.4 This method allows visual inspection of a medium’s pH with a tube in the incubator and is accurate to 0.1–0.2pH units (Table 16.1). When using bicarbonate buffered media, the concentration of CO2 has a direct impact on medium pH. Although most media work over a wide range of pH (7.2 to 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 6–7%. The amount of CO2 in the incubation chamber can be calibrated with a Fyrite, although such an approach is only accurate to ±1%. An alternative method is to use an infrared metering system that can be calibrated and are accurate to around 0.1%. 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 increases in pH. To facilitate this we use pediatric isolettes designed to maintain temperature, humidity, and CO2 concentration. However, should it not be feasible to use an isolette, then the media used can be buffered with either 20mM HEPES or MOPS81 together with 5mM 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. The human and F1 mouse embryo can grow at atmospheric oxygen concentration (20%) and this 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%,82,83 whereas the oxygen concentration in the oviduct of hamster, rabbit and rhesus monkey is 8%.84 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.84,85 Several studies on different

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species have demonstrated that culture at a reduced oxygen concentration (5–7%) results in enhanced embryo development in vitro, and that exposure to reduced oxygen in vitro leads to higher fetal development.86,87 In our experience 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%).87 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. INCUBATION VESSEL AND THE EMBRYO: VOLUME RATIO Culture of embryos in groups in drops of culture medium under an oil overlay is the preferred and most effective method to date of culturing embryos. Within the lumen of the female reproductive tract the developing embryo is exposed to microlitre volumes of fluid.68 In contrast, the embryo grown in vitro is subject to large volumes of medium, of up to 1ml. 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 that cleavage rate and blastocyst formation increase when embryos are grown in groups (up to 10) or reduced volumes (around 20µl).88–91 Of greatest significance is the

Table 16.1. Preparation of colour standards for pH of media. Stock A: 9.08 g KH2PO4 (0.067 M), 10mg phenol red in 1l of water. Stock B: 9.46 g Na2HPO4 (0.067 M), 10mg phenol red in 1l of water. pH at 18°C Solution A (ml) Solution B (ml) 6.6 62.7 37.3 6.8 50.8 49.2 7.0 39.2 60.8 7.2 28.5 71.5 7.4 19.6 80.4 7.6 13.2 86.8 Measure the pH with meter, and adjust pH as required, ie add solution A to lower the pH (make more acidic), add solution B to increase the pH (make more alkali). The pH standards should be filter-sterilized and can then be kept for up to six months.

Fig 16.2 Effect of incubation volume and embryo grouping on embryo development and differentiation. (a) A single embryo cultured in a 4 well plate or test tube, any factor produced by the embryo will become ineffectual owing to dilution.

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(b) Culture of embryos in reduced volumes and/or in groups increases the effective concentration of embryo derived factors, facilitating their action in either a paracrine or autocrine manner. (c) Effect of embryo grouping on bovine blastocyst development and differentiation. Bovine embryos were cultured either individually or in groups of 2 or 4 in 50µl drops of medium. Like pairs are significantly different; P<0.05. From Gardner with permission.94 observation that decreasing the incubation volume significantly increases embryo viability90 owing to an increase in inner cell mass development.92 Similar results have been obtained with sheep23 and cow embryos.93 It is therefore apparent that the preimplantation mammalian embryo produces (a) factor(s) capable of stimulating development of both itself and surrounding embryos (Fig 16.2). In order to culture in such reduced volumes (of 20–50µl) 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 or mineral oils are recommended. The benefits of using drops of medium under oil would obviously be negated should the oil be embryo toxic. 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 the container supplied. 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 lot of oil prescreened with a mouse embryo bioassay before clinical use. Oil toxicity may not necessarily show up by simply culturing mouse embryo 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. Amino acids are relatively stable in solution at 4°C. However, when amino acids are placed at 37°C they spontaneously breakdown and release ammonium.

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Glutamine is the most labile amino acids and, owing to the presence of two amine groups, it produces the highest levels of ammonium of any amino acid. The significance of this is that ammonium impairs embryos development in vitro48 and subsequent development in utero after transfer.54 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. Glutamine can be replaced with alanylglutamine, a dipeptide that is stable at 37°C. Vitamins are light sensitive and therefore care should be taken to 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. The types of bioassays conducted for this have been the focus of much discussion.4 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 embryos in protein free media. There has been a lot of conflicting data regarding the use of this bioassay, but with just a few conditions, one can not only increase the sensitivity of the assay, one can also attempt to quantitate quality with it. First, the stage from which the embryos is cultured has an impact on development. Embryos collected at the pronucleate stage do not tend to fare as well in culture as those collected at the 2 cell stage. Second, 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 random bred strain of mice provides greater genetic diversity. Third, 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 water95,96 should be interpreted carefully after taking into account the types of media used and the supplementation of medium with protein. Silverman et al96 used Ham’s F-10. This medium contains amino acids, which can chelate any possible toxins present in the tap water, for example, heavy metals. George et al95 included high levels of bovine serum albumin (BSA) in their zygote cultures to the blastocyst. Albumins can chelate potential embryo-toxins and thereby mask the effect of any present in the culture medium.97,98 Furthermore, all studies used blastocyst development as the sole criterion for assessing embryo development. Blastocyst development is a poor indicator of embryo

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quality and does not accurately reflect developmental potential. 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. When new components of certain culture media that can affect the development of the inner cell mass directly, such as essential amino acids, a differential nuclear stain should be performed in order to determine the extent of inner cell mass (ICM) development. Using such an approach makes it possible to identify potential problems in culture media before they are used clinically.

ON WHAT DAY SHOULD EMBRYO TRANSFER BE PERFORMED? For the past two decades the majority of embryos conceived through IVF have been transferred between days 1 and 3 at either the pronucleate or cleavage stage. 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 newer culture systems (described above) it is feasible to perform day 5 blastocyst transfers as a matter of routine in an IVF clinic.9 This now raises the question which day of embryo development should embryos be transferred on. Before answering this question, one must first consider the potential advantages and disadvantages of blastocyst culture and transfer. BLASTOCYST TRANSFER: ADVANTAGES AND DISADVANTAGES The potential advantages of blastocyst culture and transfer have been well documented.7,56,99–102 Briefly the advantages include the following. (1) Synchronizing embryonic stage with the female tract. This is important as the levels of nutrients within the fallopian tube and uterus do differ. Furthermore, the uterine environment during a stimulated cycle cannot 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.103–105 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 8 cell stage the embryonic genome has only just begun to be transcribed,106–107 and therefore it is not possible to identify from within a given cohort the embryos 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

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developmental potential. Comparatively recently, there has been a move to assess pronucleate stage embryos in order to select embryos for transfer,108–109 with the report that implantation rates can be increased by the assessment of pronuclear morphology. Similarly, Gerris et al110 have used a scoring system for use on day 3 to increase implantation rates. However, assessment of the embryos at either the pronuclear or the cleavage stage 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,111 but it provides limited information regarding true embryo developmental potential. (3) Not all fertilized oocytes are normal, and therefore a percentage always exists that is not destined to establish a pregnancy or go to term. Most such abnormalities are chromosomal. It has been determined that around 25% of oocytes are aneuploid,112,113 and that this problem is exacerbated with maternal age.114 Similarly, there is an age related increase in chromosomal abnormalities in embryo karyotypes.115 Patients aged between 20 and 34 exhibit a 16% chromosomal abnormality in their embryos. However, this figure increases to 53% in patients 40 and over. Other factors contributing to embryonic attrition include an insufficiency of stored oocyte coded gene products, and a failure to activate the embryonic genome.116 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. (4) Uterine contractions have been negatively correlated with embryo transfer outcome, possibly by the expulsion of embryos from the uterine cavity.117 Uterine junctional zone contractions have been quantitated and found to be strongest on the day of oocyte retrieval.118 All patients exhibited such contractions on day 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 of embryonic expulsion and loss.119 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 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 5.9 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, this due to a significant increase in implantation rates. However, this increase in the percentage of patients not having an embryo transfer can be avoided if a different approach is taken to selecting which patients have blastocyst transfer (see below). Edwards and Beard109 have proposed that the implantation rates of blastocysts are not greater than that of cleavage stage embryos when implantation rates are expressed per pronucleate embryo. These authors determined that as around half of pronucleate embryos form blastocysts the corrected implantation rate is 25%—similar to that obtained with day

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3 transfers. However, this proposal assumes that all pronucleate embryos form day 3 embryos of equal developmental potential, which is certainly not the case. Of the patients having day 3 embryo transfer in a prospective randomized trial,119 around 60% of pronucleate embryos from patients having a transfer on day 3 were ≥6 cells with only minor fragmentation— good quality embryos. So using this as a correction factor, the value for day 3 transfer per pronucleate embryo would give an implantation rate of around 18%, below that obtained per blastocyst. However, it is difficult to draw such lines in the sand regarding embryo quality and developmental potential as embryos with less than six cells and with fragmentation can establish a pregnancy. Conversely, embryos that do not reach the blastocyst stage by day 5 but do compact can also implant (see below).

INTRODUCTION OF BLASTOCYST CULTURE AND TRANSFER INTO A CLINICAL PROGRAM AND PATIENT SELECTION It has been shown in a prospective randomized trial that in patients who respond well to gonadotropins, with 10 or more follicles, blastocyst culture and transfer results in higher implantation rates than embryo transfer on day 3 (Table 16.2119). Therefore in such patients blastocyst culture should be the primary treatment. Certainly blastocyst culture and transfer can be employed to successfully eliminate high order multiple gestations in any group of patients at risk of conceiving a multiple gestation. After over 300 cases in our

Table 16.2. Effect of day of transfer on implantation and pregnancy outcome. Variable Day 3 Day 5 No of patients 47 45 Mean age (±SEM) in years 34.5±0.6 33.6±0.7 Age range 26–43 26–43 Previous no of cycles (mean ± SEM) 0.21±0.07 0.62±0.14 FSH (mean±SEM) 7.7±0.3 7.3±0.4 Patients with ICSI (%) 34 33 No. of pronuclear embryos(mean ± SEM) 10.9±0.7 11.6±0.8 No. of embryos transferred (mean ± 3.7±0.1 2.2±0.1 SEM) Patients with embryo freezing (%) 30 64 Mean no. of frozen embryos (±SEM) 3.0±0.7 3.2±0.5 Implantation rate (fetal sac) (%) 37.0 55.4 Implantation rate (fetal heart) (%) 30.1 50.5

P value NS <0.05 NS NS NS <0.01 <0.01 NS <0.01 <0.01

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Clinical pregnancy rate (%)* 66 71 NS *Includes two patients in the blastocyst culture group who did not have an embryo transferred on day 5 owing to embryonic arrest at the cleavage stages. From Gardner et al.119

Fig 16.3 Relationship between the number of pronucleate embryos and the number of blastocysts formed. There is a significant linear relationship; r2=0.495, P<0.0001. Where two or more patients overlap, only one point is shown on the plot. n=301 patients. From Gardner et al with permission.120

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Fig 16.4 Distribution of blastocyst development in 301 patients. All embryos were cultured for the first 48 h in medium G1.2, and then washed three times in medium G2.2, followed by culture in medium G2.2. From Gardner et al with permission.120 program an implantation rate of 50% has been maintained.120 Analysis of these 301 cases revealed that there was no relation between percentage blastocyst development and the number of either oocytes or pronucleate embryos. In contrast, there was a significant linear relation between the number of either oocytes or pronucleate embryos and the number of resultant blastocysts (Fig 16.3). These data therefore indicate that around 50% of embryos will form blastocysts, irrespective of the number of oocytes and embryos within a given cohort. However, this 50% blastocyst development is an average value, and there is considerable variation between patients regarding the percentage of embryos that form blastocysts (Fig 16.4). It can be seen in Fig 16.4 that eight patients did not form blastocysts. Five of these patients had embryos that arrested at the 8 cell stage and did not have an embryo transfer; the remaining three patients had embryos that developed to the morula stage and did have an embryo transfer. After the transfer of morulae (mean of 2 replaced) two patients became pregnant. Therefore in the “no blastocyst” group a 25% pregnancy rate was obtained.

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Fig 16.5 Relation between day 3 cell number and blastocyst development. There is a linear relation with cell number and blastocyst development up to the 8 cell stage (P<0.01). Solid circles represent IVF patients. Open circles represent oocyte donors. Data from Langley et al. 122 For patients having oocyte donation, blastocyst culture, and transfer is the most effective course of treatment.121 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 not only reach the blastocyst stage at a higher frequency than those from IVF patients, they also tend to be of higher quality.121 It is possible to attain an implantation rate of 65% when transferring blastocysts to recipients whose mean age is 41.3. Certainly oocyte donors represent as close to a gold standard as one can have in an infertility clinic. With this in mind ensuring one can attain blastocyst development of greater than 50% and implantation rates of over 50% when using donated oocytes is a good potential starting point for introducing blastocyst culture clinically. An alternative approach is to wait until day 3 of embryo development to decide whether a patient should have extended culture. Fig 16.5122 shows the relation between day 3 cell number and subsequent blastocyst

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formation in both IVF patients and oocyte donors. It can be seen that there is linear relationship between day 3 cell number and percent blastocyst development, so that an 8 cell embryo has close to an 80% chance of forming a blastocyst, whereas a 6 cell embryo has a 55% chance. Using this information, physicians and scientists may readily counsel patients on day 3 regarding the extended culture of their embryos to the blastocyst stage. This approach has been used successfully123 and reduces the chance that patients will end up without an embryo transfer after extended culture. However, it is important to reiterate the findings of Marek et al9 in that, even though the percentage of patients not receiving an embryo transfer after extended culture was above that for those patients having a transfer on day 3, the overall pregnancy rates were significantly higher after extended culture. This therefore raises the issue of patient counseling and physician’s comfort level. Behr et al66 used a further approach to the introduction of blastocyst culture. Rather than transferring blastocysts immediately, they continued with day 3 transfers but cultured the supernumerary embryos for a further two or three days. Embryos were subsequently cryopreserved at the blastocyst stage. This approach has the advantage that neither the laboratory nor physician feels extra stress when trying a new procedure. It ensures that the culture system employed is able to facilitate blastocyst development and that a suitable cryopreservation system is in place for when one makes the move to fresh blastocyst transfers. MALE INFERTILITY In our experience there is no disadvantage to using blastocyst culture in ICSI patients,120 although other groups have reported negative results with such patients and therefore only perform day 3 transfers. In our clinic slightly fewer blastocysts are formed after ICSI (48.1%) compared with conventional insemination (51.9%), but the resultant blastocysts have the same implantation rates.120 TOWARD SINGLE EMBRYO TRANSFER Gardner and Leese124 and Sakkas (Chapter 17) have discussed the development of scoring systems used in clinical IVF and their significance in identifying the most viable embryo(s) for transfer. 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 an appreciable 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.125

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CONCLUSIONS The culture system in the clinical laboratory is just one part of the overall treatment cycle. Good oocytes, derived from appropriate stimulation regimes, are able to give rise to good embryos. However, it is not feasible to obtain good embryos from poor oocytes.111 Subsequently the embryo transfer technique and subsequent luteal support administered have an impact on cycle outcome (see Chapters 43 and 50). Therefore, should extended culture not result in the expected blastocyst development and implantation rates, it is important to scrutinize all procedures within an IVF cycle. Blastocyst culture and transfer should not be perceived as a panacea for all of the problems of IVF. Indeed, should part of an IVF procedure be compromised, then moving to extended culture may actually exacerbate the situation. The increasing evidence would support the move to day 5 transfers for many patients attending an infertility program.125 However, there are certain exceptions to this, such as clinics in such countries as Germany, which are only able to culture as many pronucleate embryos as they will transfer and this can be no more than 3. 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. However, given the information in Fig 16.5, it would be possible to counsel patients on day 3 on whether they should consider extended culture.

EMBRYO CULTURE The protocol described below is that in use in our laboratory. It has been validated using the media and consumables listed. Any change to the protocol, whether it be a different source of oil, media, or pipette, should be validated carefully, as it is our experience that any such change can have a significant effect on the culture outcome. We routinely work in paediatric isolettes that maintain both temperature and CO2, and therefore circumvent the use of other buffer systems such as HEPES and MOPS. The protocols below have been modified to include the use of media buffered to maintain pH outside of a CO2 environment. For use of other media such as P1 and blastocyst medium, universal IVF medium, and M3, see the specific instructions from the manufacturer. Embryo cultures should be performed in 5% O2 and 6% CO2 when using medium G1.2 and G2.2. This gas environment can be created using either a tri-gas incubator or a modular incubator chamber/dessicator and a cylinder of special gas mix. It is advisable to minimize the number of observation made during embryo development, and to minimize the number of cases per incubator. Human embryos will grow at 20% O2, but development appears superior at lower oxygen tensions.

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PRONUCLEATE STAGE EMBRYOS TO DAY 3 CULTURE EMBRYO MANIPULATION (FOLLOWING FERTILIZATION ASSESSMENT) Once the cumulus is removed (see Chapter 7) then all manipulations should be performed using a pulled Pasteur pipette or a displacement pipette such as a Stripper. Should a pulled Pasteur be used then control of fluid can be achieved using a syringe attached through tubing. It is important to use a pipette with the appropriate size tip (day 1 to 3; around 200µM). Using the appropriate size tip minimizes the volumes of culture medium moved with each embryo, which typically should be less than a microliter. Such volume manipulation is a prerequisite for extended culture. SETTING UP CULTURE DISHES At around 4 pm on the day of oocyte retrieval label 60mm Falcon Primaria dishes with the patient’s name. Using a single wrapped biopure tip (Eppendorf, NJ) rinse the tip once first then, place 6×25µl drops of G1.2 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 9ml of oil. Prepare no more than two plates at one time. Oil should be prescreened before use. Using a new tip for each drop, first rinse the tip and then add a further 25µl of medium to each original drop. Immediately place the dish in the incubator at 5% O2 and 6% CO2. 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 correct pH under oil). For each patient, set up a wash dish at the same time as the culture dishes. Place 1ml of medium G1.2 into the center of an organ well dish. Place 2ml 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. MORNING OF DAY 1 CULTURE IN G1.2 After 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 two to three times and

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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 G1.2. Four is the maximum number of embryos that can be cultured in each drop due to the 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 low O2 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. On day 3, embryos can be transferred to the uterus in medium G2.2. DAY 3 EMBRYOS TO THE BLASTOCYST SETTING UP G2.2 DISHES On day 3 before 8.30 am label 60mm dishes with the patient’s name. Using a single wrapped biopure tip rinse the tip once first then, place 6×25µl drops of G2.2 into the plate. Immediately cover with 9ml 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 25µl of medium to each original drop. Immediately place the dish in the low incubator 5% O2, 6% CO2. Gently remove the lid and set on the side of the plate. For each patient, set up one wash dish per 10 embryos. Place 1ml of medium G2.2 into center of an organ well dish. Place 2ml of medium into the outer well. Place immediately

Fig 16.6 Human blastocysts the morning of day 5 (four days of culture from the pronucleate stage). Embryos were cultured from the

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pronucleate stage until early afternoon on day 3 in medium G1.2. Cleavage stage embryos were then washed in medium G2.2 and cultured in G2.2 for around 48 h before transfer. 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 am. Place 1ml of medium G2.2 into the center of an organ well dish. Place 2ml of medium into the outer well. Place immediately into the incubator. If working outside an isolette use HE PES/MOPS buffered medium with amino acids. This should not be placed in a CO2 incubator, but rather warmed on a heated stage. CULTURE IN G2.2 Moving embryos from G1.2 to G2.2 should occur between 12.00 pm and 2.30 pm. Wash embryos in the organ well very well (this step is crucial to remove the EDTA). Washing entails picking up the embryo two to three 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 G2.2. 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. The morning of day 5 embryos should be scored (Fig 16.6; Gardner and Schoolcraft111; Chapter 17) and the two top scoring embryos selected for transfer. Transfers should be performed in medium G2.2. 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 G2.2 for 24 h and assessed on day 6.

REFERENCES 1 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. 2 Leese HJ. Metabolism of the preimplantation mammalian embryo. In: Milligan SR, ed. Oxford reviews of reproductive biology. Oxford: Oxford University Press (1991): 35–72.

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3 Gardner DK, Lane M. Embryo culture systems. In: Trounson A, Gardner DK, eds. Handbook of in vitro fertilization. 1st ed. Boca Raton: CRC Press (1993): 85–114. 4 Gardner DK, Lane M. Embryo culture systems. In: Trounson A, Gardner DK, eds. Handbook of in vitro fertilization. 2nd ed. Boca Raton: CRC Press (1999): 205–64. 5 Hardy K, Hooper MAK, Handyside AH, Rutherford AJ, Winston RML, Leese HJ. Non-invasive measurement of glucose and pyruvate uptake by individual human oocytes and preimplantation embryos. Hum Reprod (1989); 4:188–91. 6 Gardner DK, Lane M, Calderon I, Leeton J. Environment of the preimplantation human embryo in vivo: metabolite analysis of oviduct and uterine fluids and metabolism of cumulus cells. Fertil Steril (1996); 65:349–53. 7 Bolton VN, Wren ME, Parsons JH. Pregnancies after in vitro fertilization and transfer of human blastocysts. Fertil Steril (1991); 55:830–2. 8 Huisman GJ, Fauser BCJM, Eijkemans MJC, Pieters MHEC. Implantation rates after in vitro fertilization and transfer of a maximum of two embryos that have undergone three to five days of culture. Fertil Steril (2000); 73:117–22. 9 Marek D, Langley M, Gardner, DK, et al. Introduction of blastocyst culture and transfer for all patients in an in vitro fertilization program. Fertil Steril (1999); 72:1035–40. 10 Gardner DK, Lane M. Culture of viable human blastocysts in defined sequential serum-free media. Hum Reprod (1998); 13 (S3):148–59. 11 Bavister BD. Culture of preimplantation embryos: facts and artifacts. Hum Reprod Update (1995); 1:91–148. 12 Leese HJ. Metabolic control during preimplantation mammalian development. Hum Reprod Update (1995); 1:63–72. 13 Pool TB, Atiee SH, Martin JE. Oocyte and embryo culture: basic concepts and recent advances. Infert Reprod Med Clinics of N A (1998); 9:181–203. 14 Chatot CL, Ziomek CA, Bavister BD, Lewis JL, Torres I. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J Reprod Fertil (1989); 86:679–88. 15 Schini SA, Bavister BD. Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose. Biol. Reprod (1988); 39:1183–92. 16 Thompson JG, Simpson AC, Pugh A, Tervit HR. Requirement for glucose during in vitro culture of sheep preimplantation embryos. Molec Reprod Dev (1992); 31:253–7. 17 Takahashi Y, First NL. In vitro development of bovine one-cell embryos: influence of glucose, lactate, pyruvate, amino acids and vitamins. Theriogenology (1992); 37:963–78.

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18 Rosenkrans CF Jr, Zeng GQ, McNamara GT, Schoff PK, First NL. Development of bovine embryos in vitro as affected by energy substrates. Biol Reprod (1993); 49:459–62. 19 Matsuyama K, Miyakoshi H, Fukui Y. Effect of glucose levels during in vitro culture in synthetic oviduct fluid medium on in vitro development of bovine oocytes matured and fertilized in vitro. Theriogenology (1993); 40:595–605. 20 Conaghan J, Handyside AH, Winston RM, Leese HJ. Effects of pyruvate and glucose on the development of human preimplantation embryos in vitro. J Reprod Fertil (1993); 99:87–95. 21 Quinn P. Enhanced results in mouse and human embryo culture using a modified human tubal fluid medium lacking glucose and phosphate. J Assist Reprod Genet (1995); 12:97–105. 22 Gardner DK, Lane M. The 2-cell block in CF1 mouse embryos is associated with an increase in glycolysis and a decrease in tricarboxylic acid (TCA) cycle activity: alleviation of the 2-cell block is associated with the restoration of in vivo metabolic pathway activities. Biol Reprod (1993); 49 (S1):52. 23 Gardner DK, Lane M, Spitzer A, Batt PA. Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the absence of serum and somatic cells: amino acids, vitamins and culturing embryos in groups stimulate development. Biol Reprod (1994); 50:390–400. 24 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. 25 Barnett DK, Bavister BD. Inhibitory effect of glucose and phosphate on the second cleavage division of hamster embryos: is it linked to metabolism? Hum Reprod (1996); 11:177–83. 26 Lane M, Gardner DK. Amino acids and vitamins prevent culture induced metabolic perturbations and associated loss of viability of mouse blastocysts. Hum Reprod (1998); 13:991–7. 27 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. 28 Aoki K, Nakamura M, Namiki H, Okinaga, S, Arai K. The effect of glucose and phosphate on mouse two-cell embryos to develop in vitro. Zool Sci (1990); 7:973–7. 29 Carrillo AJ, Lane B, Pridman DD, et al. Improved clinical outcomes for in vitro fertilization with delay of embryo transfer from 48 to 72 hours after oocyte retrieval: use of glucose- and phosphate-free media. Fertil Steril (1998); 69:329–34. 30 Ludwig TE, Lane M, Bavister BD. Increased fetal development after transfer of hamster embryos cultured with glucose. Biol Reprod (1998); 58 (S1):306.

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31 Dan-Goor M, Sasson S, Davarashvili A, Almagor M. Expression of glucose transporter and glucose uptake in human oocytes and preimplantation embryos. Hum Reprod (1997); 12:2508–10. 32 Rieger D. Relationship between energy metabolism and development of the early embryo. Theriogenology (1992); 37:75–93. 33 Gardner DK. Embryo development and culture techniques. In: Clark J, ed. Animal Breeding: Technology for the 21st Century. London: Harwood Academic Publishers (1998):13–46. 34 Rogers PAW, Murphy CR, Gannon BJ. Absence of capillaries in the endometrium surrounding the implanting rat blastocyst. Micron (1982); 13:373. 35 Rogers PAW, Murphy CR, Gannon BJ. Changes in the spatial organisation of the uterine vasculature during implantation in the rat. J Reprod Fertil (1982); 65:211–4. 36 Lane M, Gardner DK. Selection of viable mouse blastocysts prior to transfer using a metabolic criterion. Hum Reprod (1996); 11:1975–8. 37 Perkins JL, Goode L. Free amino acids in the oviduct fluid of the ewe. J Reprod Fertil (1967); 14:309–11. 38 Menezo YJR. Amino constituents of tubal and uterine fluids of the eostrous ewe: comparison with blood serum and ram seminal fluid. In Hafez ESE, Thibault CG, eds. The biology of spermatozoa. New York: Basel Press (1972):174–81. 39 Casslen BG. Free amino acids in human uterine fluid. Possible role of high taurine concentration. J Reprod Med (1987); 32:181–4. 40 Miller JGO, Schultz GA. Amino acid content of preimplantation rabbit embryos and fluids of the reproductive tract. Biol Reprod (1987); 36:125–9. 41 Gardner DK, Leese HJ. Concentrations of nutrients in mouse oviduct fluid and their effects on embryo development and metabolism in vitro. J Reprod Fertil (1990); 88:361. 42 Van Winkle LJ. Amino acid transport in developing animal oocytes and early conceptuses. Biochim Biophys Acta (1988); 947:173–208. 43 Gardner DK, Clarke RN, Lechene CP, Biggers JD. Development of a noninvasive ultramicrofluorometric method for measuring net uptake of glutamine by single mouse preimplantation embryos. Gamete Res (1989); 24:427–38. 43 Schultz GA, Kaye PL, McKay DJ, Johnson MH. Endogenous amino acids pool sizes in mouse eggs and preimplantation embryos. J Reprod Fertil (1981); 61:387–93. 44 Chatot CL, Tasca RJ, Ziomek CA. Glutamine uptake and utilization by preimplantation mouse embryos in CZB medium. J Reprod Fertil (1990); 89:335–46. 45 Eagle H. Amino acid metabolism in mammalian cell cultures. Science (1959); 130:432–7.

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46 Dumoulin JCM, Evers JLH, Bakker, JA, Bras M, Pieters MHEC, Geraedts JPM. Temporal effects of taurine on mouse embryo development in vitro. Hum Reprod (1992); 7:403–7. 47 Dumoulin JCM, Evers JLH, Bras M, Pieters MHEC, Geraedts JPM. Positive effect of taurine on preimplantation development of mouse embryos in vitro. J Reprod Fertil (1992); 94:373–80. 48 Gardner DK, Lane M. Amino acids and ammonium regulate mouse embryo development in culture. Biol Reprod (1993); 48:377–85. 49 Bavister BD, McKiernan SH. Regulation of hamster embryo development in vitro by amino acids. In Bavister BD, ed. Preimplantation embryo development. New York: Springer (1993):57– 72. 50 McKiernan SH, Clayton MK, Bavister BD. Analysis of stimulatory and inhibitory amino acids for development of hamster one-cell embryos in vitro. Mol Reprod Dev (1995); 42:188–99. 51 Steeves TE, Gardner DK. Temporal and differential effects of amino acids on bovine embryo development in culture. Biol Reprod (1999); 61:731–40. 52 Devreker F, Winston RM, Hardy K. Glutamine improves human preimplantation development in vitro. Fertil Steril (1998); 69:293–9. 53 Lane M, Gardner DK. Differential regulation of mouse embryo development and viability by amino acids. J Reprod Fertil (1997); 109:153–64. 54 Lane M, Gardner DK. Increase in postimplantation development of cultured mouse embryos by amino acids and induction of fetal retardation and exencephaly by ammonium ions. J Reprod Fert (1994); 102:305–12. 55 Gardner DK, Sakkas D. Mouse embryo cleavage, metabolism and viability: role of medium composition. Hum Reprod (1993); 8:288–95. 56 Gardner DK. Mammalian embryo culture in the absence of serum or somatic cell support. Cell Biol Int (1994); 18:1163–79. 57 Gardner DK, Lane M. Culture and selection of viable blastocysts: a feasible proposition for human IVF? Hum Reprod Update (1997); 3:367–82. 58 Ho Y, Wigglesworth K, Eppig JJ, Schultz RM. Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression. Molec Reprod Devel (1995); 41:232–8. 59 Edwards RG. Test-tube babies. Nature (1981); 293:253–6. 60 Abramczuk J, Solter D, Koprowski, H. The beneficial effect of EDTA on development of mouse one-cell embryos in chemically defined medium, Devel Biol (1977); 61:378–83. 61 Mehta TS, Kiessling AA. Development potential of mouse embryos conceived in vitro and cultured in ethylenediaminetetraacetic acid with or without amino acids or serum. Biol Reprod (1990); 43:600–6.

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62 Erbach GT, Lawitts JA, Papaioannou VE, Biggers JD. Differential growth of the mouse preimplantation embryo in chemically defined media. Biol Reprod (1994); 50:1027–33. 63 Gardner DK, Lane MW, Lane M. EDTA stimulates cleavage stage bovine embryo development in culture but inhibits blastocyst development and differentiation . Mol Reprod Devel (2000); 57:256– 61. 64 Hoshi M, Toyoda Y. Effect of EDTA on the preimplantation development of mouse embryos fertilized in vitro. Jpn J Zootech Sci (1985); 56:931–7. 65 Hewitson LC, Leeese HJ. Energy metabolism of the trophectoderm and inner cell mass of the mouse blastocyst. J Exp Zool (1993); 267:337–43. 66 Behr B, Pool TB, Milki AA, et al. Preliminary clinical experience with human blastocyst development in vitro without co-culture. Hum Reprod (1999); 14:454–7. 67 Barnes D, Sato G. Methods for growth of cultured cells in serum-free medium. Anal Biochem (1980); 102:255–70. 68 Leese HJ. The formation and function of oviduct fluid. J Reprod Fertil (1988); 82:843–56. 69 Walker SK, Heard TM, Seamark RE In vitro culture of sheep embryos without co-culture: success and perspectives . Theriogenology (1992); 37:111–26. 70 Thompson JG, Gardner DK, Pugh PA, et al. Lamb birth weight is affected by culture system utilized during in vitro pre-elongation of ovine embryos. Biol Reprod (1995); 53:1385–91. 71 Dorland M, Gardner DK, Trounson A. Serum in synthetic oviduct fluid causes mitochondrial degeneration in ovine embryos. J Reprod Fertil Abstract Series (1994); 13:70. 72 Menezo YJ, Chouteau J, Girard A, Veiga A. Birth weight and sex ratio after transfer at the blastocyst stage in humans. Fertil Steril (1999); 72:221–4. 73 Gray CW, Morgan PM, Kane MT. Purification of an embryotrophic factor from commercial bovine serum albumin and its identification as citrate. J Reprod Fertil (1992); 94:471–80. 74 Batt PA, Gardner DK, Cameron AWN. Oxygen concentration and protein source affect the development of preimplantation goat embryos in vitro. Reprod Fert Devel (1991); 3:601–7. 75 McKiernan SH, Bavister BD. Different lots of bovine serum albumin inhibit or stimulate in vitro development of hamster embryos. In vitro cell dev biol (1992); 28A: 154–6. 76 Kane MT. Variability in different lots of commercial bovine serum albumin affects cell multiplication and hatching of rabbit blastocysts in culture. J Reprod Fertil (1983); 69:555–8.

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77 Gardner DK, Lane M. Recombinant human serum albumin and hyaluronan can replace blood-derived albumin in embryo culture media. Fertil Steril (2000); 74: (Suppl 1):0–086. 78 Phillips KP, Leveille MC, Claman P, Baltz JM. Intracellular pH regulation in human preimplantation embryos. Hum Reprod (2000); 4:896–904. 79 Lane M, Baltz JM, Bavister BD. Regulation of intracellular pH in hamster preimplantation embryos by the sodium hydrogen (Na+/H+) antiporter. Biol Reprod (1998); 59:1483–90. 80 Edwards LE, Williams DA, Gardner DK. Intracellular pH of the preimplantation mouse embryo: effects of extracellular pH and weak acids. Mol Reprod Devel (1998); 50:434–42. 81 Good NE, Winget GD, Winter W, et al. Hydrogen ion buffers for biological research. Biochemistry (1996); 5:467–77. 82 Mastroianni L Jr, Jones R. Oxygen tension in the rabbit fallopian tube. J Reprod Fertil (1995); 9:99–102. 83 Ross RN, Graves CN. O2 levels in the female rabbit reproductive tract. J Anim Sci (1974); 39:994. 84 Fischer B, Bavister BD. Oxygen tension in the oviduct and uterus of rhesus monkeys, hamsters and rabbits. J Reprod Fertil (1993); 99:673– 9. 85 Mass DHA, Storey BT, Mastroianni L Jr. Oxygen tension in the oviduct of the rhesus monkey (Macaca mulatta). Fertil Steril (1976); 27:1312–7. 86 Quinn P, Harlow GH. The effect of oxygen on the development of preimplantation mouse embryos in vitro. J Exp Zool (1978); 206:73– 80. 87 Gardner DK, Lane M, Johnson J, Wagley L, Stevens J, Schoolcraft WB. Reduced oxygen tension increases blastocyst development, differentiation and viability. Fertil Steril (1999); 72: (Suppl 1): 0–079. 88 Wiley LM, Yamami S, Van Muyden D. Effect of potassium concentration, type of protein supplement, and embryo density on mouse preimplantation development in vitro. Fertil Steril (1986); 45:111–9. 89 Paria BC, Dey SK. Preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors. Proc Natl Acad Sci USA (1990); 87:4756–60. 90 Lane M, Gardner DK. Effect of incubation volume and embryo density on the development and viability of mouse embryos in vitro. Hum Reprod (1992); 7:558–62. 91 Salahuddin S, Ookutsu S, Goto K, Nakanishi T, Nagata Y. Effects of embryo density and co-culture of unfertilized oocytes on embryonic development of in-vitro fertilized mouse embryos. Hum Reprod (1995); 10:2382–5.

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92 Gardner DK, Lane MW, Lane M. Development of the inner cell mass in mouse blastocysts is stimulated by reducing the embryo: incubation volume ratio. Hum Reprod (1997); 12, Abstract Book 1:132. 93 Ahern TJ, Gardner DK. Culturing bovine embryos in groups stimulates blastocyst development and cell allocation to the inner cell mass. Theriogenology (1998); 49:194. 94 Gardner DK. Improving embryo culture and enhancing pregnancy rate. In: Shoham Z, Howles C, Jacobs H, eds. Female infertility therapy: current practice. London: Martin Dunitz (1998):283–99. 95 George MA, Braude PR, Johnson MH, Sweetnam DG. Quality control in the IVF laboratory: in-vitro and in-vivo development of mouse embryos is unaffected by the quality of water used in culture media. Hum Reprod (1989); 4:826–31. 96 Silverman IH, Cook CL, Sanfilippo JS, Yussman MA, Schultz GS, Hilton FH. Ham’s F-10 constituted with tap water supports mouse conceptus development in vitro. JIVET (1987); 4:185–7. 97 Fissore RA, Jackson KV, Kiessling AA. Mouse zygote development in culture medium without protein in the presence of ethylenediaminetetraacetic acid. Biol Reprod (1989); 41:835–41. 98 Flood LP, Shirley B. Reduction of embryotoxicity by protein in embryo culture media. Molec Reprod Devel (1991); 30:226–31. 99 Menezo Y, Guerin J-F, Czyba J-C. Improvement of human early embryo development in vitro by coculture on monolayers of Vero cells. Biol Reprod (1990); 42:301–6. 100 Eopata A. The neglected human blastocyst. J Assist Reprod Genet (1992); 9:509–12. 101 Olivennes F, Hazout A, Lelaider C, et al. Four indications for embryo transfer at the blastocyst stage. Hum Reprod (1994); 9:2367–73. 102 Scholtes MCW, 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. 103 Ertzeid G, Storeng R. Adverse effects of gonadotrophin treatment on pre- and postimplantation development in mice. J Reprod Fert (1992); 96:649–55. 104 Ertzeid G, Storeng R, Lyberg T. Treatment with gonadotropins impaired implantation and fetal development in mice. J Assist Reprod Genet (1993); 10:286–91. 105 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. 106 Braude PR, Bolton VN, Moore S. Human gene expression first occurs between the four-and eight-cell stages of preimplantation development. Nature (1988); 332:459–61. 107 Taylor DM, Ray PF, Ao A, et al. Paternal transcripts for glucose-6phosphate dehydrogenase and adenosine deaminase are first detectable

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in the human preimplantation embryo at the three- to four-cell stage. Mol Reprod Dev (1997); 48:442–8. 108 Scott LA, Smith S. The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum Reprod (1998); 13:1003–13. 109 Edwards RG, Beard HK. Is the success of human IVF more a matter of genetics and evolution than growing blastocysts? Hum Reprod (1999); 14:1–4. 110 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. 111 Gardner DK, Schoolcraft WB. Human embryo viability: what determines developmental potential and can it be assessed? J Ass Reprod Genet (1998); 15:455–8. 112 Kola I, Sathanathan AH, Gras L. Chromosomal analysis of preimplantation mammalian embryos. In: Trounson A, Gardner DK, eds. Handbook of in vitro fertilization, 2nd ed. Boca Raton: CRC Press (1993): 173–93. 113 Van Blerkom J. Developmental failure in human reproduction associated with chromosomal abnormalities and cytoplasmic pathologies in meiotically mature oocytes. In: Van Blerkom J, ed. In the biological basis of early human reproductive failure. New York: OUP (1994):283–326. 114 Janny L, Menezo YJ. Maternal age effect on early human embryonic development and blastocyst formation. Mol Reprod Dev (1996); 45:31–17. 115 Munne S, Alikani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities. Fertil Steril (1995); 64:382–91. 116 Tesarik J. Developmental failure during the preimplantation period of human embryogenesis. In: Van Blerkom J, ed. In the biological basis of early human reproductive failure. New York: OUP (1994):327–44. 117 Fanchin R, Righini C, Olivennes F, Taylor S, de Ziegler D, Frydman R. Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization. Hum Reprod (1998); 13:1968–74. 118 Lesney P, Killick SR, Tetlow RL, et al. Uterine junctional zone contractions during assisted reproduction cycles. Hum Reprod Update (1998); 4:440–5. 119 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. 120 Gardner DK, Lane M, Schoolcraft WB. Culture and transfer of viable blastocysts: A feasible proposition for human. Hum Reprod (2000); (suppl) In press.

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121 Schoolcraft WB, Gardner DK. Blastocyst culture and transfer increases the efficiency of oocyte donation. Fertil Steril (2000); 74:482–6. 122 Langley M, Marek D, Gardner DK, et al. Rate of blastocyst formation from day three multi-cell embryos. Proc Am Soc Reprod Med (1999) P-028. 123 Milki AA, Fisch JD, Behr B. Two-blastocyst transfer has similar pregnancy rates and a decreased multiple gestation rate compared with three-blastocyst transfer. Fertil Steril (1999); 72:225–8. 124 Gardner DK, Leese HJ. Assessment of embryo metabolism and viability. In: Trounson A, Gardner DK, eds. Handbook of in vitro fertilization. 2nd ed. Boca Raton: CRC Press (1999):347–71. 125 Gardner DK, Lane M, Stevens J, et al. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril (2000); 73:1155–8.

17 Evaluation of embryo quality: a strategy for sequential analysis of embryo development with the aim of single embryo transfer Denny Sakkas

INTRODUCTION The first successful pregnancy after in vitro fertilization (IVF)1 initiated an unforeseen increase in both private and public clinics treating fertility. The ability to stimulate women so that a greater number of eggs could be obtained led to a second major breakthrough in the field of IVF.2 This second achievement however, generated a further problem in IVF as it meant that each couple was more likely to have more embryos at their disposal. Although freezing the embryos was a viable option,3 the preferred choice in many clinics was to transfer an increased number of embryos, hence increasing the chances of infertile couples to achieve pregnancy. This has led to one of the major criticisms of IVF treatment in that it leads to a recognized increase in multiple pregnancy.4 The dangers of multiple pregnancy for the mother and children are extremely high. In a number of countries these dangers have been allayed by legally restricting the number of embryos that it is possible to transfer. For example, in the United Kingdom (UK) the Human Fertilisation and Embryology Authority (HFEA) has restricted the number of transferred embryos to a maximum of three. In the UK and in some other countries there is already discussion to restrict the number of embryos transferred to two, and the transfer of only a single embryo is already being attempted by some clinics.5 In many other countries no legal restriction is in place, and the onus is on individual clinics to decrease the number of embryos transferred so that they achieve a balance between the dangers of multiple pregnancy with a perceived decline in overall pregnancy rates. Further pressures have now come into play in that health insurance companies have realized that the financial costs of supporting infants born from multiple pregnancies after IVF are in many cases higher than the cost of supporting IVF.6 In this case the health insurers may choose not to support clinics that maintain a high multiple pregnancy rate and will only support clinics transferring low numbers of embryos. The current indications are that in the future we will be compelled by a legal, financial, or moral

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obligation to restrict the number of embryos transferred to minimize the risk of multiple pregnancy. This situation leads to fears that there will be a concurrent decline in pregnancy rates for many clinics. Faced with the possibility that in the future we will have to select only one or two embryos for transfer we will be asked to make certain choices. The first would be to rely on less aggressive stimulation protocols, hence generating a lower number of eggs at collection. The second choice is to define a more rigorous selection process for defining the quality of individual embryos so that the ones we choose for transfer are more likely to be viable. This chapter will discuss various selection criteria that will help us achieve this second choice.

EMBRYO SELECTION METHODS Many methods have been suggested to evaluate embryo viability in IVF programs. A limiting factor is that these measurements need to be noninvasive and not time consuming. Routinely, the embryos selected for transfer are chosen on the basis of their morphology and rate of development in culture. In one such study, Cummins et al7 established an embryo quality and development rating and found that good ratings for both were more likely to result in clinical pregnancies. Other studies have also found advantages in transferring embryos on the basis of a morphological and developmental assessment.8–10 In addition to the above methods, the measurement of several metabolic parameters of the embryos by using non-invasive procedures has also been proposed.11,12 In one such study Conaghan et al13 showed that an inverse relation existed between pyruvate uptake and human embryo viability. Jones et al14 have, however, reported that the measurement of glucose metabolism could not be used as a biomarker of viability to select human blastocysts for transfer. Although ideally these methods are potentially of great benefit, they unfortunately require additional expertise and cost to IVF centers, which may be prohibitive in smaller centers. The aim is to find techniques that are simple to perform, convey no deterioration on the embryo, and are highly discriminating. In 1995 Bavister15 highlighted the problem that most clinics have in selecting their best embryos. He stated that the examination of embryos at arbitrary time points during development can be quite misleading with respect to categorizing the stage of development reached and “timeliness” of development. The selection of a critical timepoint is essential so as to maximize the differences between embryos. Observations of embryo development in culture are sometimes made infrequently (commonly at 16–18 hours after insemination to check for pronuclei and again at ~40 hours just before transfer) so that precise data on cleavage timing is usually not available.

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In this chapter a series of simple selection methods will be discussed that in combination will hopefully lead to a selection schedule that will allow us to select one or two embryos of high viability. The selection criteria adopt two key methods similar to those proposed by earlier studies but apply a sequential analysis. The two key criteria are the assessment of morphological markers at different stages and the selection of critical time points. Three key developmental points will be highlighted as markers of selection. The first is assessment of the pronuclear stage embryo, the second the cleavage rate, and the third development to the blastocyst stage. THE PRONUCLEAR STAGE EMBRYO The many transformations that take place during the fertilization process make this a dynamic stage to assess. The oocyte contains most of the developmental materials, maternal mRNA, for ensuring 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.16 The quality of the oocyte therefore plays a crucial part for determining embryo development and subsequent viability. A number of studies have postulated that embryo quality can be predicted from the pronuclear stage embryo. Two recent studies have concentrated on the predictive value of polar body placement and the presence of nucleolar precursor bodies (NPB). Tesarik and Greco17 postulated that the normal and abnormal morphology of the pronucleus was related to the developmental fate of human embryos. They retrospectively assessed the number and distribution of NPB in each pronucleus of fertilized zygotes that led to embryos that implanted. The characteristics of these zygotes were then compared with those that led to failures in implantation. The features that were shared by zygotes that had the 100% implantation success were: (1) that the number of NPB in both pronuclei never differed by more than 3 and, (2) that the NPB were always polarized or not-polarized in both pronuclei but never polarized in one pronucleus and not in the other. Zygotes 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 that had shown the above criteria at the pronuclear stage in those transferred led to a pregnancy rate of 22/44 (50%) compared with only 2/23 (9%) when none were present. A further criterion of pronuclear embryos that may effect embryo morphology is the orientation of pronuclei relative to the polar bodies. Oocyte polarity is clearly evident in non-mammalian 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.18 In human oocytes a differential distribution of various factors within the oocyte has been described and anomalies in

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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.19,20 Following from this hypothesis Garello et al21 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 Smith22 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 with only 4/49 (8%) and 4/178 (2%) in the low embryo score group. CLEAVAGE STAGE EMBRYOS The most widely used criteria for selecting the best embryos for transfer have been based on cell number and morphology.7 A vast number of variations on the theme have been published, however, some recent studies by Gerris et al23 and Van Royen et al5 used 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.5 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 used 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. Most studies that have used and report 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 Bavister15 one of the most critical factors in determining selection criteria

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Fig 17.1 Ideal features shared by pronuclear embryos that have high viability as described by Tesarik and Greco,17 Garello et al,21 and Scott and Smith.22 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. In our own studies we have used cleavage to the 2 cell stage at 25 hours after insemination or microinjection as the critical timepoint for selecting embryos.24,25 In a larger series of patients we have 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 (Table 17.1). Furthermore, nearly 50% of the patients who have two early cleaving 2 cell embryos transferred achieve a clinical pregnancy (Fig 17.2). Interestingly, in a small series of patients where new

Table 17.1. Parameters and pregnancy rates in treatment cycles according to whether embryos had or had not undergone early cleavage to the 2 cell stage by 25 to 26 hours after in vitro fertilization or intracytoplasmic sperm injection. Parameter No early Early cleavage cleavage No of cycles 159 129 No of oocytes collected (mean±SD) 2010 (12.7±8.4) 1701 (13.0±7.9) N° of 2 PN (mean±SD) 1087 (6.9±5.1) 1099 (8.5±5.1) Early 2 cells (mean±SD) 0 355 (2.8±2.0)

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No of embryos transferred (mean±SD) 357 (2.2±0.6) 280 (2.2±0.4) Implantation rate (%) (fetal heart beat/ embryo 58/357 (16.2) 66/280 (23.4)* transferred) No of clinical pregnancies (%) 47 (29.6) 58 (45.0)* *significantly different compared to no early cleavage by P<0.05 and P<0.01 respectively

Fig 17.2 The percentage of clinical pregnancies (light columns) and implantation rate (dark columns) in relation to whether patients had zero, one or two early cleavage embryos transferred. The numbers in parentheses indicate the number of cycles per group. (*−p<0.05 compared to O group).

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Table 17.2. Pregnancy rates achieved when selecting early cleaving 2 cell embryos at 25 to 26 hours after in vitro fertilization or intracytoplasmic sperm injection comparing new sequential complex culture media with a simple medium. Culture medium Simple Complex Complex Parameter Scandinavian IVF G1.2* Mmedium* Cleave** No of cycles 43 30 34 No of cycles with early cleaving 14 (32.6) 19 (63.3) 22 (64.7) 2-cell embryos N° of clinical pregnancies (%) 15 (34.9) 16 (53.3) 18 (52.9) *-Scandinavian IVF Science, Gothenburg, Sweden. **-Ellios BioTek, Paris, France. complex media were used more patients had early cleaving 2 cell stage embryos available for transfer and pregnancy rates were similar to those observed when only early cleaving embryos were transferred (Table 17.2). The embryos that cleave early to the 2 cell stage have also been reported to have a significantly higher blastocyst formation rate.26 It is also interesting to note that, in the embryo scoring system described by Scott and Smith,22 embryos that had already cleaved to the 2 cell stage by 25–26 hours after insemination were assigned an additional score of 10. This score is a sizeable part when the high quality embryos were judged to be those scoring ≥15. DEVELOPMENT TO THE BLASTOCYST STAGE With the advent of new sequential culture media systems our confidence in obtaining blastocysts has increased. 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 Schoolcraft27 is based on the expansion state of the blastocyst and on the consistency of the inner cell mass and trophectodermal cells (Fig 17.3). Examples of high quality blastocysts are shown in Fig 4. In using their grading system when two high scoring blastocysts (>3AA), i.e. expanded blastocoelic cavity with compacted inner cell mass and cohesive trophectodermal epithelium, are transferred a clinical pregnancy an implantation rate of 59/68 (86.7%) and 95/136 (69.9%) can be achieved.28 When two blastocysts not achieving these

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Fig 17.3 The blastocyst grading system. Modified from Gardner and Schoolcraft.27 scores (<3AA) are transferred the clinical pregnancy and implantation rate are significantly lower, 7/16 (43.8%) and 9/32 (28.1%).28 The time of blastocyst formation is also crucial. When we compared cases where only day 5 and 6 frozen blastocysts were transferred with those frozen on or after day 7 and transferred the pregnancy rates were 7/18 (38.9%) and 1/16 (6.2%) respectively.29 In these cases expanded blastocysts with a definable inner cell mass and trophectoderm were frozen. These results showed that even though blastocysts could be

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obtained, the crucial factor was when they became blastocysts. When taking this into account, the best blastocysts would be those that develop by day 5. Selecting the fastest blastocysts may, however, create a bias in sex selection, as Menezo et al30 reported that blastocysts transferred after development in coculture gave rise to the birth of more male offspring. The use of sequential culture media to develop blastocysts does not, however, seem to lead to a bias in the gender of the offspring (DK Gardner, personal communication). This may be related to the rate of development of blastocysts in coculture being slightly slower when compared to the new generation media.

A STRATEGY FOR SELECTING THE BEST EMBRYO The above selection criteria all 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 following hypothetical plan could be followed. 18–19 hours after insemination/ICSI: (Fig 1) The pronuclei are examined for: (a) symmetry (b) the presence of even numbers of NPB (c) the positioning of the polar bodies 25–26 hours after Insemination/ICSI: (Fig 5) (a) embryos that have already cleaved to the 2 cell stage (b) zygotes that have progressed to nuclear membrane breakdown 42–44 hours after insemination/ICSI: (Fig 5) (a) number of blastomeres should be greater than or equal to four (b) fragmentation of less than 20% (c) no multinucleated blastomeres

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Fig 17.4 Blastocyst scores for human blastocysts using the grading system reported by Gardner and Schoolcraft27 (reproduced by kind permission). Embryos considered to have an expanded cavity and well developed inner cell mass and trophectodermal layer would be embryos 1, 2, and 3. Of these blastocysts number 1 has a more expanded blastocoelic cavity, the inner cell mass is tightly packed, and the trophectodermal cells form a cohesive epithelium. This would score as a 4AA. Embryos 4 and 5 are examples of blastocysts that have not yet obtained a well developed blastocoelic cavity and would be scored as grade 1 and 2 blastocysts respectively. 66–68 hours after insemination/ICSI: (Fig 5) (a) number of blastomeres should be greater than or equal to eight (b) fragmentation of less than 20% (c) no multinucleated blastomeres 106–108 hours after insemination/ICSI: (Figs 3 and 4) (a) the blastocoelic cavity should be full (b) inner cell mass should be numerous and tightly packed

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(c) 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

Fig 17.5 Ideal features of embryos scored at 25–26 hours, 42–44 hours and 66–68 hours after insemination/ICSI. For greater details on the scoring criteria see Sakkas et al,24 Shoukir et al,25 and Van Royen et al.5 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 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

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of selection appear to be the selection of a high quality blastocyst on day 5 of development.28 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

Fig 17.6 A strategy for the sequential analysis of embryo development with the aim of selecting a single embryo for transfer. of culturing embryos in groups.31 A further practical issue is that embryos will need to be observed more often, however using drop culture systems under oil should allay this. The extra observations, if performed under a

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controlled heated and gassed climate, should not be detrimental to the embryo. 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 early cleaving 2 cell embryos and assessment of embryos on day 2. One optional observation would be that of the polar body placement, as described by Garello et al.21 This assessment criterion 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.

CONCLUSION For many years we considered the preimplantation embryo as a static entity. By performing embryo culture in a single medium we became complacent about the impact of the culture media on pregnancy and implantation rates. The advent of sequential culture media systems showed us that the embryo is a dynamic entity, changing its needs as it develops. Learning from this lesson we must now adopt a similar strategy in selecting for the best embryo or embryos. We cannot make single static assessments on the day of transfer and believe that this is indicative of a whole series of complex developmental processes. The embryo passes numerous hurdles as it develops; we must assess each of these hurdles so that we can select the most viable embryo. In adopting this strategy we will be able to maintain high pregnancy rates and minimise the risks for our patients.

ACKNOWLEDGEMENTS I would like to thank the members of the Assisted Conception Unit, Birmingham Women’s Hospital, Birmingham, UK for their support in performing some of the studies included in this chapter.

REFERENCES 1 Steptoe PC, Edwards RG. Birth after the reimplantation of a human embryo. Lancet (1978); 2:366. 2 Trounson AO, Leeton JF, Wood C, Webb J, Wood J. Pregnancies in humans by fertilization in vitro and embryo transfer in the controlled ovulatory cycle. Science (1981); 212:681–2. 3 Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature (1983); 305:707– 9.

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4 Martin PM, Welch HG. Probabilities for singleton and multiple pregnancies after in vitro fertilization. Fertil Steril (1998); 70:478–81. 5 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. 6 Wolner-Hanssen P, Rydhstroem H. Cost-effectiveness analysis of invitro fertilization: estimated costs per successful pregnancy after transfer of one or two embryos. Hum Reprod (1998); 13:88–94. 7 Cummins JM, Breen TM, Harrison KL, Shaw JM, Wilson LM, Hennessey JF. 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. 8 Edwards RG, Fishel SB, Cohen J, et al. Factors influencing the success of in vitro fertilization for alleviating human infertility. J In Vitro Fert Embryo Transf (1984); 1:3–23. 9 Hill GA, Freeman M, Bastias MC, et al. The influence of oocyte maturity and embryo quality on pregnancy rate in a program for in vitro fertilization-embryo transfer. Fertil Steril (1989); 52:801–6. 10 Steer CV, 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); 7:117–9. 11 Leese HJ. Analysis of embryos by non-invasive methods. Hum Reprod (1987); 2:37–40. 12 Leese HJ, Hooper MA, Edwards RG, Ashwood-Smith MJ. Uptake of pyruvate by early human embryos determined by a non-invasive technique. Hum Reprod (1986); 1:181–2. 13 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. 14 Jones GM, Trounson A, Vella PJ, et al. Glucose metabolism cannot be used as a biomarker of viability to select human blastocysts for transfer, 11th World Congress on In Vitro Fertilization and Human Reproductive Genetics, Sydney, Australia 1999; 0–163. [Abstract.] 15 Bavister BD. Culture of preimplantation embryos: facts and artefacts. Hum Reprod Update (1995); 1:91–148. 16 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. 17 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.

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18 Gardner R. The early blastocyst is bilaterally symmetrical and its axis of symmetry is aligned with the animalvegetal axis of the zygote in mouse. Development (1997); 124:289–301. 19 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. 20 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. 21 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. 22 Scott LA, Smith S. The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum Reprod (1998); 13:1003–13. 23 Gerris J, De Neubourg D, Mangelschots K, Van Royen E, Van de Meerssche M, Valkenburg M. Prevention of twin pregnancy after invitro fertilization or intracytoplasmic sperm injection based on strict embryo criteria: a prospective randomized clinical trial. Hum Reprod (1999); 14:2581–7. 24 Sakkas D, Shoukir Y, Chardonnens D, Bianchi PG, Campana A. Early cleavage of human embryos to the twocell stage after intracytoplasmic sperm injection as an indicator of embryo viability. Hum Reprod (1998); 13:182–7. 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 Platteau P, Fenwick J, Herbert C, et al. Early cleavage of human embryos to the two-cell stage: Pregnancy, implantation rate and blastocyst formation, 11th World Congress on In Vitro Fertilization and Human Reproductive Genetics, Sydney, Australia 1999;0–161. [Abstract.] 27 Gardner DK, Schoolcraft WB, Jansen R, Mortimer D, eds. Towards reproductive certainty: infertility and genetics beyond. Carnforth: Parthenon Press; 1999; In vitro culture of human blastocysts. p. 378. 28 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. 29 Shoukir Y, Chardonnens D, Campana A, Bischof P, Sakkas D. The rate of development and time of transfer play different roles in influencing the viability of human blastocysts. Hum Reprod (1998); 13:676–81.

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30 Menezo YJ, Chouteau J, Torello J, Girard A, Veiga A. Birth weight and sex ratio after transfer at the blastocyst stage in humans. Fertil Steril (1999); 72:221–4. 31 Lane M, Gardner DK. Effect of incubation volume and embryo density on the development and viability of mouse embryos in vitro. Hum Reprod (1992); 7:558–62.

18 Oocyte cryopreservation Eleonora Porcu

Our knowledge of the cryopreservation of human embryos in liquid nitrogen has made great steps forward in recent times. The excess number of human embryos that are often present in the programs of in vitro fertilization/embryo transfer (IVF/ET) were a force in perfecting this process. Not all the embryos developed can be transferred, owing to the risk of multiple pregnancies, thus necessitating the storage of the surplus embryos in liquid nitrogen. However, doctors, patients, legislators, and, above all, the public have legal, moral and religious problems with the cryopreservation of human embryos. The use of this technique has been restricted or even forbidden in some countries, such as Germany, Austria, Switzerland, Denmark and Sweden.1 One solution to these problems could be the cryopreservation of female gametes. The condition of iatrogenic sterility after chemo- or radiotherapy in neoplastic pathologies would be avoided by the preservation of oocytes, as in the cryostorage of sperm. In addition, even women who suffer from pathologies of the reproductive system such as the functioning of the ovaries (premature ovarian failure, endometriosis, cysts, and pelvic infections) could insure potential fertility using this technique, which was unheard of until recently. The use of frozen oocytes in a program of assisted fertilization would be able to guarantee the maintenance of fertility in patients with these pathologies. The cryopreservation of oocytes could also allow women who delay maternity due to career demands, the lack of a partner, or to pathologies that momentarily prevent pregnancy, another choice in family planning. And, as a last point, the use of frozen oocytes could be included in a program of oocyte donation. The storage of male gametes or human embryos has faced fewer problems than the cryopreservation of oocytes. This is due to the biological features of the oocytes, and various questions have been raised about inducing aneuploidy after the gametes have been exposed to cryoprotectants and the freezing and thawing process. The oocytes are, in fact, blocked at ovulation at the metaphase of the second meiotic division, where 23 dichromatidic chromosomes are bound to the microtubules of the meiotic spindle. In this phase, when the oocytes are extremely sensitive to changes in temperature and the eventual depolymerization of the microtubules of the spindle caused by cryoprotectants or ice crystals formed during the freezing and thawing process, the normal separation of the chromatids at the moment of fertilization could be impaired, thus inducing aneuploidy after the

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extrusion of the second polar body. As cited in the literature, the low number of pregnancies after the cryopreservation of oocytes2–7 shows the important technical difficulties this procedure faces. There are five important stages in the cryopreservation procedure: (1) initial exposition to cryoprotectants, substances that reduce cellular damage caused by the crystallization of water, (2) freezing to temperatures below 0°C, (3) storage, (4) thawing, (5) dilution and removal of the cryoprotectants, and the return to a physiological microenvironment, thus allowing further development. The most potentially critical moments for cellular survival are the initial phase of freezing at a very low temperature and the final return to physiological conditions. If a sufficiently low temperature is reached (normally −196°C, the temperature of liquid nitrogen), storage, even for a lengthy period of time, has no effect on the successive survival rate. At this temperature, in fact, there is not sufficient energy available for most physiological reactions and water molecules are aligned in a glassy, crystalline structure. The breakdown of DNA caused by cosmic radiation is the only potential damage for gametes and embryos stored at this temperature. When an oocyte is cooled at a temperature between −5°C and −15°C, ice formation is first induced in the extracellular medium as a result of a process called seeding. When the temperature decreases, the amount of ice increases and the solutes concentrate in the extracellular medium. The result is the creation of an osmotic gradient. As a result of this gradient, water is drawn from the cytoplasm to the extracellular medium, and the cell becomes smaller. If this process is sufficiently slow, the passage of considerable water out of the cell will decrease the probable nucleation of ice within the cell, to approximately −15°C. The rate of the modification of the cell volume as a function of its permeability, membrane area, and temperature8 is represented by a mathematical model. For those cells with a low surface/volume ratio, such as gametes, a low freezing rate is necessary in order to allow enough water to move out of the cell. In this way, the intracellular ice crystals that form would be small enough not to damage the intracellular components. It must be emphasized, however, that an increase in the freezing rate reduces the survival rate of any type of cell. The optimal freezing rate depends on various variables: the cytosolic water content, the changing permeability constant of the membrane, the area of the membrane, and the temperature. The intracellular water content, besides causing mechanical damage at the moment of freezing, may also cause material damage at thawing, as the result of an increase in volume during this process. Indeed, if thawing occurs slowly, the survival rate decreases because the crystals formed in the cytosol have enough time to grow, thus damaging intracellular structures. Recrystallization and osmotic shock, which occur during the thawing of frozen oocytes, may effectively reduce the survival rate.

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Recrystallization is the process by which water goes back into the cell taking on a solid state around small ice crystals previously formed in the cytosol. When the temperature is raised to −40°C, some water molecules may return along the track followed during freezing, thus returning to the cytosol and reforming hydrogen bonds with the ice crystals already present which significantly increases the dimension of the cell. Both the thawing and the freezing rates influence the probability of recurrence of this phenomenon. Cellular dehydration is probably insufficient after rapid freezing, allowing the formation of large intracellular masses if the thawing process is carried out very slowly. The formation of intracellular ice can be avoided if rapid thawing takes place at the nucleation point of the ice. Osmotic shock may take place during rapid thawing. In fact, if the cryoprotectant previously put into the cell does not diffuse rapidly enough to prevent an influx of water, the oocyte will swell and burst. In this phase, two opposing needs must be faced: on the one hand, the contact time between the cell and the cryoprotectant at room temperature must be reduced to a minimum since the cryoprotectant provokes a temperature dependent cytotoxity; on the other hand, the process of dilution of the cryoprotectant in the cytosol must be done very slowly in order to avoid excessive reduction of the extracellular osmotic potential, thus causing a large influx of water into the cell with consequent cellular lysis. When examining the literature, we noted that research performed until now on the cryopreservation of human oocytes has offered contrasting information as to the ideal method which does the least damage to cellular integrity. The oocyte itself and the technique used are the principal factors involved in the success of cryopreservation.

OOCYTE RELATED VARIABLES The size of the oocyte influences the overall survival rate, just as the probability of intracellular ice formation depends on it. Human sperm offers a good example of the influence of cytoplasmic volume on survival after cryopreservation; male gametes are 180 times smaller than female gametes, and their survival rate is much higher. Optimal quality of the oocyte is essential in order to guarantee its survival upon freezing. Frequently, low quality supernumerary oocytes are frozen which results in low survival rates. For this reason, some authors, such as Chen,2 chose to freeze all the best oocytes available. Regarding this parameter, the four most important aspects involved in the evaluation of oocyte quality are: nuclear stage, cytoplasmic characteristics, aspect of the corona radiata, and expansion of the cumulus cells. Different authors argue both for and against the maintenance of the cumulus oophorus to optimize the survival rate. Chen and Van Uem affirmed that its absence facilitates the penetration of the cryoprotectant into the cytoplasm; in effect the first pregnancies were achieved after

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thawing cryopreserved oocytes without cumulus.2,4 Even Gook et al reported an increased survival rate of frozen oocytes without the cumulus with respect to those maintaining cumulus (69% v. 48%).9 On the other hand, several studies showed the importance of the presence of the cumulus which guarantees greater cellular survival at the end of the cryopreservation process.11–13 Some authors hypothesized that the presence of cumulus cells is able to act as a protective shield against sudden osmotic modifications and stress caused by the sudden concentration and dilution of cryoprotectants during the process of equilibrium and removal after thawing. In our experience, the presence of the cumulus does not seem to significantly condition oocyte survival7 and agrees with the experience of Mandelbaum et al.14 All oocytes should be frozen shortly after being harvested, between 38 and 40 hours after human chorionic gonadotrophin (hCG).3 The older oocytes, cultured in vitro before freezing, present a significantly reduced fertilization potential, and an increase in anomalous fertilizations and polyploidy.15 All the pregnancies achieved with frozen oocytes come from metaphase II oocytes. In fact, oocytes which are mature at pickup have higher survival and fertilization rates.5 The cryopreservation of oocytes in prophase I has been proposed as an alternative approach in the storage of female gametes. In these oocytes, meiosis is arrested, and the chromosomes are inside the nucleus, not aligned along the spindle. Furthermore, in this stage, the cells are small and undifferentiated, lacking a zona pellucida, and are comparatively quiescent from a metabolic point of view. Given these theoretical data, various groups of researchers have examined the potential of cryopreserved oocytes in prophase I to mature, to become fertilized, and to develop into embryos. Mandelbaum et al obtained discouraging results.14 In fact, given the low survival rate (37%) and the rate of in vitro maturation on thawing (20%), these researchers deduced that prophase I, although theoretically not susceptible to the damage of “cold shock,” is not the best stage in which to freeze oocytes. Toth et al have studied fresh and cryopreserved immature oocytes again at prophase I in order to compare the maturation rate at metaphase II,15 fertilization and maturation. The authors showed that immature human oocytes are capable of surviving cryopreservation and maturing to MII. Moreover, frozen oocytes maintain the same possibilities of fertilization and maturation when compared to unfrozen control oocytes. Two different methods for the cryopreservation of immature oocytes were compared in a subsequent study.16 Differences in freezing and thawing rates, in the temperature chosen for the seeding, and in the utilization or not of sucrose as a cryoprotectant distinguished one technique from the other. Method I consisted of the slow freezing/slow thawing rate, and a seeding at −6°C and method II involved rapid freezing and rapid thawing, with the use of sucrose as cryoprotectant and seeding at −7°C. The results reported confirmed that oocytes at prophase I are capable of surviving cryoconservation and maturing to metaphase II after thawing. Both

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protocols result in the same amount of mature oocytes. Important clinical applications may be observed even if the study performed by the authors did not consider the fertilization capacity and the development of such treated oocytes. Those patients who desire to maintain their own reproductive potential despite chemoor radiotherapy or ovariectomy, may benefit from this technique combined with IVF. A recent study by an Australian group was conducted to evaluate the survival rate after freezing and thawing immature oocytes using PROH as a cryoprotectant, where dosages and exposure time to the cryoprotectant were the same as those used for storing mature oocytes.17 Encouraging results were obtained, showing the efficacy of this method and proposing the strategy of freezing immature oocytes as a feasible therapeutic method for the future. Innovative strategies have recently been suggested in order to preserve fertility in those patients undergoing antineoplastic treatments or removal of the ovaries. A new freezing technique to store thin slices of ovarian parenchyma has been investigated by Hovatta et al.18 The ovarian cortex is rich in follicles at different stages of maturation, in particular primordial follicles. It has been possible to store slices of ovarian cortex for a period varying from 24 hours to five weeks using two different freezing protocols (DMSO 1.5 M+ PROH 1.5 M+sucrose 0.1M). A water bath at 37°C was used in both cases to quickly carry out the thawing procedure. On histological examination before and after freezing in either protocol, the authors did not find any difference. The authors affirm that, because of the normal state of follicles after freezing and thawing, the oocyte should be capable of in vitro maturation and fertilization, under adequate stimuli. Newton et al19 and Gosden et al20 demonstrated that thin slices of ovarian parenchyma, frozen and successively thawed, could be grafted in the abdomen to permit the maturation of primitive and primordial follicles. They showed that the cryoprotectants ethylene glycol and DMSO effectively reduced the damage done to the parenchyma by ice crystals, and high survival rates for the follicles were obtained. The authors concluded that, although further improvement of the technique of cryopreservation and transplantation is necessary, the results are sufficiently encouraging, and banking of ovarian tissue is suggested as a valid method of preserving fertility in selected cases. If orthotopic autograft is capable of re-establishing ovulatory menstrual cycles, the necessity of ovulation induction and in vitro fertilization is eliminated. Pediatric patients may also benefit from this technique. In fact, most primordial follicles and the prepubertal quiescent state of the ovary may improve the possibility of success, and in certain cases, ovarian tissue banking is the only option of maintaining fertility that is available.

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TECHNICAL VARIABLES Cryoprotectants are substances that have different chemical compositions. They share a high water solubility associated with toxicity, which is directly proportional to their concentration and temperature. Their role is to protect cells from any damage, known as “cold shock,” which may occur during the procedures of freezing, storing, and thawing. Cryoprotectants are divided into two categories according to their capacity of penetrating the cells: intracellular and extracellular agents. Biochemically, it is possible to distinguish three classes of cryoprotectant compounds: Alcohols (methanol, ethanol, propanol, 1,2 propanediol (PROH), glycerol), sugars (glucose, lactose, sucrose, starch) and dimethylsulfoxide (DMSO). DMSO AND GLYCEROL DMSO and glycerol, both of which have a low molecular weight, have been recognized as cryoprotectants against freezing damage for the past 30 years. They have both been used in different protocols. In 1988, Friedler demonstrated that DMSO was more effective than glycerol.21 1,2 PROPANEDIOL (PROH) PROH has been used, for the most part, in blastocysts and pre-embryo cryopreservation both in humans and other species. In combination with other agents that reduce its toxicity and osmotic power, PROH seems to obtain a better oocyte survival rate after thawing;22 this characteristic may be due to the fact that PROH penetrates the oolemma more rapidly; it is also more water soluble21 and less toxic.12 SUCROSE Sucrose is often used together with other cryoprotectants. It is not able to penetrate through the cellular membrane, and its presence in extracellular media can exert a significant osmotic effect. Sucrose is protective during the dilution phase or after rapid thawing, when cell begin to rehydrate and swell. This risk may be reduced by removing the intracellular cryoprotectants (for example, PROH) in a stepwise dilution (1.5; 1.0; 0.25 M) in order to reduce the amount of cellular swelling. An alternative and more rapid method of removing permeating cryoprotectants is tied to the addition of nonpermeating molecules such as sucrose to the thawing solution. The elevated extracellular concentration of these molecules balances the high concentration of intracellular cryoprotectant, reducing the osmolarity differences on the two sides of the plasmatic membrane.

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Today, sucrose is the only non-penetrating cryoprotectant routinely utilized in human oocyte cryopreservation. The mechanism of action of cryoprotectants is quite complex and is due to a series of properties. First of all, the presence of cryoprotectants in the solution determines a slight lowering of the cryoscopic point of the solution, approximately −2°C or −3°C. The protective effects are principally a result of the capacity of these molecules to form hdyrogen bonds which alter the normal crystal structure of water, thus reducing its dimension. Through their −OH groups, glycerol and PROH, for example, may form hydrogen bonds with water as does DMSO through its oxygen atoms. Cryoprotectant agents reduce the damaging effects of the high concentration of electrolytes in the liquid water portion. In systems that are constituted by two phases at a constant pressure, such as ice and water, the total concentration of solutes in the liquid phase is constant for each concentration. Since the total concentration of the solutes must be constant, the addition of cryoprotectants reduces the amount of water that crystallizes.21 The efficacy of these compounds is directly related to the temperature at which they are added to the culture medium. In 1991, Pickering demonstrated that human oocytes exposed to DMSO at a temperature of 37°C lose the capacity to be fertilized;23 but at 4°C this capacity is maintained. Even though Pickering did not freeze the oocytes, the results of this study suggest that the addition of a cryoprotectant to the medium must take place at a temperature below 10°C in order to avoid fertilization failure. Optimal cryoprotectant concentration varies according to the cell type and the species type under examination.24 In 1988, Sathananthan demonstrated that exposure time to DMSO can influence the amount of damage to the meiotic spindle;25 only 60 minutes of exposure at a concentration of 1.5 M is sufficient to determine important modifications of its structure in the majority of the oocytes and this is not reversible, while after 10–20 minutes the spindle still presented structural integrity. Van der Elst et al found that the exposition of the oocytes to PROH 1.5M for a brief period was harmless.26 An important step in the process of cryopreservation is the removal of the permeating cryoprotectant from the cytoplasm.23 The procedure consists the passage of the oocyte through a series of solutions containing gradually diminishing concentrations. As a result of the effect of osmotic pressure, the cells would explode if placed in a medium without cryoprotectant immediately upon thawing. The freezing and thawing rates condition the diffusion of water through the cellular membrane. Furthermore, the choice of the optimal thawing rate depends on the rate at which freezing has taken place as described above. By means of varying the rates of freezing and thawing, several protocols for oocyte cryopreservation have been used. Oocyte storage is often performed with a slow freeze-rapid thaw procedure. Chen achieved the first worldwide pregnancy with this

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protocol.2 The same strategy was adopted by Siebzehnruebl et al in 1989.27 Although uncommon and rarely reported in literature, slow freezing/slow thawing resulted in the second pregnancy with a frozen oocyte.4 The authors used DMSO 1.5M as a cryoprotectant and oocyte thawing was performed at room temperature. With the use of high concentrations of cryoprotectants, ultrarapid freezing/rapid thawing prevents the formation of ice crystals and induces a glassy, amorphous medium. Trounson first applied this strategy to human oocyte cryopreservation by the direct immersion of ova in liquid nitrogen (ultrarapid freezing).28 Rapid thawing was performed at 37°C in a water bath. Nine of 18 mature human ova thus treated survived to thawing, but all of them degenerated in culture. In another process called vitrification, a highly concentrated solution of cryoprotectants solidifies during freezing without the formation of ice crystals, in a supercooled, highly viscous fluid. It shows some clear advantages compared with simple freezing because the damage caused by intracellular ice crystal formation is avoided. The combination of a high cooling rate (nearly 1500°C/min) and high concentrations of cryoprotectants such as DMSO, acetamide, propyleneglycol and polyethyleneglycol are required for vitrification. The theoretical basis of vitrification was clearly expressed by Rall and Fahy as a technique for preserving the embryos.29 However, the results are not in accord, and the toxicity of the cryoprotectants is confirmed by experimental studies.21 Trounson reported acceptable survival and fertilization rates but low cleavage rates.28 The cleavage block may be related to the irreversible damage induced in the cytoskeleton by the association of cooling and vitrification solutions. Hunter et al investigated human oocyte vitrification in order to demonstrate the feasibility of this procedure.30 According to their data, mature human oocytes are able to tolerate vitrification at room temperature but survival rates decrease if vitrification is performed at −196°C. The researchers obtained good survival and fertilization rates, but the oocytes treated in this way showed a strongly impaired cleavage rate. This phenomenon may be due to irreversible cytoskeletal damage when freezing is associated with vitrification.

EFFECT OF FREEZING ON OOCYTE STRUCTURE The process of freezing/thawing and the cryoprotectants can damage several cell structures.

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CHROMOSOME AND MEIOTIC SPINDLE The meiotic spindle is made up of fragile fibers originating from the opposing poles of the cell, from a structure called the centriole, and extending to the chromosomes. Any loss of the microtubules during freezing could separate the chromosomes and cause aneuploidy.11,21,25,26,31 Gook et al showed in 1994 that normal fertilization can be achieved in cryopreserved oocytes, suggesting that reasonable integrity is preserved after cryopreservation.13 As a result of karyotyping and DNA staining, chromosomes were shown not to have been lost from the spindle during the fertilization of frozen oocytes. Hence the suggestion that oocyte cryopreservation would result in a high rate of aneuploid embryos, owing to the loss of chromosomes from the spindle, is unfounded for human oocytes. Probably the chromosomes are anchored via the associated kynetochores and are not free to move in the cytoplasm. It is probable that the human oocyte spindle is less sensitive to freezing compared with the mouse spindle. Chromosomal loss from the spindle is minimal in human oocytes after freezing/thawing and fertilization, suggesting that the cryodamage documented in animals is not as common in human oocytes. CYTOSKELETON The cytoskeleton is constituted of a complex fibrillary cytoplasmic structure, the purpose of which is to maintain and modify the form, allowing the movement of cytoplasmic organelles, exocitosis as well as that of the intrinsic membrane proteins. Microtubules, microfilaments of actin, and intermediate filaments are the major components of the cytoskeleton. The totality of components is quite sensitive to various stimuli and is capable of rapid depolymerization of the subunits. Vincent et al showed that the cryoprotectant DMSO produces notable damage in the microfilaments of murine oocytes, which is directly proportional to its concentration.32 When DMSO is used at a temperature close to 0°C, this effect seems to be reduced. Alterations in the components of the cytoskeleton, produced by ice crystals or cryoprotectants in frozen/thawed oocytes have also been postulated by Hunter et al33 and Van Blerkom and Davis.34 Younis et al obtained identical results,35 and even if it was not possible to affirm whether the cytoskeleton damage caused by the cryoprotectant results directly from the components used or as a result of osmotic modifications, it is clear that the alterations are directly proportional to the concentration of the cryoprotectants, their exposure time and the temperature at which they are added to the culture medium.

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CORTICAL GRANULES According to some investigations, the oocytes that survive thawing demonstrate a high aneuploidy rate when fertilized in vitro.36 Normally, cortical granules in mature oocytes are aligned immediately under the oolemma. The zona reaction takes place after the movement of these granules to the periphery of the cytoplasm and is responsible for the block of polyspermy. When using the electron microscope to study human and murine oocytes,37 a significant reduction was found in the number of and morphological alterations of cortical granules after thawing. This observation might explain the high incidence of aneuploidy in frozen/thawed oocytes. Van Blerkom and Davis noticed that the premature exocytosis of cortical granules may lead to a sudden zona hardening and, as a consequence, to a reduction of the fertilization rate (IVF).34 The premature release of cortical granules might be due to the damage caused by ice crystals or by cryoprotectants on the microfilaments of actin present just below the oolemma.24,34 In their study, Gook et al found an elevated number of cortical granules in frozen thawed oocytes, indicating that neither the cryoprotectants nor the low temperature reduce the release of these organelles.9 ZONA PELLUCIDA A common characteristic of all mammarian oocytes is the presence of a glycoprotein layer, the zona pellucida, just external to the oolemma. The functions of the zona pellucida are multiple and only partly understood. The best known ones include: the presentation of receptors to sperm, the induction of the zona reaction, the block of polyspermy and the physical protection of the embryo. Various researchers have warned of the risk of damaging the zona pellucida during cryopreservation.1,38 In particular, lesions have been observed in 20–29% of the oocytes. The damage to the zona pellucida is thought to result from the formation of cleavage planes in the ice or to the formation of large crystals, which may trap and perforate the cell during the freezing/thawing process. PARTHENOGENETIC ACTIVATION Since 1940 it has been shown that parthenogenetic activation can be induced by physical conditions such as freezing. Successively, it was found that thermal shock in the form of heat and cold could be effective as a parthenogenetic activator in some animal species. Gook et al observed that fresh and aged cryopreserved human oocytes underwent parthenogenesis in 27 and 29% of oocytes, respectively.39

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SURVIVAL AND FERTILIZATION RATES OF FROZEN/THAWED OOCYTES There is considerable variance in the literature concerning the survival rate of human oocytes at thawing. The results of Chen, who reported a 76% survival rate, are classified among the highest.3 It must be emphasized that this author has frozen only oocytes of optimum quality in metaphase II. Mean survival rates inferior to those of Chen were reported by AI-Hasani et al (25%),5 who froze only supernumerary oocytes and not always those of good quality. Substantially low percentages were obtained by Kazem et al (34.4%)1 and Tucker et al (24.7%).6 Gook et al documented a variable survival rate between 48% and 95%.40 This author used 1,2 propanediol and observed a greater survival rate freezing oocytes without the cumulus (69%) with respect to those with the cumulus oophorus (48%). The variability of the results can be partly accounted for by the substantially low number of thawed oocytes in all the studies. Our center has presented the largest case study of thawed oocytes reported in the literature until now, and our mean survival rate varies from 57% to 58%.7 The in vitro fertilization rate of frozen/thawed oocytes is quite variable and ranges from little more than 13%1 to 71%.3 In most studies, the variability is between 30% and 55%, which is inferior to the fertilization rate of “fresh” oocytes. Anomalous fertilizations, these being generally polyploids, range from 5%40 to 15.3% and are calculated on the number of inseminated oocytes.5 The often reduced percentage of fertilization and the rather high incidence of anomalous fertilizations in cryopreserved oocytes have been related to possible damage of the zona pellucida and cortical granules, which interfere with the correct interaction with the spermatozoa. Intracytoplasmic sperm injection (ICSI) has recently been proposed as a solution for these problems. In 1995, Gook’s group obtained normal fertilization in 50% of cases using this technique.40 This was associated, however, with a 21% abnormal fertilization rate. It is important to note that with respect to those embryos obtained with traditional IVF, the embryos derived from the ICSI technique demonstrated a greater capacity of cell division for a number of days. Analogous experiences were conducted by Kazem et al, who documented 43.2% of normal fertilizations with ICSI1 and by Tucker et al, who obtained 65% of normal fertilizations and three pregnancies, all of which resulted in abortions.6 After a preliminary experience with IVF involving frozen oocytes and resulting in a fertilization rate of 46%, we undertook a study associating the ICSI technique with the freezing of female gametes. Superovulation was induced through a combination of a gonadotropin releasing hormone (GnRH) analogue and gonadotropins.41 The oocytes were cryopreserved using a slow freeze/rapid thaw protocol and PROH plus sucrose as cryoprotectants. Six hours after collection, the cumulus-corona complex

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of each oocyte was removed by briefly exposing it for 30–40 seconds to a buffered culture medium containing 40IU/ml of hyaluronidase and aspirating it through hand-drawn glass pipettes. Afterwards, the oocytes were examined under an Olympus IMT-2 inverted microscope at ×400 magnification and assessed for their nuclear maturity. The oocytes were cryopreserved using a slow freeze-rapid thaw protocol. They were equilibrated for 10 minutes in phosphate buffered saline (PBS) supplemented with 1.5M 1,2-propanediol (PROH) and 30% Plasmanate. After equilibration, the oocytes were transferred in PBS supplemented with 1.5M 1,2-propanediol, 0.2M sucrose and 30% Plasmanate, loaded into plastic straws and placed in an automated Kryo II 10/17 biological vertical freezer (Planer Product, Datamed, Milano, Italy) with the chamber temperature at 23°C. The temperature was slowly reduced from +20°C to −8°C at a rate of −2°C per minute. Ice nucleation was induced manually by seeding. Then the temperature was gradually reduced to −30°C at a rate of −0.3°C/min and rapidly lowered to −150°C at a rate of −50°C/min. After 10 minutes of temperature equilibration, the straws were transferred into liquid nitrogen tanks and stored. For thawing procedure the straws were removed from liquid nitrogen, held at room temperature for 30 seconds, and put into a 30°C water bath for 40 seconds. The cryoprotectants were removed by stepwise dilution. The oocytes were equilibrated in 1.0, 0.5 M PROH solution for five minutes, then in 0.2 M sucrose and PBS for 10 minutes, and finally transferred to a fresh culture medium at 37°C in an atmosphere of 5% CO2 for three hours before ICSI. Sperm selection was done by the minipercoll technique. The sperm suspension was kept in the 37°C incubator until the intracytoplasmic injection of the oocytes. The ICSI technique was performed as previously reported. During ICSI, technical damage occurred in 7% of the oocytes,42 a lower rate than that reported by Kazem et al in 1995 (32%)1 and by Gook et al in the same year (26%).40 The percentage of normal fertilizations obtained in our study was 64.3%,43–45 similar to those reported by Tucker et al (1996).6 Abnormal fertilizations amounted to 7.2%, which was the same as that found by our group using IVF and in the ICSI of “fresh” oocytes. Most of the embryos were of good quality and showed a satisfactory tendency for subsequent division. PREGNANCIES AND BIRTHS In 1986, Chen reported the first pregnancy from frozen thawed oocytes.2 One year later, two additional pregnancies and births were reported.34 Ten years later, our group reported the first birth of a healthy baby girl, conceived associating the techniques of freezing oocytes and ICSI.7 At the moment we have obtained 16 pregnancies and the birth of 11 normal and healthy babies.46 The best results were obtained with the transfer of the embryos in a delayed hormonal replacement cycle.

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Oocytes cryopreservation can be used for several clinical applications. A further step of our investigation was the fertilization of frozen oocytes with frozen sperms that resulted in the birth of a healthy baby boy. This case documents that both female and male cryopreserved human gametes can be safely used together to obtain healthy offspring. In addition, we obtained pregnancies from frozen oocytes and epididimal and testicular sperms.47,48 Ovarian hyperstimulation syndrome is another clinical condition in which the elective cryopreservation of all the oocytes is a valid alternative devoid of ethical implications compared with embryo freezing. We currently choose this option for all the patients at risk of developing severe ovarian hyperstimulation syndrome. Finally, we have started an oocyte cryopreservation program for oncological patients, in which 13 subjects have been included so far. The cryopreservation of oocytes in the past was considered to be an inefficient technique, involving poor survival, fertilization, and cleavage rates. With the introduction of the ICSI technique, the results in terms of fertilization, embryo development, and implantation have become similar to those obtained with fresh oocytes. The only critical step of the process currently appears to be the survival rate of the oocytes after thawing, and this should be improved upon. The safety of this technique has been widely debated. One of the most important concerns regards the possible damage to the meiotic spindle and the consequent induction of aneuploidy. However, the results obtained by Gook et al9,13,39 have reassuringly shown that, in cryoconserved oocytes, there is a normal genetic patrimony devoid of stray chromosomes. It is, therefore, most probable that in the process of freezing only the best and most resistant oocytes are selected, those able to survive different types of stress.

REFERENCES 1 Kazem R, Thompson LA, Srikantharajah A, Laing MA, Hamilton MPR, Templeton A. Cryopreservation of human oocytes and fertilization by two techniques: in-vitro fertilization and intracytoplasmic sperm injection. Hum Reprod (1995); 10:2650–4. 2 Chen C. Pregnancy after human oocyte cryopreservation. Lancet (1986); i:884–6. 3 Chen C. Pregnancies after human oocyte cryopreservation. Ann NY Acad Sci (1987); 541:541–9. 4 Van Uem JFHM, Siebzehnrubl ER, Schun B, Koch R, Trotnow S, Lang N. Birth after cryopreservation of unfertilized oocytes. Lancet (1987); i: 752–3.

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5 Al-Hasani S, Diedrich K, Van der Ven H, Reinecke A, Hartje M, Krebs D. Cryopreservation of human oocytes. Hum Reprod (1987); 2:695– 700. 6 Tucker M, Wright G, Morton P, Shanguo L, Massey J, Kort H. Preliminary experience with human oocyte cryopreservation using 1,2 propanediol and sucrose. Hum Reprod (1996); 11:1513–5. 7 Porcu E, Fabbri R, Seracchioli R, Ciotti PM, Magrini O, Flamigni C. Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes. Fertil Steril (1997); 4:724–6. 8 Mazur P. Limits to life at low temperatures and at reduced water activities. Orig Life (1980); 10:137. 9 Gook D, Osborn S, Johnston W. Cryopreservation of mouse and human oocytes using 1,2 propanediol and the configuration of the meiotic spindle. Hum Reprod (1993); 8:1101–9. 10 Pellicer A, Lightman A, Parmer TG, Behrman HR, De Cherney AH. Morphologic and functional studies of immature rat oocyte-cumulus complexes after cryopreservation. Fertil Steril (1988); 50:805–10. 11 Sathananthan AH, Kirby C, Trounson A, Philipatos D, Shaw J. The effects of cooling mouse oocytes. J Assist Reprod Genet (1992); 9:139–48. 12 Imoedmhe DG, Sigue AB. Survival of human oocytes cryopreserved with or without the cumulus in 1,2propanediol. J Assist Reprod Genet (1992); 9:323–7. 13 Gook D, Osborn S, Bourne H, Johnson W. Fertilization of human oocytes following cryopreservation; normal karyotipes and absence of stray chromosomes. Hum Reprod (1994); 9:684–91. 14 Mandelbaum J, Junca AM, Tibi C, et al. Cryopreservation of immature and mature hamster and human oocytes. In Vitro Fertil and Assist Reprod (1988); 541:550–61. 15 Toth T, Baka S, Veeck L, Jones H, Muasher S, Lanzendorf S. Fertilization and in vitro development of cryopreserved human prophase I oocytes. Fertil Steril (1994); 61:891–4. 16 Toth TL, Lazendorf SE, Sandow BA, et al. Cryopreservation of human prophase I oocytes collected from unstimulated follicles. Fertil Steril (1994); 61:1077–82. 17 Gook D, Osborn SM, Bourne H, Johnston WIH, Speirs AL. Mature and immature human oocyte cryopreservation. In: Porcu E, Flamigni C, eds. Human oocytes: from physiology to IVF. Bologna: Monduzzi (1998):279–84. 18 Hovatta O, Silye R, Krausz T, et al. Cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants. Hum Reprod (1996); 11:1268–72. 19 Newton H, Aubard Y, Rutherford A, Sharma V, Gosden R. Low temperature storage and grafting of human ovarian tissue. Hum Reprod (1996); 11:1487–91.

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20 Gosden R. Ovarian tissue banking. In: Porcu E, Flamigni C, eds. Human oocytes: from physiology to IVF. Bologna: Monduzzi (1998):265–9. 21 Friedler S, Giudice L, Lamb E. Cryopreservation of embryos and ova. Fertil Steril (1988); 49:743–64. 22 Baka SG, Toth TL, Veeck LL, Jones HW, Muasher SJ; Lanzendorf SE. Evaluation of the spindle apparatus of invitro matured human oocytes following cryopreservation. Hum Reprod (1995); 10:1816–20. 23 Pickering S, Braude P, Johnson M. Cryoprotection of human oocyte: inappropriate exposure to DMSO reduces fertilization rates. Hum Reprod (1991); 6:142–3. 24 Vincent C, Pruliere G, Pajot-Augy E, Campion E, Garnier V, Renard JP. Effects of cryoprotectants on actin filaments during cryopreservation of one-cell rabbit embryos. Cryobiology (1990); 127:9–23. 25 Sathananthan AH, Trounson A, Freeman L, Brady T. The effects of cooling human oocytes. Hum Reprod (1988); 8:968–77. 26 Van Der Elst J, Van Den Abbeel E, Nerinckx S, Van Steirteghem A. Parthenogenetic activation pattern and microtubular organization of the mouse oocyte after exposure to 1,2-propanediol. Cryobiology (1992); 29:549–62. 27 Siebzehnruebl ER, Todorow S, Van Uem J, Koch R, Wildt L, Lang N. Cryopreservation of human and rabbit oocytes and one-cell embryos: a comparison of DMSO and propanediol. Hum Reprod (1989); 4:312– 17. 28 Trounson A. Freezing human eggs and embryos. Fertil Steril (1986); 46:1–12. 29 Rall WF, Fahy GM. Ice free cryopreservation of mouse embryos at −196°C by vitrification. Nature (1985); 313:573. 30 Hunter JE, Fuller B, Bernard A, Jackson A, Shaw RW. Vitrification of human oocytes following minimal exposure to cryoprotectants; initial studies on fertilization and embryonic development. Hum Reprod (1995); 10:1184–8. 31 Pickering S, Braude P, Johnson M, 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. 32 Vincent C, Pickering SJ, Johnson MH, Quick SJ. Dimethylsulfoxide affects the organization of microfilaments in the mouse oocyte. Development (1990); 26:227–35. 33 Hunter JE, Bernard A, Fuller B, Amso N, Shaw RW. Fertilization and development of the human oocyte following exposure to cryoprotectants, low temperatures and cryopreservation: a comparison of two techniques. Hum Reprod (1991); 6:1460–5. 34 Van Blerkom J, Davis P. Cytogenetic, cellular and developmental consequences of cryopreservation of immature and human oocytes. Microscopy Res Tec (1994); 27:165–93.

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35 Younis AI, Toner M, Albertini DF, Biggers JD. Cryobiology of nonhuman primate oocytes. Hum Reprod (1996); 11:156–65. 36 Al-Hasani S, Kirsch J, Diedrich K, Blanke S, Van Der Ven H, Krebs D. Successful embryo transfer of cryopreserved and in-vitro fertilized rabbit oocytes. Hum Reprod (1989); 4:77–9. 37 Al-Hasani S, Diedrich K. Oocyte storage. In: Grudzinskas JG and Yovich JL, eds. Gametes—the oocyte. Cambridge University Press, Cambridge, UK (1995). 38 Dumoulin JCM, Marij Bergers Janssen J, Pieters HEC, Enginsu ME, Geraedts JPM, Evers JLH. The protective effects of polymers in the cryopreservation of human and mouse zonae pellucidae and embryos. Fertil Steril (1994); 62:793–8. 39 Gook D, Schiewe MC, Osborn S, Asch RH, Jansen RPS, Johnston WIH. Intracytoplasmic sperm injection and embryo development of human oocytes cryopreserved using 1,2-propanediol. Hum Reprod (1995); 10:2637–41. 40 Gook D, Osborn S, Johnston W. Partenogenetic activation of human oocytes following cryopreservation using 1,2-propanediol. Hum Reprod (1995); 10:654–8. 41 Porcu E, Dal Prato L, Seracchioli R, Fabbri R, Longhi M, Flamigni C. Comparison between depot and standard release triptoreline in in vitro fertilization: pituitary sensitivity, luteal function, pregnancy outcome and perinatal results. Fertil Steril (1994); 62:126–32. 42 Porcu E, Fabbri R, Petracchi S, et al. Microinjection of cryopreserved oocytes. In: Porcu E, Flamigni C, eds. Human oocytes: from physiology to IVF. Bologna: Monduzzi (1998):285–90. 43 Porcu E, Fabbri R, Seracchioli R, et al. Intracytoplasmic sperm injection of cryopreserved human oocytes. In: Gomel V, Leung PCK, eds. In vitro fertilization and assisted reproduction. Bologna: Monduzzi (1997): 1150–7. 44 Porcu E, Fabbri R, Petracchi S, et al. Fertilization of cryopreserved human oocytes with ICSI. In Ambrosini A, Melis GB, Dalla Pria S, Dessole S, eds. Infertility and assisted reproductive technologies. Bologna: Monduzzi (1997):173–7. 45 Porcu E, Fabbri R, Seracchioli R, et al. Birth and pregnancy after microinjection of human oocytes. Proceedings of 53rd annual meeting of the American Society for Reproductive Medicine (1997):75. 46 Porcu E, Fabbri R, Seracchioli R. Cycles of human oocytes cryopreservation and intracytoplasmic sperm injection: results of 112 cycles. Fertil Steril (1999); 72(3) Suppl. 1, 59. 47 Porcu E, Fabbri R, Ciotti PM, Petracchi S, Seracchioli R, Flamigni C. Ongoing pregnancy after intracytoplasmic sperm injection of epididymal spermatozoa into cryopreserved human oocytes. J Assist Reprod Genet (1999); 16(5):283–5. 48 Porcu E, Fabbri R, Ciotti PM, Petracchi S, Seracchioli R, Flamigni C. Ongoing pregnancy after intracytoplasmic injection of testicular

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spermatozoa into cryopreserved human oocytes. Am J Obstet Gynecol (1999); 180(4):1044–5.

19 Cryopreservation of human embryos Jacqueline Mandelbaum, Yves JR Ménézo

INTRODUCTION The use of superovulation techniques to Improve and simplify human in vitro fertilization (IVF) led to the problem that large numbers of oocytes, and consequently embryos, would be produced. A possible alternative would have been to perform IVF in spontaneous cycles after having triggered ovulation by exogenous human chorionic gonadotrophin (hCG). The results however remained poor,1,2 occasionally interesting,3 but useless in routine. Other solutions would have been to limit the number of oocytes recovered or inseminated or to discard the embryos in excess of the number appropriate for a safe transfer avoiding the risk of multiple pregnancies. These alternatives are either unethical or lead to a decrease in the overall efficiency of IVF, which, finally, is also unethical. Such considerations initiated the development of embryo freezing in humans. Mammalian embryos have been successfully frozen and stored since 1972, when Whittingham et al obtained live mice after the transfer of freeze-thawed morulae.4 Cryotechnology derived from rodents was industrially applied to cattle, representing, since 1980, more than 100000 embryo transfers per year in the world. In humans, the first pregnancy from a frozen embryo occurred in 1983, in Australia, owing to Trounson and Mohr.5 Human embryo freezing spread rapidly, with the help of optimized and simplified biological procedures such as the use of propanediol and sucrose as cryoprotectants6 and became an indispensable extension of IVF. In 1977, Whittingham obtained the first births from transfer of mice morulae, issued from mature oocytes frozen and stored at −196°C.7 About 10 years passed until Chen reported the successful cryopreservation of human oocytes.8 However, until 1997, very few births have arisen from human cryopreserved oocytes and only a handful of live babies had been reported in the literature.8,9 Despite the fact that human oocytes were frozen and thawed according to techniques successfully applied on other species and especially rodents, survival rates remained low, and fertilization rates were reduced after conventional IVF with a high incidence of polyploidy.10–12 Moreover, exposure to cryoprotective compounds and/or variations in temperature

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were claimed to have deleterious effects on oocyte structures such as zona pellucida, cortical granules, spindle microtubules, cytoplasmic microfilaments, and organelles.13 Recently, several fundamental studies arose in humans, which reported a rather good preservation of cell structures after freeze-thaw procedures carried out on mature oocytes.14–18 They were followed by a new set of clinical studies, leading to the first new births for 10 years with embryos issued from cryopreserved oocytes.19,20

EMBRYO FREEZING CRYOBIOLOGICAL PRINCIPLES At the temperature of liquid nitrogen (−196°C), most of the chemical reactions are inhibited, water does only exist as bound, crystalline, or glassy forms; the cellular time is arrested. The only risk that remains is the detrimental effect of earthly radiations on DNA structure. However, in mice, the exposition of frozen 8 cell embryos to the equivalent of 2000 years of radiation impairs neither embryonic survival and viability nor incidence of anomalies at birth.21 Embryos can therefore remain safely in liquid nitrogen but how can they reach those low temperatures and be thawed without damage? CELL CHANGES DURING FREEZING22–24 The fall in temperature has an effect on the integrated biochemistry of the cell. A decrease in temperature from 37°C to 7°C will result in an eightfold reduction in the overall rate of enzyme reactions. Whether or not this interferes with further human embryo quality is not known. As the temperature goes on decreasing, aqueous solutions surrounding the living cells undergo physical and chemical changes: • The solubility of dissolved gases decreases. This may play a certain part in cell damage by the creation of giant gas bubbles when the freezing medium contains sodium bicarbonate gassed with 5% CO2.22 • Water turns from a liquid into a solid phase by way of the formation of pure ice crystals, which coexist with the solvent and salt solution still remaining liquid (generally highly viscous). These crystals grow with the fall in temperature and amalgamate creating an ice front, endlessly modified; it can cause major lesions to the cell membranes protecting the inner cell system. • With decreasing temperature, water forms crystal and becomes less and less available in the extracelluar “milieu” as solvent for the remaining salts. Indeed, eutectic points of salt components (the moment they will crystallize from the water-salt-ice mixture) are reached after the beginning of water crystallization, and for normal culture media are kept at −10°C to −17°C; for sodium chloride the temperature is as low as −21.5°C. Therefore, in the absence of cryoprotective agents the molarity will increase, and cells

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will be exposed to large changes in the pH and the external concentration of salts. This is called “solution effects”25 causing lipoprotein denaturation and thus inducing constraints in the membranes. • The increase in salt concentration of the residual liquid compartment results in water being osmotically drawn from the cell, producing passive dehydration and shrinkage. An excess in the cell volume reduction will lead to irreversible lesions to the cell cytoskeletal structures and to cell death. Cells, especially those containing large amounts of water as oocytes and embryos, need therefore to be protected against all the above-mentioned detrimental effects of freezing if they are to be successfully cryopreserved. There may be an indication for freezing at the blastocyst stage as cell volumes are much lower at this stage. PROTECTIVE PROCEDURES DURING EMBRYO FREEZING Three main technical measures will be used to better control the unstable period of phase changes in the solution surrounding the embryo and in the embryo itself. • Control of the rates of freezing and thawing As the cooling rate increases, the probability that intracellular ice will form increases as well (Fig 19.1). For every cell there is an optimum rate of cooling (with or without the presence of a cryoprotective agent), which depends on several factors, particularly cell volume and membrane composition. Both factors influence the diffusion rates of water, ions, and metabolites. The highest survival rates are obtained with a cooling speed of 1000°C/minute for human red cells and 0.3°C/minute for zygotes and embryos. • Chemical cryoprotection Control of the temperature fall is not sufficient to ensure an optimal cell survival after freezing and thawing. In 1949, the cryoprotective action of glycerol was discovered by Polge et al, on sperm cells.26 Since then, several non-toxic chemicals have proved to be efficient in protecting

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Fig 19.1 Relationship between ice cristal formation in the cell and cell survival. cells against lesions associated with cryopreservation. Chemicals exerting this cryoprotection have been called cryoprotectants. The four most commonly used cryoprotectants for oocyte and embryo cryopreservation are glycerol, ethylene glycol, dimethyl sulfoxide (DMSO) and 1, 2-propanediol (PROH) (Fig 19.2). They are water soluble, able to form hydrogen bonds with water molecules, and able to enter cells. Their cryoprotective activity seems to be based on their ability to decrease the freezing point of solutions and to reduce the amount of salts and other solutes present in the remaining liquid phase, below the freezing point of the solution (beginning of ice formation) and above the eutectic points of the salts. Thus the incidence of lipoprotein denaturation is reduced. Cryoprotectants may also decrease the velocity of crystal formation and modify their shape into a smoother pattern. They may also act by a direct association with cell membranes. Their efficiency has still only been demonstrated empirically. So, glycerol enters and leaves embryonic cells much slower

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Fig 19.2 Chemical structure of the main cryoprotectants. than DMSO and PROH, particularly at early cleavage stages but has proved to be successful for morulae and blastocysts;27 PROH has a lower toxicity at high concentrations when compared to glycerol or DMSO.28 The choice of the best cryoprotectant will therefore rely on its intracell permeability and toxicity for a given cell and on its biological efficiency, empirically determined. After thawing, the cryoprotectant must be quickly removed from the cells in order to avoid its detrimental effect on further embryo development. Some polymers such as dextran, polyvinylpyrrolidone (PVP), and various protein components of serum have some cryoprotective action per se or in association with PROH, glycerol, or DMSO. Serum from the patients or from fetal cord is thus widely used in the composition of freezing and thawing solutions. Some sugars, mono, di-, or trisaccharides do not enter the cells but do act as cryoprotectants by increasing the osmotic pressure of the external milieu. This will help an osmotic desiccation of the cells prior to freezing, reducing the risks of internal ice formation. Moreover, movements of water inside and outside the cells are much faster than movements of solutes. Then, when embryos are first exposed to a permeable cryoprotectant then briefly to a solution containing a non-permeable agent, the partial dehydration will be associated with an increase in the concentration of the cryoprotectant inside the cell. During the thawing process this osmotic property of the non-permeable compounds can also be used to limit the entry of water during the procedure of cryoprotectant removal, thus protecting the embryo from excessive and too rapid rehydration.

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• Ice nucleation Cryoprotective solutions usually reach temperatures as low as −15°C before ice is formed. This is called supercooling (Fig 19.3), and this phenomenon produces latent heat, which can be seen on the cooling curve as a short break in the programmed linear decrease in temperature. Avoiding supercooling is essential for success of embryo freezing. Whittingham did not obtain any survival of 8 cell mouse embryos when supercooling to −12°C was followed by internal ice formation during the freezing process.29 The usual way of preventing supercooling is to induce ice formation at about −7°C by a brief contact on the surface of vials or straws containing the embryos

Fig 19.3 Schematic diagram of a freezing curve. 1: starting (room) temperature 2: seeding point 3: freezing point inducing by exothermy an increase in temperature 4–5: holding plateau before starting 2nd decrease in temperature 6: final freezing temperature (may vary according to protocol).

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with forceps, previously cooled in liquid nitrogen. The forceps are removed as soon as a first crystal is seen as a white point in the extra cellular solution and the programmed fall in temperature is restarted. This ice seeding can be automatically applied in some programmable freezers but is less reliable than the mechanical ice induction, visually controlled. EMBRYO STORAGE AND THAWING Embryos in the freezing solutions are packaged into ampoules or plastic straws (Fig 19.4) and can be stored at the end of the temperature fall in liquid nitrogen for years. The technique of thawing has to be directly related to the freezing procedure and the amount of internal ice. Indeed, when cell dehydration is insufficient an ice-water-glass mixture coexists, even at low temperatures. During thawing, ice crystals change before melting: water molecules run from small crystals to the surface of big ones, which grow in dimensions and roughness and may destroy the cells. Thawing has therefore to be very rapid, to limit the period during which the changes of the water phase may occur. When the fall in temperature is low (0.3°C/min) until −80°C, a high degree of cell dehydration is obtained and thawing must be low as well (4–8°C/min) to allow a progressive rehydration of the cells. Therefore, the whole procedure takes a long time (>6 hours). It is possible to shorten by half the duration of cooling by limiting the slow fall in temperature at −30°C to −40°C and then plunging the embryos directly in liquid nitrogen.28 Small internal ice crystals do form, but they are limited as a partial cell dehydration has already occurred during the initial cooling. Cell dehydration could also have been optimized by the addition of sucrose at room temperature before freezing, as already mentioned. Thawing will have to be very rapid (300°– 400°C/min) to avoid any recrystallization. These high thawing speeds are obtained in practice by just holding the ampoules or straws, containing the embryos at room temperature for several seconds or by directly exposing them to 37°C in a waterbath or any other form of heating. After thawing, cryoprotectants have to be removed, usually by a stepwise procedure, lowering progressively their concentrations and allowing them to leave the cells without excessive rehydration or osmotic stress. As already seen, sucrose may help during this period of cryoprotectants’ removal. Following basic cryobiological principles and taking into account numerous experimental studies, it became possible to successfully freeze and thaw embryos of various animal species: rodents for research purposes,4,29,30 cattle and sheep to select the best animals and help the animal breeding industries,31,32 and finally humans5,33 to balance the side effects of superovulation in assisted reproductive technologies (ART).

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Fig 19.4 Find set-up of the straws before starting cooling and freezing procedures. Top: early-stage embryos (with propanediol). Bottom: Blastocysts (with glycerol). VITRIFICATION PROCEDURES As the formation of internal or external ice may be detrimental for cell survival, it was tempting to obtain the freezing of samples without any ice. This could be obtained by combining the cryoprotective properties of various agents. Such mixtures allow to directly pass from ambient temperatures to −196°C while water and solutes form a glassy phase. As no ice is present, there is no possibility of lethal intracellular ice and no salts concentrate avoiding the toxic solute effects. This vitrification process can be obtained with a mixture of DMSO, acetamide, propanediol and polyethylene glycol at very high molar concentrations.34 The problem of this procedure is the high toxicity of the vitrification solutions: there are some concerns about the integrity of the chromosomes. This can actually explain why, although the first results were rather good with respect to morphology and development in vitro, the preservation of embryo viability, measured by the percentage of live births, is rather disappointing. Nevertheless, live offspring had been produced in mice, rabbits, rats, cattle, sheep, and from 2 cell embryos as well as blastocysts.35 The success is dependent upon the type of cryoprotectants used, the method of addition and dilution, the time of exposure before cooling. Cooling and warming rates should be sufficiently fast to avoid extensive devitrification and recrystallization.

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THE DIFFERENT PROTOCOLS OF HUMAN EMBRYO FREEZING (TABLE 19.1) SLOW COOLING Human embryos can be successfully cryopreserved by protocols using PROH,6 DMSO,5 or glycerol.27 In cattle, blastocyst freezing is also successfully performed with ethyleneglycol. Each of these cryoprotectants gives an optimal efficacy when used at a particular developmental stage: PROH for zygotes or early cleaved embryos, DMSO for cleaved stages,36 and glycerol for blastocysts, depending on the permeability of blastomere membranes. The protocol using PROH (1.5mol/l) and sucrose (0.1mol/l) was reported to be quite successful when embryos were slow cooled (0.3°C/min) to −30°C and was especially suitable for pronucleated and two or three day old embryos.11,37,38 Therefore, a widespread application of embryo cryopreservation was initiated in IVF centers using this procedure. However, optimal survival rates are obtained only when sucrose is

Table 19.1. Freezing and thawing procedures of human embryos (slow cooling). Cryoprotectant 1,2Dimethylsulfoxide Glycerol Propanediol (DMSO) (PROH) Embryonic stage 1 to 8 cells 4 to 8 cells Blastocyst Freezing solution PROH 1.5 M DMSO 1.5 M Glycerol 9% Sucrose 0.1 M Sucrose 0.2 M Cooling rates 1 or 2°C/mn from room temperature to −7°C; seeding (ice nucleation); then 0.3°C/min until… Rapid cooling or direct −30°C to −80°C −30°C to −40°C plunge to −196°C −40°C (temperature threshold) Thawing rates rapid slow usually rapid (300°C/mn) (8°C/mn) (300°C/min) Cryoprotectant dilution stepwise stepwise stepwise (Sucrose 0.2 (Sucrose 0.5 M M) to 0.2 M) References Lassalle et al, Trounson and Mohr, Cohen, 1985; 1995 1983 Ménézo, 1992; Ménézo and Veiga, 1997

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Table 19.2. Freezing protocol for zygote and early cleaved human embryos using PROH and sucrose. Preparation for freezing: 2 steps at RT under a stream of gas (5% O2; 5% CO2; 90% N2) 1. Exposure to 1.5M PROH in PBS or culture medium supplemented with 20% serum for 15 minutes. 2. Exposure to 1.5M PROH and 0.1M sucrose for 5 minutes. Packaging in straws (0.25ML) (Fig 19.4) Freezing curve; seeding and thawing (Table 19.1) Cryoprotectant removal: 4 steps of 5 minutes each 1. Solution of 1 M PROH—0.2 M sucrose 2. Solution of 0.5 M PROH—0.2M sucrose 3. Solution of 0.25 M PROH—0.2M sucrose 4. Solution of 0 M PROH—0.2M sucrose Washing and recovery in embryo culture medium before transfer combined with PROH. Propanediol alone leads to reduced survival rates (32% v. 62%) and only 10% of totally intact embryos as compared to 44% with the PROH-sucrose protocol (Table 19.2).39 DMSO was the first cryoprotective agent used in humans5 and was mostly applied to cleaved stages and particularly three day old embryos.40,41 It has the disadvantage to require a slow cooling until −80°C and either a slow thawing or a rapid one, on crushed ice,36 which, in any case, is a time consuming procedure when taken as a whole. It still remains difficult to determine which method produces the best results. A randomized study by Van der Elst et al on 2220 multicellular supernumerary embryos reported a higher chance of survival and implantation after Cryopreservation when DMSO had been used.36 However, the low success rate of this group with PROH compared with other reports, weakened the conclusions of the study. Glycerol is rather used for the latest stages of preimplantation development, namely the blastocyst27,42 and has proved to have a good efficiency (Table 19.3). Attempts to try new cryoprotectants and new molecules such as polymers or antifreeze proteins have barely been made in humans.43 ULTRARAPID COOLING AND VITRIFICATION The ultrarapid freezing method developed by Trounson and Sjoblom allowed embryos to be plunged into liquid nitrogen after a two to three minute equilibration in media containing 2.0–3.0M DMSO and 0.25– 0.5M sucrose, with high rates of survival and development in vitro.44 Pregnancies were reported, using this ultrarapid freezing,45–48 but the

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number of infants born remains small and marginal (n=30) with only 7% evolutive

Table 19.3. Freezing protocol used for human blastocysts (Ménézo, 1997). Preparation for freezing: 2 steps at RT, under a stream of gas (5% O2; 5% CO2; 90% N2) 1. Solution of 5% glycerol in culture medium for 10 minutes. 2. Solution of 9% glycerol in culture medium containing 0.2M sucrose—10 minutes Packaging in straws (0.25ML) (Fig 19.4) Freezing curve; seeding; thawing (Table 19.1) Cryoprotectant removal: 2 steps 1. Solution of 0.5M sucrose—10 minutes. 2. Solution of 0.2M sucrose—10 minutes. Washing and recovery in embryo culture medium before transfer pregnancies per transfer (30/430), explaining that, despite the simplicity of this procedure, it is still used for human embryo freezing in IVF programs only in exceptional cases. Vitrification procedures were applied to human embryos only in exceptional cases, by using 40% ethyleneglycol as cryoprotectant. The results are poor for multicellular embryos.49 They seem better for morulae and blastocyts.50 TECHNICAL FACTORS IMPROVING SUCCESS RATES • The efficiency of the ice seeding technique in slow cooling protocols is of major importance and may be impaired in some freezers with automatic seeding, thus leading to a failure of the whole freezing program.51 • A serum supplementation to the medium is usual in preparing freezing and thawing solutions for zygote or multicellular embryo freezing. The serum source may vary: human fetal cord, individual autologous or pooled maternal samples, fetal calf serum, or human serum albumin fraction (HSA). They have the disadvantage of an incomplete safety with respect to virus transmission, especially for pooled and cord sera and for HSA. Moreover, HSA seems to lower the pregnancy rates and implantation rates (IR) per embryo when used for the culture and freezing of embryos instead of serum (pregnancy rates: 5.6% HSA versus 11.3% serum; IR: 2.2% HSA v. 6.6% serum).52 Substitutes of serum are also used but there are no studies on the comparative efficiency of these compounds. • Storage time: Careful storage is essential and the work by Tyler et al stressed again the importance of a rapid handling of vials and especially straws containing the frozen embryos.53 Less than 40 seconds at RT may lead to potentially detrimental changes, and in one minute, a 0.25ml straw has reached −7°C, the eutectic point. When storage

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is careful there is no impairment in the viability of surviving and transferred embryos after up to eight years of storage.54–56 One study reported a small but significant decrease in survival rates after the fourth year of storage, but the ability of surviving embryos to implant and proceed to term remained optimal.54 Human embryos can, therefore, survive at least five years of cryopreservation. EMBRYONIC FACTORS INVOLVED IN THE SUCCESS OF FREEZING EMBRYO SURVIVAL The efficiency of a freezing program is evaluated, first, on the morphological integrity of the embryo at thawing, second, on its ability to further cleave in vitro and, principally, in vivo. Early stage embryos are considered surviving when they keep at least half of their initial blastomeres intact after thawing and dilution of the cryoprotectants (“survival index*”=50%). The survival rate, however, when analysing the results of a freezing program, is expressed as the percentage of “surviving” embryos among all frozenthawed embryos. It usually represents at least 65% of thawed embryos. Zygotes are considered to have survived the free-thaw process when they appear intact after thawing, with a clear cytoplasm and no zona pellucida breaches, and when they are able to cleave in vitro during the next 24 hours of culture. Blastocyst survival is more difficult to appraise, considering the number of cells and their specialization. It is usual to recommend after thawing to transfer in utero only the morphologically normal embryos that had re-expanded after three to four hours of recovery at 37°C in the culture medium. The surviving embryos are, in this case, the embryos suitable for transfer. This recovery rate is now 75–85%. CLEAVAGE STAGE EMBRYO FREEZING The parameters affecting the ability of multicellular embryos to survive cryopreservation and implant are well documented and were assessed on 716 transfers of a single frozen-thawed embryo,54 leading to the birth of 44 living new borns (6%). Indeed, embryos * Survival index: (no of intact blastomers at thawing/no of intact blastomeres at freezing)×100

having good morphology (group 1) survived in 80% of the cases compared with 67% and 62% respectively for those presenting cytoplasmic fragments (>40% of the embryo volume; group 2) or unequally sized blastomeres (group 3), (P<0.001). The birth rate per singly surviving and transferred embryo did not differ significantly

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between the fair embryos and the others despite a tendency towards reduction: 7% for group 1; 5% for group 2; 3% for group 3. Other parameters have no statistically significant effect on the freezing ability. These parameters include the age of cleaved embryos (day 2 or 3 after insemination, the number of cells before freezing, the stimulation regimen of the recovery cycle (whether comprising GnRH analogs or not),57 and the size of the oocyte cohort from which they were issued.58 Nevertheless, when looking again at the birth rate per individual transferred frozenthawed embryo, it reached 12% for an 8 cell stage (day 3) and 10% for a 4 cell stage (day 2) at freezing, which was significantly better than the 2% obtained with two day old embryos with only two or three blastomeres cryopreserved. The best embryo to freeze is a fair 4 cell at day 2 or an 8 cell at day 3 post-insemination. The best frozen-thawed embryo to transfer is undoubtedly a totally preserved embryo with 100% of intact blastomeres. In routine programs, and in large series, thawed multicellular embryos survive in 70% of cases, allowing almost 90% of patients attempting embryo thawing to have a transfer, and leading to a birth rate of 10–15% among different groups throughout the world.54,59 ZYGOTE FREEZING Pronucleated eggs an be successfully frozen by using PROH and sucrose and give similar results to early cleaved embryos (two or three days old, from two to eight cells), with implantation rates per thawed embryo reaching 6.3% and 6%, respectively.54 Precise timing appears to be important for optimal zygote freezing to avoid the risks of cooling a single cell with spindle structures or during DNA synthesis;60 the best time should be 20–22 hours after insemination. There are few reports of advantages to using zygote Cryopreservation,61 whereas the drawbacks are the time constraints and the inability to allow the selection of embryos (especially now with ICSI) for the fresh transfer on developmental criteria. Nevertheless, zygote freezing is applied routinely in many IVF centers, especially in Germany, for ethical reasons. BLASTOCYST FREEZING In domestic animals, embryos are frozen mainly at the blastocyst stage. Blastocysts have the advantages to contain numerous small cells; thus the loss of some cells during the freezing and thawing procedure is probably less capable of impairing the further development of the embryo. Human blastocyst Cryopreservation was first reported by Cohen et al using glycerol alone (10%) as cryoprotectant added in a stepwise procedure (10 steps).27 Survival rates were 52%, and the implantation rates per transferred blastocyst reached 35% on a small series (n=23). Blastocyst Cryopreservation was nevertheless abandoned for years

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because the suboptimal culture conditions in usual media allowed only 25% of human zygotes to reach the blastocyst stage in vitro. Moreover, in 1991, Hartshorne et al obtained only 9% of births per transfer of frozenthawed blastocysts (on 134 transfers), which was no better than with early cleaved embryos, although 75% of zygotes had not developed into blastocysts.62 Coculture on feeder cells (Vero) have allowed to obtain in vitro good blastocysts with higher rates reaching 50–60%.42 A simplification of the freezing program with exposure to glycerol (9%) in two steps only and addition of sucrose (0.2M) in the freezing solution, allowed to increase the survival rates to over 80%.63 After thawing, the birth rates per transferred blastocyst are around 13% (take-home baby rate per frozen blastocyst: 10%). It is difficult to compare zygote, early cleaved embryo, and blastocyst freezing to get conclusive data on the respective efficacy of each protocol for a given frozen embryo. Extended in vitro culture results in the developmental arrest of almost half of the cultured

Table 19.4. Freezing and thawing blastocysts from sequential media v. coculture. Same period of time Sequential media Coculture Significance* Number of 249 127 couples 82.3% 93.7% 0.03 Number of 205 119 transfers Number of 430 220 thawed blastocysts 74.2% 83.2% 0.01 Number of 319 183 blastocysts transferred Pregnancies 25 PR/T=12.2% 28 PR/T=23.5% 0.02 Implantations 25 lmp/blast.=7.8% 28 Imp/blast.=15.3% 0.03 24/220=10.9% 0.01 Take home 21/430=5% baby rate per frozen blastocyst *Determined by χ2

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zygotes and embryos, which may block owing to biochemical or cytogenetic problems. This allows a selection of the most viable embryos, and one should therefore expect a twofold increase in implantation rate per frozen and thawed blastocyst, which is obtained with a 13% birth rate per transferred blastocyst compared with 6% per zygote or cleaved embryo. Culture conditions may, however, affect the success of blastocyst freezing. Blastocyst are now obtained using sequential media. When comparing the results, obtained during the same period of time with both types of blastocysts, it appears that blastocysts grown in sequential media are less cryoresistant than the coculture ones (Table 19.4). It has been evoked that the cryotolerance after coculture on Vero cells could be directly related to the amount of Leukemia Inhibitory Factor (LIF) secreted by the cells.64 However, no convincing hypothesis may be actually associated to this statement. FREEZING OF MICROMANIPULATED EMBRYOS (ICSI) The treatment of couples with severe male infertility by intracytoplasmic sperm injection (ICSI) allowed patients who were previously taken off IVF programs to achieve high fertilization and implantation rates. Many supernumerary embryos with perforated zonae became available for cryopreservation. It is therefore essential to evaluate the effects of freezing on micromanipulated zygotes and embryos by comparing their survival rates and implantation potential with regard to zygotes or embryos obtained by conventional IVF. Five teams (Table 19.5),65–68 obtained identical results concerning survival rates of frozenthawed embryos in both groups. Similarly implantation rates were not different between frozen-thawed ICSI or IVF zygotes or embryos.

Table 19.5. Freezing of ICSI derived embryos. Authors Ref Stage of freezing PR/ET Van Steirteghem (1994) 65 Cleaved embyro Al Hasani (1996) 66 Zygote Hoover (1997) 67 Zygote Mandelbaum (1997) Cleaved embryo Kim (1997) 68 Zygote Cleaved embryo Lejeune (1997) 69 Cleaved embryo PR/ET: Pregnancy rate per embryo transfer

ICSI PR/ET 11% 17% 14% 14% 30% 32% 4%

FIV 13% 20% 17% 18% 31% 19% 13%

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One study, however, reported a reduction in implantation rates for ICSI cryopreserved embryos (4%) when compared with IVF frozen-thawed embryos (13%).69 More experience is needed to definitively exclude the possibility that the ICSI breach in the zona pellucida could impair the outcome of the freeze-thaw procedure applied to embryos. For blastocysts, ICSI does not seem to have a negative impact but it is still too early to clearly analyse this point. The paternal effect may interfere with this analysis.70 ASSISTED HATCHING OF CRYOPRESERVED EMBRYOS Assisted hatching (AH), either by partial zona dissection (PZD),71 or by zona drilling,72 or by laser73 had been proposed to enhance the implantation rate of cryopreserved zygotes or cleaved embryos. The only randomized study that reported significantly different results is the work by Check et al.72 They obtained a 14% implantation rate per transferred frozen-thawed embryo in 79 cycles with AH compared with a 5% implantation rate in 79 control cycles. The rationale for applying AH to cryopreserved eggs or embryos should be founded on the hypothesis that the freeze-thaw procedure leads to a zona hardening, which remains to be shown. In mice, Matson et al showed that the cryopreservation of embryos was not associated with an increase in the time of zona digestion by chymotrypsin (1864 seconds v. 2181 for control 2 cell embryos).74 Thus, in this species at least, microdissection of the zona appears unwarranted. Similar studies, as well as large randomized series, are needed in humans to evaluate the true benefit of AH after embryo freezing. MATERNAL FACTORS INVOLVED IN THE SUCCESS OF FROZEN-THAWED EMBRYO TRANSFERS • To illustrate the importance of maternal factors in the final efficiency of transfers of fresh or cryopreserved embryos, one can check the implantation rate in overall patients and in pregnant patients, where all the problems of endometrium receptivity are supposed to be set aside. Menezo and Veiga thus showed that if the ongoing implantation rate was 12.5% in overall patients for cryopreserved blastocysts, it reached 47% in pregnant women.63 Thus, two thirds of the embryos met an unsuitable uterine environment to implant. It therefore appears essential to pay a careful attention to the endometrial preparation. • Cryopreservation allows the transfer of embryos without any time relation to the stimulated cycle of oocyte recovery from which they have arisen. It thus offers a wide variety of options for the timing of embryo transfer and the method of endometrial preparation. The success of frozen embryo transfer requires a good synchronization between the age of the embryos and the age of postovulatory uterus.54

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• Frozen-thawed embryos can be successfully transferred in spontaneous cycles. In case of anovulatory or dysovulatory patients, stimulation protocols using exogenous gonadotropins were used to initiate, improve and control follicular growth and ovulation. In patients devoid of ovarian function, preparation of the endometrium with estrogen and progesterone yielded high pregnancy rates.75 This promoted the use of programmed cycles of endometrial preparation in anovulation or irregular cycles with similar pregnancy rates as compared to the natural regular cycle. Temporary hypogonadism could be induced with a GnRH agonist followed by a physiological regimen of steroid hormone replacement.76,77 But endometrial exogenous preparation without GnRH agonist pretreatment has also been reported to be successful.78 In most studies no statistically significant difference was found in the results among the various endometrial preparation protocols used. The choice has to be made according to the patient’s ovarian function and to the convenience and disadvantages of each method: daily monitoring to pinpoint ovulation in natural cycles and eventual cancellation; risk of ovarian hyperstimulation in case of stimulated cycles; need for hormonal support during the first trimester of pregnancy in case of artificially programmed cycles.

CONCLUSION The major aim of embryo cryopreservation is to provide further possibilities for conception in addition to those obtained through the initial cycle and fresh transfer. This goal is achieved with an increase in the birth rate of 8% for the women who had embryos cryopreserved and by 5% for the overall program.79 Embryo freezing has also contributed to lowering the risks of severe ovarian hyperstimulation by cancelling the fresh transfer80 and to simplifying oocyte donation.75 Embryo freezing could become an obligatory step to limit the risks of viral transmission.81 A recent report of a study in mice on the long term effects of embryo freezing confirmed that this procedure did not induce major anomalies but could be responsible for small differences, such as particularities in the mandible’s morphology, various responses to the behaviour and neurosensorial tests, or an 11% increase in body weight in males at senescence.82 All these features were depending on genotype, sex, or age. This study highlighted the importance of large surveys on the condition of human babies conceived by the use of cryopreserved embryos. Until now, comparative analyses with pregnancies issued from fresh transfers have not found any differences particularly in respect to fetal development, perinatal risk, obstetrical outcome and to the rate of congenital anomalies.83–87 Frozen-thawed blastocyst transfer does not change birth weight and sex ratio.88 Whether freezing and transfers should be made at the blastocyst stage rather than earlier is still a matter for debate. Blastocyst formation is also a selection of the best embryos. It allows to decrease the number of embryo

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transferred. However, the sequential media technology is still young, and a use on a larger scale, with more data, is needed. Freezing can be performed at earlier stages, and then the selection can be performed afterwards in vitro, especially if we consider the degree of aneuploidy detected in earlystage frozen-thawed embryos.89 After five years of storage, 85% of the frozen embryos are usually transferred back to the patients. This is acceptable.

REFERENCES 1 Foulot H, Ranoux C, Dubuisson JB, Rambaud D, Aubriot FX, Poirot C. In-vitro fertilization without ovarian stimulation: a simplified protocol applied in 80 cycles. Fertil Steril (1989); 52:617–21. 2 Cooke ID, Lenton EA. IVF in the natural cycle. J Assist Reprod Genet (1997); 14:PS26–2,385. 3 Lindheim SR, Vidali A, Ditkoff E, Sauer MV. Poor responders to ovarian hyperstimulation may benefit from an attempt at natural cycle oocyte retrieval. J Assist Reprod Genet (1997); 14:174–6. 4 Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos, frozen to −196°C and −289°C. Science (1972); 178:411–4. 5 Trounson A, Mohr L. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature (1983); 305:707– 9. 6 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); 44:645–51. 7 Whittingham DG. Fertilization in-vitro and development to term of unfertilized mouse oocytes previously stored at −196°C. J Reprod Fertil (1977); 49:89–94. 8 Chen C. Pregnancy after human oocyte cryopreservation. Lancet (1986); i:884–6. 9 Van Uem JF, Siebzehnruebl ER, Schuuh B, Koch R, Trotnow S, Lang N. Birth after cryopreservation of unfertilized oocytes. Lancet (1987); 3:752–3. 10 Al-Hasani S, Diedrich K, van der Yen H, Reinecke A, Hartje M, Krebs D. Cryopreservation of human oocytes. Hum Reprod (1987); 2:695– 700. 11 Mandelbaum J, Junca AM, Plachot M, et al. Cryopreservation of human embryos and oocytes. Hum Reprod (1988); 3:117–9. 12 Hunter JE, Bernard A, Fuller B, Amso N, Shaw RW. Fertilization and development of the human oocyte following exposure to cryoprotectants, low temperatures and cryopreservation: a comparison of two techniques. Hum Reprod (1991); 6:1460–5.

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13 Bernard A, Fuller BJ. Cryopreservation of human oocytes: a review of current problems and perspectives. Hum Reprod Update (1996); 2:193–207. 14 Imoedemhe DG, Sigue AB. Survival of human oocytes cyropreserved with or without the cumulus in 1,2-propanediol. J Assist Reprod Genet (1992); 9:323–7. 15 Gook DA, Osborn SM, Johnston WIH. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum Reprod (1993); 8:1101–9. 16 Gook DA, Osborn SM, Bourne H, Johnston WIH. Fertilization of human oocytes following cryopreservation; normal karyotypes and absence of stray chromosomes. Hum Reprod (1994); 9:684–91. 17 Gook DA, Osborn SM, Johnston WIH. Parthenogenetic activation of human oocytes following cryopreservation using 1,2-propanediol. Hum Reprod (1995); 10:654–8. 18 Van Blerkom J, Davis PW. Cytogenetic, cellular, and developmental consequences of cryopreservation of immature and mature mouse and human oocytes. Microscopy Res Tech (1994); 27:165–93. 19 Tucker MJ, Wright G, Morton P, Shanguo L, Massey J, Kort H. Preliminary experience with human oocyte cryopreservation using 1,2propanediol and sucrose. Hum Reprod (1996); 11:1513–5. 20 Porcu E, Fabbri R, Seracchioli R, et al. Intracytoplasmic sperm injection of cryopreserved human oocytes. In: 10th World Congress on In-Vitro Fertilization and Assisted Reproduction. Bologna, Italy: Monduzzi (1997): 1153–7. 21 Glenister PH, Lyon MF. Long term storage of eight-cell mouse embryos at −196°C. IVF (1997); 3:20–7. 22 Ashwood-Smith MJ. The cryopreservation of human embryos. Hum Reprod (1986); 5:319–32. 23 Mazur P. The role of intracellular freezing in the death of cells cooled at supraoptimal rate. Cryobiology (1977); 14:251–72. 24 Morris GJ, Watson PF. Cold shock injury: a comprehensive bibliography. Cryo-Letters (1984); 5:352–72. 25 Lovelock JE. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochem Biophys Acta (1953); 11:28–36. 26 Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperature. Nature (1949); 164:666. 27 Cohen J, Simons RF, Edwards RG, Fehilly CB, Fishel SB. Pregnancies following the frozen storage of expanding human blastocysts. J In Vitro Fertil Embryo Transfer (1985); 2:59–64. 28 Renard JP, Nguyen BX, Garnier V. Two-step freezing of two-cell rabbit embryos after partial dehydration at room temperature. J Reprod Fertil (1984); 71:573–80.

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29 Whittingham DG. Some factors affecting embryo storage in laboratory animals. Amsterdam: Ciba Foundation (1977):97–108. 30 Whittingham DG, Adams CE. Low temperature preservation of rabbit embryos. J Reprod Fertil (1976); 47:269–72. 31 Willadsen SM, Polge C, Rowson LEA, Moor RM. Deep-freezing of sheep embryos. J Reprod Fertil (1976); 46:1541–4. 32 Trounson AO, Shea BF, Ollis GW. Jacobson ME. Frozen storage and transfer of bovine embryos. J Anim Sci (1978); 47:677–81. 33 Zeilmaker GH, Alberda AT, Van Gent I, Rijkmans C, Drogendijk AC. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil Steril (1984); 2:293–6. 34 Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at 196°C by vitrification. Nature (1985); 313:573–5. 35 Paynter S, Cooper A, Thomas N, Fuller B. Cryopreservation of multicellular embryos and reproductive tissues. In: Reproductive tissue banking. New York, Academic Press (1997):359–97. 36 Van der Elst J, Camus M, Van den Abbeel E, Maes R, Devroey P, Van Steirteghem AC. Prospective randomized study on the cryopreservation of human embryos with dimethylsulfoxide or 1,2propanediol protocols. Fertil Steril (1995); 63:92–100. 37 Testart J, Lassale B, Belaïsch-Allart J, et al. High pregnancy rate after early human embryo freezing. Fertil Steril (1986); 46:268–72. 38 Fugger EF, Bustillo M, Katz IP, Dorfmann AD, Bender SD, Schulman JD. Embryonic development and pregnancy from fresh and cryopreserved sibling pronucleate human zygotes. Fertil Steril (1988); 50:273–8. 39 Mandelbaum J, Junca AM, Plachot M, et al. Human embryo cryopreservation, extrinsic and intrinsic parameters of success. Hum Reprod (1987); 2:709–15. 40 Marrs RP, Brown J, Sato F, et al. Successful pregnancies from cryopreserved human embryos produced by in vitro fertilization. Am J Obstet Gynecol (1987); 156:1503–8. 41 Camus M, Van den Abbeel E, Waesberghe LV, Wisanto A, Devroey P, Van Steirteghem AC. Human embryo viability after freezing with dimethylsulfoxide as a cryoprotectant. Fertil Steril (1989); 51:460–5. 42 Menezo Y, Nicollet B, Herbaut N, Andre D. Freezing cocultured human blastocysts. Fertil Steril (1992); 58:977–80. 43 Dumoulin J, Bergers-Janssen JM, Pieters M, Enginsu M, Geraedts J, Evers J. The protective effects of polymers in the cryopreservation of human and mouse zonae pellucidae and embryos. Fertil Steril (1994); 62:793–8. 44 Trounson AO, Sjoblom P. Cleavage and development of human embryos in vitro, after ultrarapid freezing and thawing . Fertil Steril (1988); 50:373–6.

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45 Gordts S, Roziers P, Campo R, Noto V. Survival and pregnancy outcome after ultrarapid freezing of human embryos. Fertil Steril (1990); 53:469–72. 46 Feichtinger W, Hochefellner C, Ferstl U. Clinical experience with ultra-rapid freezing of embryos. Hum Reprod (1991); 6:735–6. 47 Hsieh YY, Tsai HD, Chang CC, Lo Hy, Lai ACH. Ultrarapid cryopreservation of human embryos: experience with 1,582 embryos. Fertil Steril (1999); 72:253–6. 48 Lai Ach, Lin BPH, Chang CC, Tsai HD, Hwang VWS, Lo HY. Pregnancies after transfer of ultrarapidly frozen human embryos. J Assist Reprod Genet (1996); 13:625–8. 49 Mukaida T, Wada S, Takahashi K, Pedro PB, An TZ, Kasai M. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Hum Reprod (1998); 13:2874–9. 50 Vanderzwalmen P, Delval A, Chatziparasisou A, et al. Pregnancies after vitrification of human day 5 embryos. Hum Reprod (1997); Abst. book 1 12:98. 51 B.L.E.F.C.O., Denis I, Mandelbaum J, Testart J. Congélation des embryons humains: enquête sur la situation en France (1985–1993). Contracept Fertil Sex (1996); 24:229–32. 52 Warnes GM, Payne D, Jeffrey R, et al. Reduced pregnancy rates following the transfer of human embryos frozen or thawed in culture media supplemented with normal serum albumin. Hum Reprod (1997); 12:1525–30. 53 Tyler JPP, Kime L, Cooke S, Driscoll GL. Temperature change in cryo-containers during short exposure to ambient temperatures. Hum Reprod (1996); 11:1510–2. 54 Mandelbaum J, Plachot M, Junca AM, et al. Human embryo cryopreservation in an IVF program. Limits, facts, prospects. In: Mori T, Aono T, Tominaga T, Hiroi M, eds. Frontiers in endocrinology. Ares Serono Symposia (1994): 505–12. 55 Lin YP, Cassidenti DL, Chacon RR, Soubra SS, Rosen GF, Yee B. Successful implantation of frozen sibling embryos is influenced by the outcome of the cycle from which they were derived. Fertil Steril (1995); 63:262–7. 56 Avery S, Marcus S, Spillane S, Macnamee M, Brinsden P. Does the length of storage time affect the outcome of frozen embryo replacement? J Assist Reprod Genet (1995); 12:67S 57 Mandelbaum J. Human embryo cryopreservation. Contracept Fertil Sex (1990); 18:341–53. 58 Toner J, Brzyski RG, Oehninger S, Veeck LL, Simonetti S, Muasher SJ. Combined impact of the number of preovulatory oocytes and cryopreservation on IVF outcome. Hum Reprod (1991); 6:284–9. 59 Wang XJ, Ledger W, Payne D, Jeffrey R, Matthews CD. The contribution of embryo cryopreservation to in vitro fertilization/gamete

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intrafallopian transfer: 8 years’ experience. Hum Reprod (1994); 9:103–9. 60 Wright G, Wilker S, Elsner C, et al. Observations on the morphology of pronuclei and nucleoli in human zygotes and implications for cryopreservation. Hum Reprod (1990); 5:109–15. 61 Demoulin A, Jouan C, Gerday C, Dubois M. Pregnancy rates after transfer of embryos obtained from different stimulation protocols and frozen at either pronucleate or multicellular stages. Hum Reprod (1991); 6:799–804. 62 Hartshorne GM, Elder K, Crow J, Dyson H, Edwards RG. The influence of in vitro development upon post-thaw survival and implantation of cryopreserved human blastocysts . Hum Reprod (1991); 6:136–41. 63 Menezo Y, Veiga A. Cryopreservation of blastocysts. In: 10th World Congress on In-Vitro Fertilization and Assisted Reproduction. Bologna: Monduzzi (1997):49–53. 64 Carnegie JA, Morgan JA, McDiarmid N, Durnford R. Influence of protein supplements on the secretion of leukemia inhibitory factor by mitomycin-pretreated cells: possible application to the in vitro production of bovine blastocysts with high cryotolerance. J Reprod Fertil (1999); 117:41–8. 65 Van Steirteghem AC, Van der Elst J, Van den Abbeel E, Joris H, Camus M, Devroey P. Cryopreservation of supernumerary multicellular human embryos obtained after intracytoplasmic sperm injection. Fertil Steril (1994); 62:775–80. 66 Al-Hasani S, Ludwig M, Gagsteiger F, et al. Comparison of cryopreservation of supernumerary pronuclear human oocytes obtained after intra-cytoplasmic sperm injection (ICSI) and after conventional in-vitro fertilization. Hum Reprod (1996); 11:604–7. 67 Hoover L, Baker A, Check JH, Lurie D, Summers D. Clinical outcome of cryopreserved human pronuclear stage embryos resulting from intracytoplasmic sperm injection. Fertil Steril (1997); 67:621–4. 68 Kim JW, Han MH, Byun HK, et al. Comparison of transfer of cryopreserved supernumerary embryos obtained after conventional IVF and ICSI. In: 10th World Congress on In-Vitro Fertilization and Assisted Reproduction. Bologna: Monduzzi (1997):79–82. 69 Lejeune B, Vanderzwalmen P, Vandamme B, et al. Reduced implantation rate after the transfer of frozen-thawed embryos obtained by ICSI. Hum Reprod (1997); 12: Abstract book 1:3–4. 70 Shoukir Y, Chardonnens D, Campana A, Sakkas D. Blastocyst development from supernumerary embryos after intracytoplasmic sperm injection: a paternal influence? Hum Reprod (1998); 13:1632–7. 71 Tucker MJ, Cohen J, Massey JB, Mayer MP, Wiker S, Wright G. Partial dissection of the zona pellucida of frozenthawed human embryos may enhance blastocyst hatching, implantation, and pregnancy rates. Am J Obstet Gynecol (1991); 165:341–5.

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72 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. 73 Germond M, Nocera D, Senn A, Rink K, Delacretaz G, Fakan S. Microdissection of mouse and human zona pellucida using a 1.48-µm diode laser beam: efficacy and safety of the procedure. Fertil Steril (1995); 65:604–11. 74 Matson PL, Graefling J, Junk SM, Yovich JL, Edirisinghe WR. Cryopreservation of oocytes and embryos: use of a mouse model to investigate effects upon zona hardness and formulate treatment strategies in an in-vitro fertilization programme. Hum Reprod (1997); 12:1550–3. 75 Salat-Baroux J, Tibi C, Cornet D, et al. Pregnancies after replacement of frozen-thawed embryos in a donation program. Fertil Steril (1988); 49:817–21. 76 Sathanandan M, Macnamee MC, Rainsbury P, Wick K, Brindsen P, Edwards RG. Replacement of frozen-thawed embryos in artificial and natural cycles. A prospective semi-randomized study. Hum Reprod (1991); 6:85–7. 77 Queenan JT, Veeck LL, Seltman HJ,. Muasher SJ. Transfer of cryopreserved-thawed preembryos in a natural cycle or a programmed cycle with exogenous hormonal replacement fields similar pregnancy results. Fertil Steril (1994); 62:545–50. 78 Lelaidier C, de Ziegler D, Gaetano J, Hazout A, Fernandez H, Frydman R. Controlled preparation of the endometrium with exogenous oestradiol and progesterone: a novel regimen not using a gonadotrophin-releasing hormone agonist. Hum Reprod (1992); 7:1353–6. 79 Mandelbaum J. Embryo freezing in humans: an overview. In: Hedon B, Bringer J, Mares P, eds. Fertility and sterility—a current overview. Proceedings of the 15th World Congress on Fertility and Sterility 1995): 419–23. 80 Queenan JT, Veeck LL, Toner JP, Oehninger S; Muasher SJ. Cryopreservation of all prezygotes in patients at risk of severe hyperstimulation does not eliminate the syndrome, but the chances of pregnancy are excellent with subsequent frozen-thaw transfers. Hum Reprod (1997); 12:1573–6. 81 Hamer FC, Horne G, Pease EHE, Matson PL, Lieberman BA. The quarantine of fertilized donated oocytes. Hum Reprod (1995); 10:1194–6. 82 Dulioust E, Toyama K, Busnel MC, et al. Long-term effects of embryo freezing in mice. Proc Natl Acad Sci USA (1992); 92:589–93. 83 F.I.V.N.A.T. Pregnancy outcome after replacement of frozen-thawed embryos and after transfer of fresh embryos in French IVF registry. Contracept Fertil Sex (1994); 22:287–91.

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84 Wada I, Macnamee MC, Wick K, Bradfield JM, Brinsden PR. Birth characteristics and perinatal outcome of babies conceived from cryopreserved embryos. Hum Reprod (1994); 9:543–6. 85 Olivennes F, Schnieder Z, Remy V, et al. Perinatal outcome and follow-up of 82 children aged 1–9 years old conceived from cryopreserved embryos. Hum Reprod (1996); 11:1565–8. 86 Sutcliffe AG, D’Souza S, Cadman J, Richards B, McKinlay IA, Liberman B. Minor congenital anomalies, major congenital malformations and development in children conceived from cryopreserved embryos. Hum Reprod (1995); 10:3332–7. 87 Wennerholm V, Albertsson-Wickland K, Bergh C, et al. Post natal growth and health in children born after cryopreservation as embryos. Lancet (1998); 351:1085–90. 88 Menezo Y, Chouteau J, Torello MJ, Girard A, Veiga A. Birth weight and sex ratio after transfer at the blastocyst stage in human. Fertil Steril (1999); 72:221–4. 89 Iwarson E, Lundqvist M, Inzunza J, Ahrlund-Richter L, Sjoblom P, Lundkvist O, Simberg N, Nordenskjold M, Blenhow E. A high degree of aneuploidy in frozen-thawed human preimplantation embryos. Hum Genet (1999); 104:376–82.

20 Managing the cryopreserved embryo bank Philip Matson, Neroli Darlington

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 effective management of the embryos once in storage is just as important in ensuring 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 (a) prohibition of the freezing of cleaved embryos in Germany,1 and (b) the limit of storage of embryos in the first instance of three 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 (for example, 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, such as pre-implantation diagnosis). 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 United

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Kingdom. The new act came into force on 1 August 1991, and one of its effects was to introduce a maximum storage period of five 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 five year limit. However, embryos frozen before 1 August 1991 had the five year countdown begin on that day, whether the patients knew about the introduction of the Act or not. By the time that the five years had passed for the 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 1 August 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

CONSENT

(a) (b)

(c)

(d)

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: 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, neighbours, 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; and the consents of donors to be clarified within the local legal framework. Fuscaldo

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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.8 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 the donor could only withdraw consent prior to the primary use of the donated semen, ie before the fertilization of oocytes, but not thereafter.

TIMING OF THAWING

(a)

(b)

(c)

(d)

A decision should be made upon the strategy used for the timing of the replacement relative to either surge in luteinizing hormone (LH), the triggering of ovulation with human chorionic gonadotrophin (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 on the following. The stage of the embryos in storage. Embryos can be frozen at either the pronucleate, 4 cell (day 2 after oocyte collection), 8 cell (day 3), or blastocyst stage. Therefore pronucleate oocytes would need to be thawed 24 hours earlier than, say, a group of 4 cell embryos, etc. 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 do 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.9 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 whereas others simply thaw out on the day of transfer.10,11 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 favour 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 early cleavage embryos does seem to reduce the potential for implantation.12 However, if it is only on the next day after culture that the damage is discovered (as can be the case with either pronucleate or early cleavage embryos) then 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 and

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freeze a 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 had not divided.13

MANAGEMENT OF EMBRYOS IN STORAGE Embryos stored in liquid nitrogen seem 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.14 The main concerns regarding embryo storage would therefore seem to relate to matters of housekeeping and legal constraints. 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 United Kingdom (five years’ storage in the first instance), and the Human Reproductive Technology Act in Western Australia (three 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 the patients before the expiry of the storage period, eg 6 months prior to the expiry date.15 Postal communication with patients appears the easiest method but there will be a group of patients that do not respond and will require other means of contact.16 The fate of 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.17 However, more IVF patients that do not wish to retain the embryos would choose to discard them rather than donate them to another couple.15,18,19 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.

RISK OF INFECTION The storage of ampoules or straws in liquid nitrogen with material from other patients must carry some risk of the transfer of infectious agents because (i) liquid nitrogen freezers are not sterile and will eventually become contaminated with potential pathogens, albeit at a low level,20 (ii) serological testing of IVF patients will not always detect the presence of current infection because of the lag for sero-conversion in some diseases, and (iii) many micro-organisms can survive freezing and thawing.21 Whilst there has been one recorded case of the transmission of hepatitis B

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during the storage of bone marrow due to the leakage of a storage bag,22 there does not seem to have been any report on 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, eg hepatitis B, but patients should be aware that their embryos will be kept with the embryos of other infected people. If the 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 high security straws may well reduce the risk of cross infection further.23

SUMMARY The storage of human embryos is now often governed by local legislation, or has the potential for legal proceedings should something go wrong. 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 (either the husband, the wife, or the donor) are known and adhered to wherever possible. The strategy for timing the thaw of embryos should be clearly laid out in the laboratory manual for the various kind 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 (24 October 1990); Gesetz zum Schutz von Embryonen (EmbryonenshutzgesetzEschG). 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 Transfer (1996); 13:197–200. 3 Wise J. Storage period ends for 4000 embryos. BMJ (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); 12:10–1. 7 Dickey RP, Krentel JB. Storage of sperm and embryos. Couples having IVF should be asked their wishes about spare embryos before egg retrieval. BMJ (1996); 313:1078–9.

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8 Fuscaldo G. Gamete donation: when does consent become irrevocable? Hum Reprod (2000); 15:515–9. 9 Mandelbaum J, Junca AM, Plachot M, Cohen J, Salat-Baroux J. Timing of embryo transfer and success of pregnancy in the human. Reprod Nutr Dev (1988); 28:1763–71. 10 Van der Elst J, Van den Abbeel E, Vitrier S, Camus M, Devroey P, Van Steirteghem AC. Selective transfer of cryopreserved human embryos with further cleavage after thawing increases delivery and implantation rates. Hum Reprod (1997); 12:1513–21. 11 Ziebe S, Bech B, Petersen K, Mikkelsen AL, Gabrielsen A, Andersen AN. Resumption of meiosis during post-thaw culture: a key parameter in selecting the right embryos for transfer. Hum Reprod (1998); 13:178–81. 12 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. 13 Horne G, Crithclow JD, Newman MC, Edozien L, Matson PL, Lieberman BA. A prospective evaluation of cryopreservation strategies in a two-embryo transfer programme. Hum Reprod (1997); 12:542–7. 14 Cohen J, Inge KL, Wiker SR, Wright G, Fehilly CB, Turner TG Jr. Duration of storage of cryopreserved human embryos. J In Vitro Fert Embryo Transfer (1988); 5:301–3. 15 Darlington N, Matson P. The fate of cryopreserved human embryos approaching their legal limit of storage within a West Australian invitro fertilization clinic. Hum Reprod (1999); 14:2343–4. 16 Brzyski RG. Efficacy of postal communication with patients who have cryopreserved pre-embryos. Fertil Steril (1998); 70:949–51. 17 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. 17 Lornage J, Chorier H, Boulieu D, Mathieu C, Czyba JC. Six year follow-up of cryopreserved human embryos. Hum Reprod (1995); 10:2610–6. 19 Hounshell CV, Chetkowski RJ. Donation of frozen embryos after in vitro fertilization is uncommon. Fertil Steril (1996); 66:837–8. 20 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. 21 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. 22 Tedder RS, Zuckerman MA, Goldstone AH, et al. Hepatitis B transmission from contaminated cryopreservation tank. Lancet (1995); 346:137–40.

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23 Letur-Konirsch H, Devaux A, Collin G, et al. Viral risk and straw for cryopreservation. A preliminary experimental study with HIV1. Presented at 11th World Congress on In Vitro Fertilization and Human Reproductive Genetics, Sydney, 9–14 May (1999).

21 Cryopreservation and storage of sperm Eileen A McLaughlin

OVERVIEW The history of human semen Cryopreservation stretches back over 200 years with the first recorded experiments involving cooling followed by successful rewarming of human spermatozoa in snow.1 Despite this early success it was not until the fortuitous discovery of glycerol as a cryoprotectant2 and subsequent live birth of a calf3 in the early 1950s that Cryopreservation of human semen for assisted reproduction became a feasible option. The ability to store human semen greatly improved the flexibility of donor insemination treatment, resulting in the first live human births in 1953.4 Artificial insemination with donor semen as a method of circumventing severe male infertility became a mainstay of fertility treatment for the next 40 years.5 As Cryopreservation of human semen results in a significant loss of motility and viability,6–8 with considerable variation between ejaculates of different individuals, only semen from a highly selected population of men is suitable for treatment purposes after Cryopreservation.8,9 Reasons for the differences between individuals in cryosurvival rates are unknown10 and the ability to predict post thaw survival is limited.11 With availability of fresh semen and in view of the fact that some human spermatozoa survive freezing and thawing tolerably well,12 little pressure to optimise semen Cryopreservation protocols existed until the mid-1980s. Following the infection of four recipients with HIV infection after insemination with semen from a seropositive donor,13 the use of quarantined cryopreserved semen became mandatory.14–16 A small number of studies suggest that while cryopreserved semen was equally fertile to fresh semen when used in in vitro fertilization systems17,18 a significant reduction in conception rates was observed in vivo.19 This was linked largely to the reduction in the number of motile sperm inseminated,20–22 and confirmed by analysis of donor fecundity related to numbers of motile sperm per straw.23 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

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studies on the effects of freezing and thawing on human spermatozoa have been undertaken.24 These have culminated with the investigation of semen preparation methods, complex cryoprotectants, and the use of programmable freezing machines as methods of optimizing the recovery of motile morphologically normal spermatozoa post thaw.

METHODS SEMEN PREPARATION PRE-FREEZE Traditionally whole semen has been diluted with an appropriate volume of cryoprotectant prior to packaging and freezing. This method is largely applicable to good quality donor semen or patient semen to be used for intracervical insemination (ICI).25 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.26 This involves the separation of the spermatozoa from seminal plasma either by a simple washing technique or by the use of density gradients such as Percoll27 or Isolate and resuspension of the sperm in a suitable culture media such as Hams F10.28 In general, semen preparation improves the concentration and the motility characteristics of spermatozoa available for insemination,29 and several studies have suggested that donor insemination pregnancy rates have benefited.26 In addition the advancements in oncology treatment with improved after 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.30 As these men often have impaired semen characteristics concentration of the spermatozoa into a small volume may be warranted prior to cryopreservation. The advent of a consistently successful assisted fertilization technique, namely intracytoplasmic sperm injection (ICSI)31 means that men with severe oligozoospermia or azoospermia can achieve pregnancies in vitro. In order to minimize unnecessary medical treatment of the female partner (such as superovulation) 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.32 While seminal plasma seems to confer a beneficial effect during cryopreservation33 studies comparing the fertilizing potential of prepared spermatozoa have not demonstrated an impaired ability of the thawed sperm to bind to homologous zona pellucida34 or to fertilise after ICSI.35

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SEMEN PREPARATION POST THAW During direct insemination of the semen cryoprotectant mixture in simple intracervical insemination 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 by slow dilution with medium prior to washing or density centrifugation.36 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.37 These have been divided into two classes, (1) those that function as permeating cryoprotectants such as dimethyl sulphoxide, propylene glycol and glycerol, and (2) non-permeating cryoprotectants, for example, sucrose raffinose and glycine. Glycerol has remained the cryoprotectant of choice for most species38 despite evidence that the optimal cryoprotectant for human spermatozoa is ethylene glycol.36 As any change in cryoprotectant will have to be evaluated for consequences on fertility many units continue to use glycerol despite the known toxic effects of this compound.39,40 If glycerol is the only cryoprotectant added, then historically a 5–10% v/v final concentration has been used,41,42 with 7.5% thought to be optimal,43 although one study with higher concentrations (12–16%) had best postthaw motility rates.44 In order to improve cryosurvival rates, more complex diluents containing other mainly non-permeable cryoprotective agents such as glycine, zwitterions, citrate, and egg yolk were developed. Among the earliest and best known extenders for human semen is glycerol egg yolk citrate (GEYC).43 Modifications45–47 are still used today (appendix 1), although evidence based on post-thaw sperm motility rates suggests that GEYC and its derivatives are only marginally better than glycerol alone.48,49 During the early eighties, influenced by changes in animal semen programmes, two other complex cryoprotective diluents both containing organic buffers were introduced. The first termed human sperm preserving medium (HSPM)46 was a modified tyrodes 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- (2hydroxyethyl) piperazine-N′ (2-ethanesulphonic acid) (appendix 2). In the original studies,46 HSPM was as good as GEYC at maintaining post-thaw spermatozoal motility with a slightly higher (although not statistically significant) pregnancy rate obtained after intracervical insemination.

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HSPM is available commercially as SpermFreeze (Medicult, Hamburg, Germany). The second complex cryoprotective buffer was a zwitter ion buffer system termed TESTCY developed by Jeyendran et al.50 This buffer contains TES (N-tris (hydroxymethyl) methyl 2-amino ethane sulphonic acid), TRIS (Tris (hydroxymethyl) amino methane), sodium citrate, and egg yolk but no glycerol, and in the initial report it proved superior to glycerol alone as a cryoprotectant.50 This remarkable result was due largely to the very rapid freezing protocol employed and was impossible to duplicate using standard methods.51 TESTCY (now containing 12% glycerol) gave satisfactory cryosurvival rates and is also now 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.11,51–53 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 groups making comparison difficult. After dilution with extender semen should be packaged and cooled immediately as evidence suggests that exposure of human spermatozoa to cryoprotectant prior to freezing should be less than 10 minutes in order to have optimal cryosurvival rates.54 PACKAGING Until recently only three forms of packaging have been routinely used in human semen cryopreservation: (1) cryovials or ampoules,55 (2) straws;5 and (3) 1.0ml syringes.56 There are advantages and disadvantages to all three types of packaging. Cryovials are easy to fill aseptically and hold a maximum of 1.0ml of semen plus cryoprotectant. Storage on canes in goblets in 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 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.57 The packaging of semen into traditional 0.25ml or 0.5ml straws requires the use of a vacuum pump and filling nozzle to aspirate the semen cryoprotectant mixture. Aseptic filling is not possible except by manually injecting semen with a hypodermic needle and syringe. Straws are available in a variety of colours suitable for the easy identification of individuals, and many thousands can be stored in plastic goblets in canisters in liquid nitrogen vessels. Overfilled straws are prone to cracking and expelling the powder sealing plugs into the liquid nitrogen.

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Syringes are also difficult to fill aseptically and impossible to seal safely. 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. COOLING AND FREEZING Many laboratories continue to use the simple rapid method of suspending straws or ampoules in the uncirculated liquid nitrogen vapour phase for a set time period, before plunging the straws into liquid nitrogen for long term storage. This method requires no specialized equipment and, although not ideal, can give satisfactory cryosurvival rates.58–60 Problems include non-uniform cooling rates both within and between aliquots of the same ejaculate61 and difficulties in maintaining reproducible freezing

Fig 21.1 Donor insemination, clinical pregnancy, and live birth rates per treatment cycles69 11/8/91–31/3/98

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conditions. Programmable freezers which circulate liquid nitrogen vapour in a controlled rate give much more reproducible cooling curves.61–63 The more sophisticated computer controlled freezing machines allow for several different freezing rates during one cooling curve and holding temperatures to permit manual seeding.7 THAWING The cooling rate determines the optimum thawing rate.64 In practice this means a slow cooling rate (1°C/minute) 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) with removing straws from liquid nitrogen and placing them on the bench top at 22°C.64 ASSESSMENT OF POST-THAW FERTILITY Traditionally success of cryopreservation is measured by the number of motile spermatozoa recovered post-thaw.47 However other effects of cryopreservation such as ultrastructural damage to the plasma membrane, loss of acrosomal contents, and other biochemical markers may contribute to a loss of fertility.65–68

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 10% or less69 (Fig 21.1) and prospective studies from the French CECOS group have confirmed that conception rates from ICI of frozen or 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 reaching 17.2% when >8×106 motile sperm in a 0.25ml 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,70 suggesting that concentration of motile spermatozoa semen prior to cryopreservation might improve conception rates. The use of insemination devices such as the intracervical cap has not contributed to improved success in routine donor insemination.71 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

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concentration of sperm prior to insemination. Overall donor insemination pregnancy rates are higher when IUI is compared with ICI25,72 with pregnancy rates only dropping when <0.5×106 motile sperm were inseminated similar to results obtained with washed fresh patient semen.73 The number of motile sperm per straw of cryopreserved donor semen does not correlate with either fertilization rates or pregnancy rates in in vitro fertilization (IVF),18 reflecting the highly selected nature of the semen stored. However as the quality of cryopreserved semen stored 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 after ICSI.35 In comparison very few sperm are required for assisted conception and in order to maximize the number of cycles the scientist may opt to freeze a lower number of sperm per straw (circa 40000).

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.74 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.75 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. However, owing to the problems in applying these guidelines to reproductive tissues, many aspects of gamete and embryo banking still require modification to ensure the safety of recipients and offspring. 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.76 Guidelines issued by the British Department of Health recommend that liquid nitrogen storage containers and cooling machines be subject to regular cleaning and disinfection.77 Two problems exist—firstly, the lack of suitable chemical cleaning agents for use with gametes, and, secondly, the inaccessability of some potentially contaminated parts of programmable freezing machines. The manufacturers following

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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 harbour pathogenic organisms. PACKAGING AND LEAKAGE Cracking and leakage from bone marrow Cryopreservation bags has been documented, and the 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.77 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 vessels78 and, theoretically, other ejaculates in storage. Similarly conclusive evidence of the leakage of liquid nitrogen into screw top cryovials, within three hours of placement into storage57 is another possible route of cross contamination. Both the Royal College of Pathologists79 and the manufacturers recommend that vials containing biological material be secondarily sealed using cryoflex tubing (Cryoflex Nalge Nunc International), before placing them in liquid nitrogen. An alternative form of

Fig 21.2 A newly developed (CBS) straw for human semen designed for aseptic loading and permanent sealing, developed in France (Cryo Bio systems, IMV Technologies, L’Aigle, France).

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straw for human semen storage (Fig 21.2), which takes into account the difficulties of aseptic loading and permanent sealing, has been developed in France (Cryo Bio systems, IMV Technologies, L’Aigle, France). Promising results of microbiological testing, and field trials conducted by the French ministry of agriculture suggest that this may substantially reduce the risk of cross infection in liquid nitrogen storage. 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 be screened for the presence of HIV 1 and 2 antibody, hepatitis B surface antigen (HbsAg), hepatitis C (HVC) 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. VAPOUR PHASE STORAGE While in theory vapour phase as an alternative to liquid nitrogen storage should minimize the risk of contamination with infective organisms, recent research detected bacterial pathogens within the vapour phase of storage tanks.76 Coupled with concerns regarding maintenance of all samples at a satisfactory temperature and the risk of partial thawing during sample retrieval, vapour phase storage has not received universal endorsement. However Clarke has demonstrated good short-term survival of human semen stored in cryovials in vapour phase.57

CONCLUSIONS 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.38,80 Latest developments in human beings include the introduction of new cryoprotective agents such as glutamine81 and pentoxifylline,82 investigation of novel linear cooling protocols,83 and the assessment of post-thaw sperm washing regimes84 to restore membrane lipid fluidity. An understanding of these processes will influence the design of the next generation of cryopreservation protocols for human spermatozoa. The major challenge today is the introduction of suitable modifications to

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ensure the safety of recipients and offspring85 while maintaining the efficacy of cryopreservation protocols and storage.

APPENDICES APPENDIX 1 COMPOSITION GLYCEROL EGG YOLK CITRATE (GEYC)61

Weigh out Trisodium citrate Glucose Fructose Glycine Double distilled water Glycerol

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)

For 2.5 litre stock buffer 50 g 20 g 20 g 5g 1966ml (approx.) 533ml

mM 68 44.4 44.4 26.7 – 2900

For stock buffer (Final glycerol concentration 1.09M) Dissolve solids in 1900ml water in sterile 3 litre flask with continuous mixing Add glycerol and mix thoroughly Adjust pH to 7.4 with 1 N NaOH Adjust final volume to 2500ml Filter (0.22µm) into sterile containers in 30ml portions Freeze and store at −80°C (maximum one year) For use Thaw out 30ml portion buffer on bench top arefully clean shell of fresh hen’s egg with sterile water Open egg and separate yolk from white in sterile petri dish Using sterile syringe aspirate 10ml egg yolk and add to 30ml buffer and mix well Dilute semen with GEYC 1:1v/v dropwise while continuously mixing GEYC will store at −20°C for up to one week—discard if evidence of bacterial growth, if it becomes translucent, or if it begins to separate APPENDIX 2 COMPOSITION HUMAN SPERM PRESERVING MEDIUM (HSPM)46

Constituents for 1 litre Sodium chloride (NaCl) Potassium chloride (KCl) Magnesium chloride (MgCl2.6H2O) Sodium dihydrogen phosphate (NaH2PO4.2H2O) Calcium chloride (CaCl2.2H2O)

g/l 5.8 0.4 0.1 0.05 0.4

mM 100 5.37 0.49 0.32 2.72

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Sodium hydrogen carbonate (NaHCO3) Sodium lactate (70–72% syrup) Glucose Kanamycin sulphate Glycine Sucrose Human serum albumin (HSA) HEPES (1 M stock solution) Glycerol

434

2.6 g 30.95 2.066ml 12.86 1.0 5.5 0.05 – 10.0 133.21 17.18 50.00 4.0 – 20ml 20.00 150ml 1700

Weigh out constituents (i) Dissolve solids (except HSA) in 500ml double distilled water by stirring continuously (ii) Add HEPES and sodium lactate and dissolve (iii) Add HSA and dissolve (iv) Add glycerol and make up to 1 litre with ddH20 (v) Adjust pH 6.5 with 1 N HCl (vi) Filter (0.22µm) into sterile containers (vii) Freeze and store at −20°C (maximum 3 months) (viii) Thaw on bench top at room temperature (ix) Add dropwise 1 part HSPM:1 part semen mixing continuously APPENDIX 3 DILUTION OF SEMEN WITH CRYOPROTECTIVE MEDIA AND PACKAGING

(1) (2) (3) (4) (5)

(6)

It is recommended that handling of semen, cryoprotectant, and packaging should be conducted within class II safety cabinet to ensure sterility. Scientific staff should wear appropriate protective clothing and gloves, and handle all semen samples as if potentially infective and to avoid the use of sharps such as needles. Only one sample (clearly labeled) should be handled at any one time to avoid possibility of confusion. Record patient details in laboratory register and assign unique identifier to ejaculate, for example, sample number and colour coding Ask patient/donor to collect semen into labelled sterile pot provided (preferably by masturbation) in a room close to the laboratory Allow semen to liquefy in incubator at 37°C and perform semen analysis86 as soon as possible preferably within one hour of production Allow pre-prepared cryoprotectant media to warm to room temperature 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 Add appropriate volume of cryoprotectant drop wise over 2–5 minute period with

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(7)

(8) (9) (10)

435

continuous gentle “swirling” to ensure thorough mixing 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 2). Do not allow straw to come in contact with semen or sides of container Remove filling device and heat seal end of straw using thermal sealer. Repeat steps 7–8 until sufficient straws are filled or entire sample is packaged Insert printed rod(s) labelled with patient/donor details and unique sample number into opposite end of straw and heat seal with thermal sealer Transfer straws to liquid nitrogen vapour phase or programmable freezer as soon as possible (i) VAPOUR PHASE FREEZING Suspend straws 5cm above liquid nitrogen horizontally on metal platform in uncirculated liquid nitrogen vapour for 10–20 minutes. Ensure straws are evenly spread and not touching. After freezing plunge into liquid nitrogen and transfer to labelled liquid nitrogen filled visitube before transferring to goblet in liquid nitrogen storage tanks—record location of sample in laboratory records

• • •

• •

(ii) 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 protocol7 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 1cm of top of semen within straw, taking care not to remove remainder of straw from nitrogen vapour 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 labelled liquid nitrogen filled visitube before transferring to goblet in liquid nitrogen storage tanks—record location of sample in laboratory records Store at −196°C. Avoid contamination of liquid nitrogen with environmental organisms and protect straws from chemical and radiation exposure

(iii) THAWING Identify location of straws in storage bank and confirm with laboratory records and patient/donor records (1) 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

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temperature for two to three minutes) (2) First wipe straws dry with paper tissue, then wipe semen end of straw with sterile alcohol swab and cut open with sterile scissors (3) Then wipe labelled end of straw with sterile swab and cut open with sterile scissors—remove labelled rod (4) Transfer straw to insemination device for intracervical insemination if required— alternative empty contents of straw into sterile container for post-thaw analysis and preparation

REFERENCES 1 Spallanzani L. Opuscoli di Fisica Anamale e Vegitabile Opuscola 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 DL. Storage of bull spermatozoa at low temperatures. Vet Rec (1951); 63:65–6. 4 Bunge RG, Sherman JK. Fertilizing capacity of frozen human spermatozoa. Nature (1953); 172:767–8. 5 Le Lannou D, Lansac J. Artificial procreation with frozen donor semen: experience of the French Federation CECOS. Hum Reprod (1989); 4 (7):757–61. 6 Keel BA, Webster BW, Roberts DK. Effects of cryopreservation on the motility characteristics of human spermatozoa. J Reprod Fertil (1987); 81 (1):213–20. 7 Critser JK, Huse-Benda AR, Aaker DV, Arneson BW, Ball GD. Cryopreservation of human spermatozoa. I. Effects of holding procedure and seeding on motility, fertilizability, and acrosome reaction [published erratum appears in Fertil Steril (1987); 48 (3):575]. Fertil Steril (1987); 47 (4):656–63. 8 McLaughlin EA, Ford WC, Hull MG. Motility characteristics and membrane integrity of cryopreserved human spermatozoa. J Reprod Fertil (1992); 95 (2):527–34. 9 Heuchel V, Schwartz D, Czyglik F. Between and within subject correlations and variances for certain semen characteristics in fertile men. Andrologia (1983); 15 (2): 171–6. 10 Leibo SP, Bradley L. Comparative cryobiology of mammalian spermatozoa. In: Gagnon C, ed. The male gamete: from basic science to clinical applications. Vienna: Cache River Press, 1999:501–16. 11 Centola GM, Raubertas RF, Mattox JH. Cryopreservation of human semen. Comparison of cryopreservatives, sources of variability, and prediction of post-thaw survival. J Androl (1992); 13 (3):283–8.

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12 Watson PF. Artificial insemination and the preservation of semen. In: Lamming GE, editor. Marshall’s Physiology of Reproduction. 4th ed. Edinburgh: Churchill Livingstone; 1990:747–869. 13 Stewart GJ, Tyler JP, Cunningham AL, et al. Transmission of human T-cell lymphotropic virus type III (HTLV-III) by artificial insemination by donor. Lancet (1985); 2 (8455):581–5. 14 ASRM. Guidelines for gamete and embryo donation. The American Society for Reproductive Medicine. Fertil Steril (1998); 70 (4 Suppl 3):1S-13S. 15 Barratt CL, Matson DL, Holt W. British Andrology Society guidelines for the screening of semen donors for donor insemination. Hum Reprod (1993); 8 (9):1521–3. 16 British Andrology S. British Andrology Society guidelines for the screening of semen donors for donor insemination (1999). Hum Reprod (1999); 14 (7):1823–6. 17 Mahadevan MM, Trounson AO, Leeton JF. Successful use of human semen cryobanking for in vitro fertilization. Fertil Steril (1983); 40 (3):340–3. 18 Hull MG, Williams JA, Ray B, McLaughlin EA, Akande VA, Ford WC. The contribution of subtle oocyte or sperm dysfunction affecting fertilization in endometriosis-associated or unexplained infertility: a controlled comparison with tubal infertility and use of donor spermatozoa. Hum Reprod (1998); 13 (7):1825–30. 19 Richter MA, Haning RV, Jr, Shapiro SS. Artificial donor insemination: fresh versus frozen semen; the patient as her own control. Fertil Steril (1984); 41 (2):277–80. 20 Bordson BE, Ricci ER, Dickey RP, Dunaway H, Taylor SN, Curole DN. Comparison of fecundability with fresh and frozen semen in therapeutic donor insemination. Fertil Steril (1986); 46 (3):466–9. 21 Hammond MG, Jordan S, Sloan CS. Factors affecting pregnancy rates in a donor insemination program using frozen semen. Am J Obstet Gynecol (1986); 155 (3):480–5. 22 Keel BA, Webster BW. Semen analysis data from fresh and cryopreserved donor ejaculates: comparison of cryoprotectants and pregnancy rates. Fertil Steril (1989); 52 (1):100–5. 23 Barratt CL, Clements S, Kessopoulou E. Semen characteristics and fertility tests required for storage of spermatozoa. Hum Reprod (1998); 13 (Suppl 2):1–7; discussion 8–11. 24 Royere D, Barthelemy C, Hamamah S, Lansac J. Cryopreservation of spermatozoa: a 1996 review. Hum Reprod Update (1996); 2 (6):553–9. 25 Le Lannou D, Gastard E, Guivarch A, Laurent MC, Poulain P. Strategies in frozen donor semen procreation. Hum Reprod (1995); 10 (7):1765–74. 26 Ford WC, Mathur RS, Hull MG. Intrauterine insemination: is it an effective treatment for male factor infertility? Bailliere’s Clin Obstet Gynaecol (1997); 11 (4):691–710.

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27 Kobayashi T, Kaneko S, Hara I, et al. Concentrating human sperm before Cryopreservation. Andrologia (1991); 23 (1):25–8. 28 Srisombut C, Morshedi M, Lin MH, Nassar A, Oehninger S. Comparison of various methods of processing human cryopreservedthawed semen samples. Hum Reprod (1998); 13 (8):2151–7. 29 Larson JM, McKinney KA, Mixon BA, Burry KA, Wolf DP. An intrauterine insemination-ready Cryopreservation method compared with sperm recovery after conventional freezing and post-thaw processing. Fertil Steril (1997); 68 (1):143–8. 30 Pfeifer SM, Coutifaris C. Reproductive technologies 1998: options available for the cancer patient. Med Pediatr Oncol (1999); 33 (1):34– 40. 31 Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet (1992); 340 (8810): 17–8. 32 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. 33 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 low-quality semen. Int J Androl (1999); 22 (3):190–6. 34 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): 114–7. 35 Tournaye H, Merdad T, Silver 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. 36 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): 112–8. 37 Karow AM, Jr. Cryoprotectants—a new class of drugs. J Pharm Pharmacol (1969) 21 (4):209–23. 38 Curry MR. Cryopreservation of semen from domestic livestock. Rev Preprod (2000); 5 (1):46–52. 39 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. 40 Gao DY, Ashworth E, Watson PF, Kleinhans FW, Mazur P, Critser JK. Hyperosamotic tolerance of human spermatozoa: separate effects of glycerol, sodium chloride, and sucrose on spermolysis. Biol Reprod (1993); 49 (1):112–23.

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41 Sherman JK. Improved methods of preservation of human spermatozoa by freezing and freeze drying. Fertil Steril (1963); 14:49–64. 42 Behrman SJ, Sawada Y. Heterologous and homologous inseminations with human semen frozen and stored in a liquid nitrogen refrigerator. Fertil Steril (1966); 17:457–66. 43 Behrman SJ, Ackerman DR. Freeze preservation of human sperm. Am J Obstet Gynecol (1969); 103 (5):654–64. 44 Pilikian S, Czyba JC, Guerin JF. Effects of various concentrations of glycerol on post-thaw motility and velocity of human spematozoa. Cryobiology (1982); 19 (2): 147–53. 45 Richardson DW. Factors influencing the fertility of frozen semen. In: Richardson D, Joyce D, Symonds M, eds. Frozen human semen. London: RCOG, 1979:33–54. 46 Mahadevan M, Trounson AO. Effect of cryoprotective media and dilution methods on the preservation of human spermatozoa. Andrologia (1983); 15 (4):355–66. 47 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. 48 Friberg J, Gemzel 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. 49 Harrison RF, Sheppard BL. A comparative study in methods of cryoprotection for human semen. Cryobiology (1980); 17 (1):25–32. 50 Jeyendran RS, Van der Yen HH, Kennedy W, Perez-Pelaez M, Zaneveld 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. 51 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. 52 Peek JC, Gilchrist SJ, Kelso CM, Quinn PJ. Comparison of three cryoprotective solutions for human semen. Clin Reprod Fertil (1982); 1 (4):301–5. 53 Prins GS, Weidel L. A comparative study of buffer systems as cryoprotectants for human spermatozoa. Fertil Steril (1986); 46 (1):147–9. 54 Fink K, Zech H. [Effect of incubation time in deep freezing human sperm]. Wien Klin Wochenschr (1991); 103 (23):707–9. 55 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.

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56 Kremer J, Dijkhuis JR, Jager S. A simplified method for freezing and storage of human semen [published erratum appears in Fertil Steril (1988); 49 (2):382]. Fertil Steril (1987); 47 (5):838–42. 57 Clarke GN. Sperm Cryopreservation: is there a significant risk of cross-contamination? Hum Reprod (1999); 14 (12):2941–3. 58 Thachil JV, Jewett MA. Preservation techniques for human semen. Fertil Steril (1981); 35 (5):546–8. 59 Wolf DP, Patton PE. Sperm Cryopreservation: state of the art. J In Vitro Fert Embryo Transf (1989); 6 (6):325–7. 60 Verheyen G, Pletincx I, Van Steirteghem A. Effect of freezing method, thawing temperature and post-thaw dilution/washing on motility (CASA) and morphology characteristics of high-quality human sperm. Hum Reprod (1993); 8 (10):1678–84. 61 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. 62 Cyzba JC, Pinatel MC, Geurin JF. Preservation and storage of human sperm. Acta Med Pol (1978); 19:133–45. 63 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. 64 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):911–8. 65 Mahadevan MM, Trounson AO. Relationship of fine structure of sperm head to fertility of frozen human semen. Fertil Steril (1984); 41 (2):287–93. 66 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. 67 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. 68 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. 69 Human Fertilisation and Embryology Authority (HFEA). HFEA. Eighth annual report and accounts. London: Dept of Health, 1999 (31 August 1999.) 70 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.

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71 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. 72 Hurd WW, Randolph JF, Jr, Ansbacher R, Menge AC, Ohl DA, Brown AN. Comparison of intracervical intrauterine, and intratubal techniques for donor insemination [see comments]. Fertil Steril (1993); 59 (2):339–42. 73 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. 74 Tedder RS, Zuckerman MA, Goldstone AH, et al. Hepatitis B transmission from contaminated Cryopreservation tank. Lancet (1995); 346 (8968):137–40. 75 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 Methods (1996); 60 (1):81–8. 76 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. 77 Department of Health. Guidance on the processing, storage and issue of bone marrow and blood stem cells. London: HMSO, 1997. 78 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. 79 Party RCoPW. HIV and the practice of pathology. London: Royal College of Pathologists, 1995. 80 Phelps MJ, Liu J, Benson JD, Willoughby CE, Gilmore JA, Critser JK. 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. 81 Renard P, Grizard G, Griveau JF, Sion B, Boucher D, Le Lannou D. Improvement of motility and fertilization potential of postthaw human sperm using glutamine. Cryobiology (1996); 33 (3):311–9. 82 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. 83 Morris GJ, Acton E, Avery S. A novel approach to sperm cryopreservation. Hum Reprod (1999); 14 (4):1013–21.

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84 James PS, Wolfe CA, Mackie A, Ladha S, Prentice A, Jones R. Lipid dynamics in the plasma membrane of fresh and cryopreserved human spermatozoa. Hum Reprod (1999); 14 (7): 1827–32. 85 Avery SM, McLaughlin EA, Dawson KJ. Safe cyropreservation of sperm and embryos. Hum Fertil (1998); 1:84–6. 86 WHO. Laboratory manual for the examination of human semen and sperm cervical mucus interaction. 4th ed. Cambridge: Cambridge University Press, UK, 1999.

22 Handling and cryopreservation of testicular sperm William W Lin, Benjamin Hendin, Dolores J Lamb, Larry I Lipshultz

With the advent of intracytoplasmic injection (ICSI), men with nonobstructive azoospermia who were previously considered hopelessly infertile now can potentially initiate pregnancy, if mature spermatozoa can be harvested from the testes.1–5 Schoysman et al first demonstrated that spermatozoa extracted from the testis are able to successfully fertilize human oocytes, leading to pregnancy.6–8 Patients with a predominant pattern of germinal cell aplasia on previous testis biopsies have been found to have mature spermatozoa in 25% of the cases in which repeat biopsy is performed. Similarly, close to 50% of patients with spermatogenic maturation arrest are also found to have mature spermatozoa on testicular biopsy.9 The corollary, of course, is that mature sperm will not be found in 50–75% of patients with spermatogenic failure, despite meticulous dissection at the time of attempted testicular sperm extraction (TESE). Unless testicular tissue and testicular spermatozoa are successfully cryopreserved, procurement procedures will necessarily have to be timed to coincide with oocyte aspiration for couples undergoing TESE-ICSI. In those cases in which no mature spermatozoa can be isolated from the testicular tissue, couples will have undergone significant physical, emotional, and financial burdens without a positive outcome. It is therefore advantageous to have viable sperm available in advance of ovulation induction for those couples who suffer from spermatogenic failure. Also, without cryopreservation, the testicular tissue and testicular sperm can only be used for one cycle of ICSI. With pregnancy rates of 35–40% per ICSI cycle, successful pregnancy outcomes may necessitate repeat cycles of micromanipulation in a significant number of couples. Lacking cryopreserved sperm, each cycle of ICSI in these couples would require repeated procurement procedures. Testicular sperm procurement is not innocuous, and with each successive procedure, identifying healthy testicular parenchyma will become more difficult as a consequence of fibrosis. Furthermore, repeated testicular surgery can cause permanent testicular damage, including partial testicular devascularization, irreversible atrophy, deterioration of spermatogenic development, and even loss of endocrine function, which would necessitate exogeneous

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testosterone replacement.10 However, with the ability to cryopreserve the testicular tissue, procurement procedures can be performed at the time of diagnostic biopsy. Sufficient testicular tissue may be obtained to provide the spermatozoa for multiple cycles of micromanipulation, thereby minimizing the number of invasive testicular procedures. Furthermore, the tissue harvest does not need to be timed to coincide with ovulation induction. Freeze-thawed testicular sperm have been shown to be capable of fertilizing human oocytes. Gates et al described 10 men with nonobstructive azoospermia who underwent cryopreservation of testicular tissue; spermatozoa obtained from these procedures were used in 19 cycles of ICSI, with an overall fertilization rate of 48%, comparable to ICSI fertilization rates using fresh testis derived spermatozoa.11

SPERM PREPARATION Prior to cryopreservation, the harvested testicular tissue needs to be processed. Gates et al proposed a method of mechanical homogenization using a loose fitting glass pestle.11 The homogenate is then further processed by repeated aspiration through a 16 gauge hypodermic needle. The final homogenate is then placed in polypropylene tubes for cryopreservation. Other methods of mechanical testicular tissue homogenization include the use of 22 gauge hypodermic needles for tissue dissection, as described by Tucker et al.12 Besides mechanical homogenization, testicular sperm may also be extracted enzymatically. Salzbrunn et al described a protocol using type IV collagenase (Sigma, Heidelberg, Germany) and a trypsin inhibitor (Sigma).13 The protocol yielded viable spermatozoa after the specimens were thawed. In addition to different methods of sperm extraction, the testicular tissue and spermatozoa obtained from TESE can be cryopreserved by using several different techniques. One method is to place testicular tissue homogenate in 1 ml aliquots using polypropylene ampules. However, it is not an easy task to identify mature spermatozoa, even in the fresh TESE specimen. A frozen specimen is certainly not easier to process. It often takes hours of meticulous dissection under the microscope to find a handful of mature spermatozoa. Areas of spermatogenesis in testes with a predominant histology of either germinal cell aplasia, or of spermatogenic arrest, are often focal.14 In these cases especially, it is advantageous to cryopreserve individual sperm as they are dissected from the testicular parenchyma. When the testicular tissue homogenate is cryopreserved, there is a considerable risk of losing spermatozoa through the conventional addition and removal of cryoprotectant in a relatively large volume of media. Hewitt et al showed that conventional sperm freezing protocols would not work with a limited number of spermatozoa.15 In 1991, Cohen et al described a method whereby cryopreservation and subsequent recovery of single spermatozoa can be performed even in men

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who have fewer than 100 spermatozoa present in the final testicular tissue homogenate.16 A porous capsule, such as the emptied zona pellucida, is used as a vessel to contain individual spermatozoa. Empty zona pellucida are prepared by microscopic dissection; small incisions are made in the zona using microdissection instruments, and the ooplasm is emptied using a microsuction device. The emptied zona pellucida are then maintained in HEPES buffered human serum albumin-supplemented human tubular fluid. A sperm suspension in 10% polyvinylpyrrolidone (PVP) is then injected into the zona by using micromanipulation techniques. The injected zona are then placed in an 8% glycerol solution and cryopreserved using standard freeze protocols with sterile plastic straws. Greater than 75% of spermatozoa have been reported to be recovered, and ICSI fertilization rates of 78% have been described. The use of empty zona pellucida also reduces the loss of motility associated with post-thaw dilution and sperm washing, which is observed with frozen donor semen.17 Craft and coworkers have proposed an alternate method of testicular sperm cryopreservation by using paraffin oil.18 Spermatozoa are suspended in glycerol and then placed in a droplet of paraffin oil, which is placed in the bottom of a microcentrifuge tube. The droplet is then frozen in the standard fashion. Motile sperm have been recovered using this frozen-thawed paraffin oil droplet procedure.

CRYOPRESERVATION All living cells function within a very narrow temperature range. Approximately 50% of donor sperm, which are presumed to be normal, are typically lost in a single freeze-thaw cycle. The lethality of the freezethaw process is in part consequent to plasma membrane damage and loss of its integrity, which leads to cellular permeabilization and subsequent loss of the homeostatic intracellular solute composition that is vital to sperm function. Sawada et al demonstrated that phase transition from the liquid to the frozen state is the chief mechanism of plasma membrane damage.19 Jeyendran et al have since demonstrated that membrane damage may also be a consequence of enhanced lipid peroxidation, which causes the loss of membrane phospholipids.20 It is an inherent risk that lethal damage may occur to some sperm during a freeze-thaw cycle. Several physiologic factors determine a cell’s ability to survive the freezethawing process. Many different cell types can be successfully supercooled well below the freezing point of water, as long as a phase transition does not occur. Marzur et al have demonstrated that yeast cells isolated in an oil droplet can survive cooling to −40°C, as long as there is no formation of ice crystals.21 Such observations indicate that exposure to subzero temperatures alone is not necessarily lethal.

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Cells are destroyed when ice is present in their environment. However, cell death is not inevitable, even when ice is present. Leibo et al demonstrated that the addition of various compounds to the cryopreservation solution could further alter the resilience of given cells.22 Cellular survival is dependent on the composition of the cryopreservation solution. In vitro stem-cell studies have demonstrated that cell survival increases as higher concentrations of glycerol are used. The mechanism of glycerol’s cryoprotective effect is unclear; but, it has been postulated that glycerol may modulate the membrane phase transition.20 Numerous other protective agents have been developed, including dimethyl sulfoxide, glucose, sucrose, polyvinyl pyrolidone, polyethylene glycol, and dextran. Egg yolk buffer containing glycerol (Irvine Scientific, Irvine, CA, USA) is the most commonly used cryoprotectant. Several investigators have shown the beneficial effect of the egg yolk on sperm cryosurvival. Conversely, Mahadevan et al found that a cryoprotective medium without egg yolk yielded better cryosurvival than media with egg yolk.23 Nevertheless, most cryobanks in the world now use glycerol/egg-yolk buffer. Although the addition of antibiotics to the cryoprotectant buffer might seem empirically worthwhile, cytotoxic effects of antibiotics, such as penicillin and streptomycin, have been documented.24 Although these agents are ineffective against clinically significant bacterial contamination, most cryolaboratories still use glycerol/egg-yolk buffer containing antibiotics. The cooling rates used during cryopreservation significantly influence cellular survival. Mazur et al demonstrated that the survival of yeast cells is a function of the rate at which they are cooled.21 At slow cooling rates, a large number of ice crystals nucleate and form large ice crystals. On the other hand, at rapid cooling rates, the ice crystals formed are much smaller and grow at such a rapid rate that cells are trapped between the crystals, enabling a greater proportion to survive. However, exceeding the optimum cooling rate results in a decrease in sperm survival. The optimum cooling rate is cell type dependent.21 Warming rates also significantly impact on the cell viability during the thawing process.25 At a slow warming rate, ice crystals will tend to aggregate into much larger crystals by a process of “migratory recrystallization.” These large crystals may disrupt the delicate cellular cytostructure. At a rapid warming rate, this process is significantly less likely to occur. Finally, the damaging effect of the warming rate may also depend on the prior freeze rate. When frozen at rates exceeding 100°C/min, cultured cells are extremely dependent on the subsequent warming rate. However, survival of those cells frozen at rates less than 100°C/min are virtually independent of the warming rate. When one takes into account these basic principles of cryobiology, cryopreservation protocols have undergone significant evolution over the last several decades, particularly since the advent of programmable

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freezing units. Sherman and Kim introduced the simple liquid nitrogen vapor freezing protocol in 1967.26 This technique has now been supplanted by stepwise, electronically controlled, freezing protocols. With the ability to configure different freezing rates, the protocol can now be tailored according to cell type to achieve optimal cryosurvival. A commonly used protocol is the two step approach: the specimen is lowered into a nitrogen vapor chamber and stabilized at −80°C for 20 minutes at a cooling rate of −10°C/minute; the specimen is then immersed into liquid nitrogen for permanent storage at −196°C.

TECHNIQUES OF TESTICULAR TISSUE AND TESTICULAR SPERMATOZOA CRYOPRESERVATION At our institution, testicular tissue is harvested by an open technique, as described by Coburn et al, at the time of diagnostic biopsy.27 Conscious sedation and local anesthesia are employed, by using intravenous midazolam and a spermatic cord block with 2% midazolam. A transverse scrotal incision is carried through the dartos fascia and then through the tunical vaginalis. The partially exposed tunica vaginalis is then incised. Extruded seminiferous tubules are excised from the testis. A wet preparation is performed using modified sperm washing media (Irvine Scientific) and a phase contrast microscope to identify mature sperm. A portion of the tissue is then placed in Bouin’s solution for formal histological examination. If mature spermatozoa are identified, additional testicular tissue is harvested through the same tunical incision and this tissue is placed in 1 ml of test-yolk buffer (TYB, Irving Scientific). If no spermatozoa are identified, seminiferous tubules from other regions of the ipsilateral testis are systematically sampled, and the contralateral testis is subsequently explored in a similar fashion, stopping if and when mature spermatozoa are identified. Testicular tissue is dissected in a shallow Falcon dish using fine jeweler’s forceps under an inverted dissecting microscope. Individual tubules are separated, and the homogenate fluid is examined for mature spermatozoa. When sperm are found, the homogenate is adjusted to a final volume of 5.0ml of TYB:glycerol. Aliquots of 1 ml are transferred to sterile polypropylene ampoules and cryopreserved using standard donor semen protocols.

CONCLUSION Cryopreservation of testicular tissue and its ultimate effective use with ICSI has revolutionized the way we practice reproductive medicine. Sperm can be identified, stored, and used at the time of oocyte maturation.

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In this way, couples are spared the disappointment of undergoing the process of controlled ovarian hyperstimulation and oocyte retrieval, only to learn that pregnancies could not be achieved owing to lack of available sperm. Because there is never an absolute guarantee that frozen sperm will thaw with appropriate viability, counseling regarding the use of “backup” donor sperm should always be considered.

REFERENCES 1 Devroey P, Liu J, Nagy Z, Tournaye H, Silver SJ, Van Steirteghem AC. Normal fertilization of human oocytes after testicular sperm extraction and intracytoplasmic sperm injection. Fertil Steril (1994); 62:639–41. 2 Devroey P, Liu J, Nagy Z, et al. Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum Reprod (1995); 10:1457–60. 3 Gil-Salom M, Minguez Y, Rubio C, Remohi J, Pellicer A. Intracytoplasmic sperm injection: An effective treatment for otherwise intractable obstructive azoospermia. J Urol (1995); 154:2074–7. 4 Gil-Salom M, Minguez Y, Rubio C, De los Santos MJ, Remohi J, Pellicer A. Efficacy of intracytoplasmic sperm injection using testicular spermatozoa. Hum Reprod (1995); 10:3166–70. 5 Kahraman S, Ozgur S, Alatas C, et al. Fertility with testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermic men. Hum Reprod (1996); 11:756–60. 6 Schoysman R, Vanderzwalmen P, Nijs M, Segal-Bertin G, van de Casseye M. Successful fertilization by testicular spermatozoa in an invitro fertilization programme. Hum Reprod (1993); 8:1339–40. (Letter.) 7 Silber SJ, Van Steirteghem AC, Liu J, Nagy Z, Tournaye H, Devroey P. High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy. Hum Reprod (1995); 10:148:52. 8 Tournaye H, Camus M, Goossens A, et al. Recent concepts in the management of infertility because of nonobstructive azoospermia. Hum Reprod (1995); 10(Suppl 1): 115–9. 9 Su LM, Palermo GS, Goldstein M, Veeck LL, Rosenwaks Z, Schlegel PN. Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia: Testicular histology can predict success of sperm retrieval. J Urol (1999); 161:112–6. 10 Schlegel PN, Su LM. Physiological consequences of testicular sperm extraction. Hum Reprod (1997); 12:1688–92. 11 Gates 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 nonobstructive azoospermia. Hum Reprod (1997); 12:734–9.

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12 Tucker MJ, Morton PC, Wright G, Ingargiola PE, Jones AE, Sweitzer CL. Factors affecting success with intracytoplasmic sperm injection. Reprod Fertil Dev (1995); 7:229–36. 13 Salzbrunn A, Benson DM, Holstein AF, Schulze W. A new concept for the extraction of testicular spermatozoa as a tool for assisted fertilization (ICSI). Hum Reprod (1996); 11:752–5. 14 Jow WW, Steckel J, Schlegel PN, Magid MS, Goldstein M. Motile sperm in human testis biopsy specimens. J Androl (1993); 14:194–8. 15 Hewitt J, Cohen J, Mathew T, Rowland G. Cryopreservation of semen in patients with malignant disease: role of in-vitro fertilisation. Lancet (1985); 2:446–7. 16 Cohen J, Garrisi GJ, Congedo-Ferrara TA, Kieck KA, Schimmel TW, Scott RT. Cryopreservation of single human spermatozoa. Hum Reprod (1991); 12:994–1001. 17 Verheyen G, Pletinex I, Van Steirteghem A. Effect of freezing method, thawing temperature and post-thaw dilution/washing on motility (CASA) and morphology characteristics of high-quality human sperm. Hum Reprod (1993); 8:1678–84. 18 Craft I, Tsirigotis M. Simplified recovery, preparation and cryopreservation of testicular spermatozoa. Hum Reprod (1995); 10:1623–6. 19 Sawada Y, Ackerman DR, Behrman SJ. Motility and respiration of human spermatozoa after cooling to various low temperatures. Fertil Steril (1967); 18:775–81. 20 Jeyendran RS, Van der Yen HH, Kennedy W, Perez-Pelaez M, Zaneveld LJ. Comparison of glycerol and zwitter ion buffer system as cryoprotective media for human spermatozoa. J Androl (1984); 5:1–7. 21 Mazur P, Schmidt JJ. The survival of yeast cells as a function of the rate at which they were cooled. Cryobiology (1968); 5:1. 22 Leibo SP, Mazur P. The role of cooling rates in low temperature cryopreservation. Cryobiology (1971); 8:447–52. 23 Mahadevan MM, Trounson AO. Biochemical factors affecting the fertility of cryopreserved human semen. Clin Reprod Fertil (1983); 2:217–27. 24 Timmermans L. Influence of antibiotics on spermatogenesis. J Urol (1974); 112:348–9. 25 Leibo SP, Mazur P, Jackowski SC. The survival rates of different cell types as a function of the rate at which they were warmed from the frozen state. Exp Cell Res (1974); 89:79. 26 Sherman JK, Kim KS. Correlation of cellular ultrastructure before freezing, while frozen, and after thawing in assessing freeze-thawinduced injury. Cryobiology (1967); 4:61–74. 27 Coburn M, Wheeler, T, Lipshultz, LI. Testicular biopsy. Its use and limitations. Urol Clin North Am (1987); 14:551–61.

23 Ovarian tissue cryopreservation and transplantation Kutluk H Oktay

OVERVIEW Studies performed on laboratory animals proved the feasibility of cryopreservation and transplantation of ovarian tissue.1,2 In this chapter, we will discuss the experience from sheep autograft and human ovarian xenograft models, and we will present our preliminary findings on the application of this technology in humans. For the first time, ovarian function after human ovarian transplantation will be reported. We will also illustrate the practical points on ovarian tissue cryopreservation, which may enhance the survival of primordial follicles and aid in the success of ovarian transplantation.

BACKGROUND Ovarian tissue banking relies on the principle of resistance of the primordial follicles to cryotoxicity.3 Because of its relatively inactive metabolism, absence of zona pellucida, and lack of metaphase spindle, primordial follicles are more durable than the larger, growing follicles when exposed to extreme changes in ambient temperature.4 Cryoprotectants are still needed to preserve the viability of primordial follicles; relatively smaller cell size makes it easier for cryoprotectants to penetrate. In larger follicles, it takes longer for the cryoprotectants to diffuse and distribute evenly.5 Comparison of the “pros and cons” of freezing ovarian follicles and oocytes at various stages are shown in Table 23.1.

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Table 23.1. Comparison of available options for banking follicles, oocytes and ovarian tissue. Cell/tissue Size Advantage Disadvantage type Primordial 30–50µm Least differentiated Difficult to isolate follicle No ZP Damage to basement No cell spindle membrane Culture not yet possible Preantral 60–200µm Easier to isolate ZP damage follicle No cell spindle More differentiated Culture possible Prophase 1 80–100µm No spindle ZP damage oocyte Short in vitro Low IVM and maturation fertilization Very few live births Metaphase II 80–100µm Easy to obtain ZP damage oocyte Cell spindle damage Organelle damage Few live births Experimental Ovarian cortex 1mm×1mm to Easy to obtain Risk of cancer cell 1cm×3cm strips Preserves stroma Can restore fertility reseeding Preventive before cancer treatment ZP: zona pellucida IVM: in vitro maturation 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 would have 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 will be 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 the ovarian cortical pieces for which there has been considerable success in achieving follicle development, after grafting in animals.

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ANIMAL MODELS OF OVARIAN TRANSPLANTATION 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. Gosden has championed the sheep ovarian transplantation model. In the first study, frozenbanked strips of ovarian cortical pieces were autotransplanted on the infindibulopelvic ligament.6 Each animal also had a fresh transplant on the opposite site serving as a control. Four months after the transplant, first signs of ovulation were detected. Two pregnancies had occurred, one from a fresh another from a frozen-thawed graft. In the second study, autotransplants were performed with frozenthawed tissue in eight sheep and the animals were monitored for up to 22 weeks.7 All the animals resumed cyclicity and showed hormone production. In that study, baseline follicle stimulating hormone (FSH) concentrations were elevated, but the luteal phase progesterone measurements were normal. However, serum inhibin-A levels were found to be low in the luteal phase. Nevertheless, these studies proved that pregnancies could be achieved after transplantation of cryopreserved ovarian tissue, and the luteal phase resulting from transplanted ovaries was comparatively normal. Doors were now open to studying human ovarian transplantation.

XENOGRAFTING USING HUMAN OVARIAN TISSUE SCID-mice carry a genetic mutation, which results in T cell and B cell immunodeficiency.8 This allows xenografts to revascularize and survive in these animals without being rejected. Gosden has adopted this model for human ovarian xenografting. In his earlier studies, both marmoset and sheep ovarian tissues were transplanted under the kidney capsule, and they grew to antral stages.9 In the first study using the human tissue as a xenograft, Gosden et al cryopreserved ovarian cortical pieces with various cryoprotectants and grafted into SCID mice.10 After 18 days, the grafts were removed, and primordial follicle counts were obtained. With the exception of glycerol, all cryoprotectants (propanediol, ethylene glycole, DMSO) did well and 44–84% of the follicles survived. On the basis of these successful results with short term xenografting, we performed the two long term studies in SCID mice by using human tissue.11,12 In the first study, one mm3 ovarian cortical pieces from a 17 year old patient were grafted under the kidney capsules of hypogonadal

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SCID mice.11 During the last six 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 5.5mm were found, estradiol levels peaked >700pg/ml, and the uteri showed clear signs of estrogenization (Table 23.2). 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.12 Because these animals were not hypogonadal, no FSH was administered. Grafts were recovered 22 weeks later. Histological examination showed that many follicles had

Table 23.2. Antral follicle development and estradiol production after transplantation of human ovarian tissue in the SCID mice. Reproduced from Reference 11: (1998); 13:1133–8. Graft Number of antral follicles Serum estradiol Uterine weight Vaginal (mm diameter) (pmol/l) (mg) introitus 1 1(5) 2070 212 (ballooned) Patent 2 2 (3,4) 780 131 Patent 3 0 35 123 Patent 4 1 (2.5, hemorrhagic) 280 126 Patent initiated growth. Interestingly, compared with controls, a higher percentage of follicles had initiated growth (5.6±2.4 v. 12.5±1.9, P<0.05), but a significant number of primordial follicles/graft (75±6.8) remained. Presumably, because no exogenous FSH was given, follicles did not develop beyond one to two layer stage. However, this study further strengthened the concept that the cryopreservation of ovarian tissue may be the future method of choice for preserving unfertilized gametes. We have recently quantified the survival rates of primordial follicles by viability stains after cryopreservation.3 2×2mm ovarian cortical pieces were cryopreserved with the slow freeze protocol (Table 23.3). After thawing, the tissues were partly digested using collagenase type IA (Sigma, USA) followed by microdissection of primordial follicles (Table 23.4). These follicles were then incubated with viability stains. We found that, about 70% of follicles had survived this process and were viable. Later electron microscopic studies, however, showed that this digestion method might damage the basement membrane of the follicle but the oocyte is rarely affected.13 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.13 These findings create another argument in favor of ovarian tissue freezing as opposed to the cryopreservation of isolated follicles.

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CLINICAL TRIALS OF HUMAN OVARIAN TISSUE CRYOPRESERVATION AND TRANSPLANTATION Encouraged by these laboratory studies, we have established a human ovarian transplantation project. In the first phase, only patients aged <35 years were enrolled, and cases with malignancies were excluded. In the 32 year old first patient, ovarian tissue was grafted in the forearm into the brachioradialis muscle, akin to the method used for parathyroid gland transplantation. We also grafted tissue in the broad ligament, adjacent to the uterine artery. Four months after the procedures, ultrasound follow up visualized the grafts, and early antral follicle development was noted in the forearm by high frequency ultrasound probes. A gradient was detected for estradiol, between the antecubital vein (which the graft drains to) and the wrist veins, indicating hormone production by the graft.

Table 23.3. Protocol for primordial follicle isolation. (1) Obtain 1–3mm thick ovarian cortical biopsies from patients <35 years of age. (2) Mince the tissue in 2mm×2mm pieces. (3) Incubate in disaggregation media containing 1mg/ml of collagenase type la and 25U/ml Dnase in L-15 medium for 1 hour 45 minutes at 37°C on a roller. (4) Wash the tissue with 1–15 with 10% serum×3. (5) Isolate primordial follicles using 27 gauge insulin needles and a siliconized mouth pipette, under 50 magnification. In the second patient, frozen banked tissue was thawed eight months after its storage. The pieces were sewn together with a microsurgical technique to form two large grafts, and they were transplanted laparoscopically in the ovarian fossa.14 This patient is currently being followed, however, early findings indicate the normalization of serum testosterone levels and resumption of ovarian function.

CLINICAL AND LABORATORY TIPS FOR CRYOPRESERVING HUMAN OVARIAN TISSUE Even though there is a limited number of studies addressing the most optimum way of cryopreserving human ovarian tissue, we have found the following points useful: (1) Age: The follicle density in frozen-thawed ovarian tissue from >35 years of age tend to have extremely low follicle densities. Therefore we do not recommend ovarian tissue freezing in women older than that. Earlier studies suggested that >60 % of the primordial follicles are lost due to the initial ischemia before revascularization after transplantation, and the freeze-thaw procedure results only in an additional 7%

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follicle loss.7 These losses are better tolerated in younger patients with 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 or better than that of a 35 year old. This point underscores the importance of age in ovarian tissue cryopreservation, perhaps better chances are offered to women of <30 years of age. (2) Tissue collection: Tissue collection is best done in the early follicular phase to avoid large ovarian follicles or a corpus luteum, which may result in hypervascularity and anatomical distortion. Ovarian cortical tissue can easily be collected via laparoscopy. This can be done using a laparoscopic punch biopsy device (CasMed, London, UK) or by oophorectomy.15 The tissues should be transported to the lab on ice. Primordial follicles have a higher tolerance for hypoxia when kept in serum supplemented media, therefore delays as long as four to six hours do not have a significant effect on follicle survival.16 (3) Optimal tissue size: The tissues should not be frozen too small. This results in excessive follicle damage during slicing and creates tissue pieces unmanageable for transplantation. In the sheep, 0.5cm×0.5–1.5cm pieces have resulted in long term follicle growth6,7 and this has also been our experience with human ovarian tissue. It is best to slice the cortex in thin (1–2mm) but long strips (1.5cm×0.5cm) (Table 23.4). (4) Choice of cryoprotectant: There is no significant difference between propanediol, ethylene glycol or DMSO.10 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 23.4.

CONCLUSIONS Ovarian tissue banking can offer hope for 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 ageing. There has been a significant progress in ovarian tissue cryopreservation and transplantation in the past five years. Now, large scale human transplantation studies are needed to test the efficacy of this procedure. In addition, research is needed to develop better cryoprotectants and cryopreservation protocols as well as new transplantation techniques to reduce follicle losses that normally occur during the freeze-thaw and grafting process.

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Table 23.4. “Oktay” modification of human ovarian tissue cryopreservation protocol. Freezing: (1) Equilibrate 1–3mm thick, 3×10mm strips of ovarian cortex for 30 minutes at 4°C in phenol free buffered medium (L-15) containing 1.5M DMSO (propanediol/ethylene glycole) 20% serum and 0.1M sucrose. Place the vials on a tissue roller during incubation to ensure 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 at 37°C water bath for two minutes. (9) Wash tissues stepwise in containing progressively lower concentrations of cryoprotectant media with 20% serum plus 0.1M sucrose, gently agitate tissue for five minutes in each step (1.5M, 1.0M, 0.5M, 0M). (10) Perform the last wash with media containing 20% serum only (11) Transfer to the operating room for transplantation in fresh media with serum, on ice. EYE TO THE FUTURE The procedure of ovarian transplantation is already futuristic. In our last review of the subject, we had proposed several theoretical approaches to transplant cryopreserved ovarian tissue in humans (Fig 23.1). At the time of writing this chapter, autologous orthotopic and heterotopic transplants have already become a reality. The first pregnancies achieved by these transplant procedures will emerge in the future and perhaps will show that the various alternatives shown in Fig 23.1 have been tried.

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Fig 23.1 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 SCID mouse). Follicles can also be isolated from the ovarian tissue and grown in vitro. In the latter instance and in the case of heterotopic grafts, in vitro fertilization will be required to achieve pregnancy. (Modified from reference 4, with permission of the American Society for Reproductive Medicine).

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REFERENCES 1 Harp R, Leibach J, Black J, Keldahl C, Karow A. Cryopreservation of murine ovarian tissue. Cryobiology (1994); 31:336–43. 2 Cox S-L, Shaw JM, Jenkin G. Transplantation of cryopreserved fetal ovarian tissue to adult recipients of mice. J Reprod Fertil (1996); 107:315–22. 3 Oktay K, Nugent D, Newton H, Salha O, Gosden RG. Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue. Fertil Steril (1997); 67:481–6. 4 Oktay K, Newton H, Aubard Y, Gosden RG. Cryopreservation of human oocytes and ovarian tissue: an emerging technology? Fertil Steril (1998); 69:1–7. 5 Oktay K, Gosden RG. Cryopreservation of human oocytes and ovarian tissue. In: Brinsden PR, ed. A textbook of in vitro fertilization and assisted reproduction. London: Parthenon Publishing (1999): 303–10. 6 Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at −196°C. Hum Reprod (1994); 9:597–603. 7 Baird DT, Webb R, Campbell BK, Harkness LM, Gosden RG. Long term ovarian function in sheep after ovariectomy and transplantation of autografts stored at −196°C. Endocrinology (1999); 140:462–71. 8 Bosma GC, Custer RP, Bosma MJ. A severe combined deficiency mutation in the mouse. Nature (1983); 301:527–30. 9 Gosden RG, Boulton MI, Grant K, et al. Follicular development of ovarian xenografts in SCID mice. J Reprod Fertil (1994); 101:619–23. 10 Newton H, Aubard Y, Rutherford A, Sharma V, Gosden R. Low temperature storage and grafting of human ovarian tissue. Hum Reprod (1996); 11:1487–91. 11 Oktay K, Newton H, Mullan J, Gosden RG. Development of human primordial follicles to antral stages in SClD/hpg mice stimulated with follicle stimulating hormone. Hum Reprod (1998); 13:1133–8. 12 Oktay K, Newton H, Gosden RG. Transplantation of banked human ovarian tissue results in follicle growth initiation in SCID mice. Fertil Steril (2000); 73:559–603. 13 Oktay K. New horizons in assisted reproduction. Assisted Reprod Rev (1998); 8:51–4. 14 Oktay K, Karlikaya G. Ovarian function after autologous transplantation of frozen, banked ovarian tissue. N Engl J Med (2000); 342:1919. 15 Gosden RG, Oktay K, Radford JA, Rutherford AJ. Ovarian tissue banking (CD-ROM). Hum Reprod Update (1997); 3:297. 16 Parkes AS. Factors affecting the viability of frozen ovarian tissue. J Endocrinol (1958); 17:337–43.

24 Severe male factor: genetic consequences and recommendations for genetic testing Ingeborg Liebaers, André Van Steirteghem, Willy Lissers

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. Genetic investigations are indicated in the case of male infertility for two major reasons. One reason is to understand more about the possible causes of azoospermia or oligoastenoteratospermia. Another reason is to be able to offer genetic counselling to the patient his partner, and his family whenever indicated. The role of genetic counseling in case of infertility has increased since the advent of assisted reproductive technology (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.1–3 In the clinic, genetic investigations are usually performed when the azoo- or oligospermia is part of a more complex disease or syndrome. On the basis of the data available today a number of genetic tests should also be performed in case of infertility in an otherwise healthy male. In most 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 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.

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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 non-obstructive azoospermia as main features.4,5 Since then many chromosomal studies were performed in series of infertile males, and the conclusions drawn from a recent review as well as from other 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 aberration such as 47,XXY and 47,XYY is proportionally higher in males with azoospermia compared with males with oligospermia, whereas structural chromosomal aberrations of autosomes such as Robertsonian (Fig 24.1a) and reciprocal (Fig 24.1 b) translocations are proportionally more frequent in oligospermic males (Table 24.1).6–8 In azoospermic males it is also possible to find a 46,XX karyotype. In roughly 80% of these Klinefelter-like males the SRY gene, normally located close to the pseudo-autosomal region of the short arm of the Y chromosome is now, thanks to a crossing over event during meiosis, present in that same region on the X chromosomes.9,10 The SRY gene referring to the sex-determining region of the Y chromosome has to be expressed to induce the sexual

Fig 24.1a 45, XY, der(13;14)(q10;q10) karyotype from a phenotypic normal male with a Robertsonian translocation of

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Fig 24.1b 46, XY, t(11;22)(q24.3;q12) karyotype from phenotypic normal male with a balanced reciprocal translocation of chromosome 11 and 22 with break points in 11q24.3 ( ) and 22q12 ( ).

Table 24.1. Incidence of chromosomal aberrations in infertile oligospermic and azoospermic males compared with newborns. Aberrations Infertile males Oligozoospermia Azoospermia Newborns (n=7876) (n=1701) (n=1151) (n=94465) Autosomes 1.3% 3.0% 1.1% 0.25% Sex chromosomes 3.8% 1.6% 12.6% 0.14% Total 5.1% 4.6% 13.7% 0.39% Summarized from 6. development of an embryo towards a male phenotype.11 In the remaining 20% of XX males most probably other genes involved in sexual development are involved. Spermatogenesis seems to be absent in these males whereas in apparently non-mosaic 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.12–18

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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 on 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.19 Nevertheless the first azoospermic male patients in 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.20 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 8%. Again it is clear that the lower the sperm count the higher the incidence of deletions.8,19,21,22,23 Careful evaluation of non-overlapping microdeletions allowed to subdivide the AZF region in 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.19 In the meantime several genes have been identified in these AZF regions. They are currently being studied to prove their role in spermatogenesis.21,24–26 It is of course clear that if these microdeletions cause the spermatogenic defect leading to a low to very low sperm count present in the ejaculate or only in the testes, these microdeletions will, through the use of ICSI, be transmitted to sons who most probably will be infertile as well.27 However, ICSI children are still too young to evaluate their fertility or their sperm count. On the other hand fertility has been described in AZFc deleted fathers who transmitted the deletion to their now infertile sons.28,29 Age at investigation may play a role as observed in one patient with an AZFc deletion being oligospermic and later on azoospermic.30 CONGENITAL BILATERAL ABSENCE OF THE VAS DEFERENS AND CYSTIC FIBROSIS Men with congenital bilateral absence of the vas deferens (CBAVD) have obstructive azoospermia. Their spermatogenesis is normal. Sperm can be obtained through microsurgical epididymal sperm aspiration (MESA) or through testicular sperm extraction (TESE) and used to fertilize oocytes in

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vitro through ICSI.31–33 CBAVD was known to be present in 95% of male cystic fibrosis (CF) patients. CF is a frequent and by now well known autosomal recessive disease in the white population with an incidence of about 1/2500. The patients now surviving into their 20s and 30s suffer from severe lung disease and pancreatic insufficiency. They are often too ill to reproduce and still die early in life. 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 10 years ago.34–36 CBAVD had also been observed in 1–2% of apparently healthy infertile males and in 6% of men with obstructive azoospermia.37 When the CFTR gene was studied in these males, mutations or splice site variants in intron 8 called the 5T variant interfering with gene expression were found in 80% of them.38–42 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.43,44 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 24.2). However since the incidence and the type of CFTR mutations vary with the ethnic origin as well as with the geographic region, counseling, and approach to treatment will have to be adjusted.

Table 24.2. Risk calculations for a CF* child or a CBAVD** child in case of CBAVD. male female risk No testing: 8/10 × 1/25 × 1/4 = 1/125 Testing female carrier: 8/10 × 1 × 1/4 = 1/5 no carrier: 8/10 × 1/150 × 1/4 = 1/750 Testing male+female female carrier: CF/CF × 1 × 1/2 = 1/2 female no carrier: CF/CF × 1/150 × 1/2 = 1/300 female carrier: CF/5T × 1 × 1/4 = 1/4 (CF) 1/8 CBAVD) *CF=cystic fibrosis. **CBAVD=Congenital bilateral absence of the Vas Deferens. If the CBAVD patient is not tested for CF mutations, his risk of having at least 1 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

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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.77 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.45 A number of these rather rare conditions which may be encountered in a fertility clinic have been summarized in Table 24.3. Myotonic dystrophy is a rather common autosomal dominant muscular dystrophy with an incidence of 1/8000. The presence of an expanded CTG trinucleotide repeat in the DMPK gene interferes with its function.46 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.47 In 60% to 80% of the male patients testicular tubular atrophy will develop and cause oligoasthenoteratospermia.

Table 24.3. Rare conditions encountered in a fertility clinic. Disease Frequency Clinic Labtests Cause Treatment Reference ICSI 46, 47, 48 AD Myotonic 1:8000 Male NormoPGD dystrophy phenotype /oligospermia LH, ‘CTG’ expansion in myotonia FSH normal or DMPK gene T normal or Azoospermia T, X-linked Hormonal 49, 50 Kallmann 1:10000 Male FSH, LH no substitution syndrome phenotype Abnormal Pubertal delay response to GnRH neuronal Anosmia migration test Point mutation in KAL1 gene AR and AD forms exist as well!! Astenozoospermia AR dynein ICSI 51, 52 Primary 1:25000 Male phenotype deficiency ciliary genetic dyskinesia heterogeneity or immotile

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cilia syndrome OligoICSI or 53 X-linked Kennedy 1:50000 Male AID (gynecomastia) /azoospermia T ‘CAG’ disease or normal or Muscular expansion in spinal atrophy androgen bulbar LH, FSH receptor gene muscular atrophy AD=autosomal dominant; AR=autosomal recessive; AID=artificial insemination with donor sperm.

Fig 24.2a Segregation of a Robertsonian translocation der(13;14) in a family: its consequences, and recommendations. OAT (our proband ) presents with infertility owing 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) (figure 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

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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,86 or a prenatal diagnosis should be offered. When such spermatozoa are used to fertilize oocytes, the risk to transmit the disease often in a more severe form due to further expansion of the trinucleotide repeat, is 1/2 or 50%. Prenatal diagnosis or preferentially preimplantation diagnosis should be offered.48 Kallmann’s syndrome is characterized by hypogonadotrophic hypogonadism due to impaired GnRH secretion and anosmia. X-linked as well as autosomal recessive and autosomal dominant inheritance exists. The X-linked form of the Kallmann syndrome is the most frequent and the best known one.49 Hormonal treatment will stimulate spermatogenesis.50 Genetic counselling is indicated (Fig 24.2b). 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.51 With the help of ICSI men with this condition can reproduce. Genetic counselling is hampered because the possibility for genetic testing is still limited.52 However, if we accept the incidence of 1/25000, 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 syndrome or spinal bulbar muscular atrophy (SBMA) is a neuromuscular disease causing muscular weakness associated with testicular atrophy leading to oligoazoospermia. It is an X-linked disease caused by an expanded CAG trinucleotide repeat in the transactivation domain of the androgen receptor gene.53 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.54 The presenting

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Fig 24.2b X-linked Kallmann syndrome in a family: its consequences and recommendations. KAL (our proband ) has Kallmann syndrome. The family history fits with an Xlinked 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 brother 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. problem here will not be male infertility. Patients with an autosomal recessive 5-αreductase deficiency and therefore being unable to synthesize dihydrotestosterone from testosterone may theoretically present at the clinic with azoospermia and pseudohermaphroditism.55 Very rarely, patients with other genetic defects may consult at a male infertility clinic. Patients with Noonan syndrome may present with oligoor 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). The autosomal dominant inheritance asks for genetic counseling.56,57 Other possible patients may be affected by

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the Aarskog-Scott syndrome with acrosomal sperm defects,58 the and Beckwith-Wiedemann syndrome with cryptorchidism59 adrenomyeloneuropathy with oligo- or azoospermia.60 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.61,62 Interesting to know is 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.63,64 Other causes of male infertility include a deficiency in enzymes involved in testosterone synthesis,55 luteinizing hormone and luteinizing hormone receptor deficiencies.65,66 Defects in energy production by the mitochondria have recently 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.67 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.68–70 Moreover, several research groups have demonstrated the presence of an almost 5kb deletion in sperm with diminished motility in otherwise healthy males.71,72,73,74

CONSEQUENCES AND RECOMMENDATIONS IN THE CLINIC GENETIC EVALUATION OF INFERTILE MALES BEFORE ART Not only should a personal history be taken; a detailed pedigree should also 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 (Figs 24.2a and 24.2b). A thorough inquiry of the proband and his partner may also pinpoint other hereditary disease 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

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associated with infertility such as Klinefelter syndrome or CF linked 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 a laboratory investigation will allow to confirm a clinical diagnosis. In case of male infertility the history, the clinical examination, a semen analysis and hormonal tests are sufficient to characterize most of the patients as being: (1) infertile in association with other physical or mental problems; or (2) infertile but otherwise healthy. These patients can be subdivided in oligospermic or eventually oligo-astheno-teratospermic males and in obstructive or non-obstructive 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 non-obstructive azoospermia a peripheral karyotype should be performed, even if the family history is not suggestive of a chromosomal disorder.6–8 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.75 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 the present day it is possible to identify 85% to 90% of the carriers in the Caucasian population by using a laboratory kit detecting 10 to 30 of the most common mutations, (ex: INNO-LiPA™ CFTR12 and CFTR 17+Tn, Innogenetics, Gent, Belgium). Depending on whether CFTR mutation have been identified in the male patient and/or his female partner the risk to conceive a child with cystic fibrosis can be calculated (Table 24.2). These figures together with the type of mutations allow to offer prenatal diagnosis or preimplantation genetic diagnosis.76–78 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 (PGD). These procedures refer to the genetic analysis 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. 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. Therefore its development

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has been rather slow over the past 10 years since its first application in the clinic.79 In reference to this chapter most of the PGDs performed were for cystic fibrosis and myotonic dystrophy but many others have been performed for either infertile or fertile couples.80–84 So far only a few have been done for chromosomal aberrations, mainly Robertsonian or reciprocal translocations.85–88 In general the take home baby rate is of the same order of magnitude of 20 to 25% as in the ICSI cycles in general.3 A number of PGDs have been performed for Klinefelter patients in whom spermatozoa found in the testes were used to fertilize oocytes.12,13,15,16 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 naturally conceived children. Preliminary results on the psychomotoric development of these children are also reassuring.3,89–95 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 fourfold increase, but the overall incidence remains low (<1%). Apart from sex chromosome anomalies also de novo balanced translocations have been observed.3,91 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.96–99

CONTROVERSIES 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 IVF cycles are necessary.100 In case of non-obstructive azoospermia about half of the males have sufficient spermatogenesis to allow ICSI after testicular sperm aspiration.101 It is probably true that in most cases a healthy although maybe infertile child will be born. Nevertheless in a number of cases, for example, in 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

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children with genetic disease that could have been avoided. Is that the price that should be paid, and would you like to be that infertile father? Another option could be to not use ICSI further and leave decisions to nature. 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 Yq11×2 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 as well chromosomal aberrations as Yq11×2 deletions have been found in patients with more than 5×106 spermatozoa/ml although to a lesser extent.22 Based on a few reports, one can also wonder whether karyotypes of the female partners should be performed.102,103 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.3,104 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 carrier of a particular gene mutation to take preventive measures. Examples are screening programmes for CF or for TaySachs disease in certain populations. Couples may want to know before having children since if both partners are carrier of such an autosomal recessive gene the risk of having an affected child is 1/4. Such screening programmes 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 diagnosis.105 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 FISH probes. Embryos diploid for the chromosomes tested

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are then transferred without of course having information on the other chromosomes. The observations reported so far are that in women aged over 37 years the IVF success rate increases,106 the rate of miscarriage decreases,107 the implantation rate per embryo increases.108 At present more data are needed to confirm the value of this aneuploidy screening performed on IVF embryos. 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.64,109,110

THE 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, only a small number of men will be unable to benefit from these advances, to have their own child. Research is ongoing to even find a solution for part of them by, for example, trying to mature sperm in vitro or to transplant testicular tissue from a fertile male into an infertile male.111 Some even envisage the possibility of reproductive cloning by transplanting a nucleus of a diploid somatic cell of the infertile male in the enucleated oocyte of his partner.112,113 This approach however is extremely controversial. ICSI has also triggered basic research in biology and genetics in order to gain more insight in 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.

CONCLUSION In case of severe male infertility, good clinical practice requests 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 defining how great the risks are to transmit infertility or possibly other genetic anomalies to the next generations.

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15 Reubinoff BE, Abeliovich D, Werner M, et al. A birth in non-mosaic Klinefelter’s syndrome after testicular fine needle aspiration, intracytoplasmic sperm injection and preimplantation genetic diagnosis. Hum Reprod (1998); 13:1887–92. 16 Palermo GDE, Schlegel PN, Hariprashad JJ, et al. Fertilization and pregnancy outcome with intracytoplasmic sperm injection in azoospermic men. Hum Reprod (1999); 14:741–8. 17 Nodar F, De Vincentiis S, Olmedo SB, et al. Birth of twin males with normal karyotype after intracytoplasmic sperm injection with use of testicular spermatozoa from a nonmosaic patient with Klinefelter’s syndrome. Fertil Steril (1999); 71:1149–52. 18 Ron-EI R, Strassburger D, Gelman-Kohan S, et al. A47,XXY fetus conceived after ICSI of spermatozoa from a patient with mosaic Klinefelter’s syndrome: case report. Hum Reprod (2000); 15:1804–6. 19 Vogt PH, Edelmann A, Kirsch S, et al. Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet (1996); 5:933–43. 20 Tiepolo L, Zuffardi O. Localization of factors controlling spermatogenesis in the nonfluorescent position of the human Y chromosome long arm. Hum Genet (1976); 34:119–24. 21 Reijo R, Lee T-Y, Salo P, et al. Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nat Genet (1995); 10:383–93. 22 Vogt PH. Human chromosome deletions in Yq11, AZF candidate genes and male infertility: history and update. Mol Hum Reprod (1988); 739–44. 23 Van Landuyt L, Lissens W, Stouffs K, et al. Validation of a simple Yq deletion screening programme in an ICSI candidate population. Mol Hum Reprod (2000); 6:291–7. 24 Ma K, Inglis JD, Sharkey A, et al. A Y chromosome gene family with RNA-binding protein homology: candidates for the azoospermia factor AZF controlling human spermatogenesis. Cell (1993); 75:1287–95. 25 Lahn BT, Page DC. Functional coherence of the human Y chromosome. Science (1997); 278:675–80. 26 Lahn BT, Page DC. Retroposition of autosomal mRNA yielded testisspecific gene family on human Y chromosome. Nat Genet (1999); 21:429–33. 27 Page DC, Silber S, Brown LG, et al. Men with infertility caused by AZFc deletion can produce sons by intracytoplasmic sperm injection but are likely to transmit the deletion and infertility. Hum Reprod (1999); 14:1722–6. 28 Chang PL, Sauer MV, Brown S, et al. Y chromosome microdeletion in a father and his four infertile sons. Hum Reprod (1999); 14:2689–94. 29 Saut N, Terriou P, Navarro A, et al. The human Y chromosome genes BPY2, CDY1 and DAZ and not essential for sustained fertility. Mol Hum Reprod (2000); 6:789–93.

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30 Girardi SK, Mielnik A, Schlegel PN. Submicroscopic deletions in the Y chromosome of infertile men. Hum Reprod (1997); 12:1635–41. 31 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. 32 Nagy Z, Joris H, Verheyen G, et al. Correlation between motility of testicular spermatozoa, testicular histology and the outcome of intracytoplasmic sperm injection. Hum Reprod (1998); 13:890–5. 33 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); 14:90–5. 34 Kerem B, Rommens JM, Buchanan JA, et al. Identification of the cystic fibrosis gene: genetic analysis. Science (1989); 245:1073–80. 35 Riordan JR, Rommens JM, Kerem B, et al. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science (1989); 245:1066–73. 36 Rommens JM, Lannuzzi MC, Kerem B, et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science (1989); 245:1059–65. 37 Dubin L, Amelar RD. Etiologic factors in 1294 consecutive cases of male infertility. Fertil Steril (1971); 22:469–74. 38 Anguiano A, Gates RD, Amos JA, et al. Congenital bilateral absence of the vas deferens. A primarily genital form of cystic fibrosis. J Am Med Ass (1992); 267:1794–7. 39 Dumur V, Gervais R, Rigot J-M, et al. Congenital bilateral absence of vas deferens (CBAVD) and cystic fibrosis transmembrane regulator (CFTR): correlation between genotype and phenotype. Hum Genet (1996); 97:7–10. 40 Lissens W, Mercier B, Tournaye H, et al. Cystic fibrosis and infertility caused by congenital bilateral absence of the vas deferens and related clinical entities. Hum Reprod (1996); 11:55–80. 41 De Braekeleer M, Férec C. Mutations in the cystic fibrosis gene in men with congenital bilateral absence of the vas deferens. Mol Hum Reprod (1996); 2:669–77. 42 Cuppens H, Lin W, Jaspers M, et al. Polyvariant mutant cystic fibrosis conductance regulator genes. The polymorphic (TG)m locus explains the partial penetrance of the T5 polymorphism as a disease mutation. J Clin Invest (1998); 101:487–96. 43 Dumur V, Gervais R, Rigot J-M, et al. Congenital bilateral absence of the vas deferens in absence of cystic fibrosis. Lancet (1995); 345:200– 1. 44 Patrizio P, Zielenski J. Congenital absence of the vas deferens: a mild form of cystic fibrosis. Mol Med Today (1996); 1:24–31.

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60 Mosser J, Douar AM, Sarde CO, et al. Putative X-linked adrenoleukodystrophy gene shares unexpected homology with ABC transporters. Nature (1993); 361:726–30. 61 Beales PL, Elcioglu N, Woolf AS, et al. New criteria for improved diagnosis of Bardet-Biedl syndrome: results of a population survey. J Med Genet (1999); 36:437–46. 62 Cassidy SB. Prader-Willi syndrome. J Med Genet (1997); 34:917–23. 63 Horsthemke B, Dittrich B, Buiting K, et al. Imprinting mutations on human chromosome 15. Hum Mutat (1997); 10:329–37. 64 Feil R, Khosla S. Genomic imprinting in mammals. Trends Genet (1999); 15:431–5. 65 Weiss J, Axelrod L, Whitcomb RW, et al. Hypogonadism caused by a single amino acid substitution in the subunit of luteinizing hormone. N Engl J Med (1992); 326:179–83. 66 Latronico AC, Segaloff DL. Naturally occurring mutations of the luteinizing-hormone receptor: lessons learned about reproductive physiology and G protein-coupled receptors. Am J Hum Genet (1999); 65:949–58. 67 Mitchell JA, Nelson L, Hafez ESE. Human semen and fertility regulation in men. Saint Louis: MO: Mosby (1996):83–106. 68 Folgero T, Bertheussen K, Lindal S, et al. Mitochondrial disease and reduced sperm motility. Hum Reprod (1993); 8:1863–8. 69 Lestienne P, Reynier P, Chrétien MF, et al. Oligoasthenospermia associated with multiple mitochondrial DNA rearrangements. Mol Hum Reprod (1997); 3:811–4. 70 Reynier P, Chrétien MF, Penisson-Besnier I, et al. Male infertility associated with multiple mitochondrial DNA rearrangements. CR Acad (1997); 320:629–36. 71 Kao SH, Chao HT, Wei YH. Mitochondrial deoxyribonucleic acid 4977-bp deletion is associated with diminished fertility and motility of human sperm. Biol Reprod (1995); 52:729–36. 72 Kao SH, Chao HT, Wei YH. Multiple deletions of mitochondrial DNA are associated with the decline of motility and fertility of human spermatozoa. Mol Hum Reprod (1998); 4:657–66. 73 St John JC, Cooke ID, Barratt CL. Mitchondrial mutations and male infertility. Nat Med (1997); 3:124–5. 74 Reynier P, Chrétien MF, Savagner F, et al. Long PCR analysis of human gamete mtDNA suggests defective mitochondrial maintenance in spermatozoa and supports the bottleneck theory for oocytes. Bioch Biophys Res Commun (1998); 252:373–7. 75 Simoni M, Bakker E, Eurling MC, et al. Laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions. Int J Androl (1999); 22:292–9. 76 Strom CM, Ginsberg N, Rechitsky S, et al. Three births after preimplantation genetic diagnosis for cystic fibrosis with sequential

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first and second polar body analysis. Am J Obstet Gynecol (1998); 178:1298–306. 77 Goossens V, Sermon K, Lissens W, et al. Clinical application of preimplantation genetic diagnosis for cystic fibrosis. Prenat Diagn (2000); 20:571–81. 78 Dreesen JC, Jacobs LJ, Bras M, et al. Multiplex PCR of polymorphic markers flanking the CFTR gene; a general approach for preimplantation genetic diagnosis of cystic fibrosis. Mol Hum Reprod (2000); 6:391–6. 79 Handyside AH, Kontogianni EH, Hardy K, et al. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature (1990); 344:768–70. 80 Liebaers I, Sermon K, Staessen C, et al. Clinical experience with preimplantation genetic diagnosis and intracytoplasmic sperm injection. Hum Reprod (1998); 13 (suppl 1):186–91. 81 Vandervorst M, Staessen C, Sermon K, et al. The Brussels’ experience of more than 5 years of clinical preimplantation genetic diagnosis. Hum Reprod Update (2000); 6:364–73. 82 Strom CM, Strom S, Levine E, et al. Obstetric outcomes in 102 pregnancies after preimplantation genetic diagnosis. Am J Obstet Gynecol (2000); 182:1629–32. 83 European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium Steering Committee. ESHRE Preimplantation genetic diagnosis (PGD) Consortium: preliminary assessment of data from January 1997 to September 1998. Hum Reprod (1999); 14:3138– 48. 84 European Society of Human Reproduction and Embryology (ESHRE) PGD Consortium Steering Committee. ESHRE Preimplantation Genetic Diagnosis (PGD) Consortium: data collection II. Hum Reprod (2000) in press. 85 Van Assche E, Staessen C, Vegetti W, et al. Preimplantation genetic diagnosis and sperm analysis by fluorescence in-situ hybridization for the most common reciprocal translocation t(11;22). Mol Hum Reprod (1999); 5:682–90. 86 Escudero T, Lee M, Carrel D, et al. Analysis of chromosome abnormalities in sperm and embryos from two t(13;14)(q10;q10) carriers. Prenat Diagn (2000); 20:599–602. 87 Munne S, Sandalinas M, Escudero T, et al. Outcome of preimplantation genetic diagnosis of translocations. Fertil Steril (2000); 73:1209–18. 88 Scriven PN, O’Mahony F, Bickerstaff H, et al. Clinical pregnancy following blastomere biopsy and PGD for a reciprocal translocation carrier: analysis of meiotic outcomes and embryo quality in IVF cycles. Prenat Diagn (2000); 20:587–92. 89 Bonduelle M, Joris H, Hofmans K, et al. Mental development of 201ICSI children at 2 years of age. Lancet (1998); 351:1553.

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90 Sutcliffe AG, Taylor B, Li J, et al. Children born after intracytoplasmic sperm injection: population control. BMJ (1999); 318:704–5. 91 Van Steirteghem A, Bonduelle M, Camus M, et al. Outcomes from intracytoplasmic sperm injection. In: Towards Reproductive Certainty. Fertility and Genetics beyond 1999. Jansen R 1 Mortimer D, eds. New York: The Parthenon Publishing Group (1999):70–6. 92 Palermo G, Colombero L, Schattman G, et al. Evolution of pregnancies and initial follow-up of newborns delivered after intracytoplasmic sperm injection. J Am Med Assoc (1996); 276:1893–7. 93 Wennerholm UB, Bergh C, Hamberger L, et al. Obstetric and perinatal outcome of pregnancies following intracytoplasmic sperm injection. Hum Reprod (1996); 11:1113–9. 94 Wennerholm UB, Bergh C, Hamberger L, et al. Incidence of congenital malformations in children born after ICSI. Hum Reprod (2000); 15:944–8. 95 Wennerholm UB, Bergh C, Hamberger L, et al. Obstetric outcome of pregnancies following ICSI, classified according to sperm origin and quality. Hum Reprod (2000); 15:1189–94. 96 Martin RH. Genetics of human sperm. J Assist Reprod Genet (1998); 15:240–5. 97 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. 98 Vegetti W, Van Assche E, Frias A, et al. Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence insitu hybridization in infertile men. Hum Reprod (2000); 15:351–65. 99 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. 100 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. 101 Silber S, Johnson L, Verheyen G, et al. Round spermatid injection. Fertil Steril (2000); 73:897–900. 102 Meschede D, Lemcke B, Exeler JR, et al. Chromosome abnormalities in 477 couples undergoing intracytoplasmic sperm injectionprevalence, types, sex distribution and reproductive relevance. Hum Reprod (1998); 13:576–82. 103 van der Ven K, Peschka B, Montag M, et al. Increased frequency of congenital chromosomal aberrations in female partners of couples undergoing intracytoplasmic sperm injection. Hum Reprod (1998); 13:48–54. 104 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.

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105 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 (1998). 106 Verlinksy 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. 107 Munné S, Magli C, Cohen J, et al. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum Reprod (1999); 14:2191–9. 108 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. 109 Manning M, Lissens W, Bonduelle M, et al. Study of DNAmethylation patterns at chromosome 15q11-q13 in children born after ICSI reveals no imprinting defects. Mol Hum Reprod (2000); in press. 110 Pfeifer K. Mechanisms of genomic imprinting. Am J Hum Genet (2000); 67:777–87. 111 Brinster RL, Nagano M. Spermatogonial stem cell transplantation, cryopreservation and culture. Cell & Develop Biol (1998); 9:401–9. 112 Campbell KH, McWhir J, Ritchie WA, et al. Sheep cloned by nuclear transfer from a cultured cell line. Nature (1996); 380:64–6. 113 Wilmut I. Cloning for medicine. Sci Am (1998); 279:58–63.

25 Chromosome abnormalities in human embryos Santiago Munné, Mireia Sandalinas, Jacques Cohen

INTRODUCTION This chapter is an update to our previous review on this subject.1 In that review we explained that when an in vitro fertilization (IVF) clinic successfully attains a high implantation rate, it can be faced with the problem of excessive multiple pregnancies. Also, women of advanced maternal age may lose many pregnancies to spontaneous abortions. Therefore, oocyte, zygote, and embryo selection becomes of central importance. Here we review the latest methods using numerical chromosome assessment as one of the main criteria for selection for these women. To study numerical chromosome abnormalities in human preimplantation embryos certain conditions need to be met. First, individual chromosomes need to be assessed to determine specific aneuploidy rates. Second, all or most blastomeres in some embryos should be analyzed to differentiate mosaicism from other abnormalities. Third, developmentally arrested embryos should also be fully analyzed, and finally, abnormalities should be assessed at different times of development (cleavage, morula, blastocyst stage). Classical cytogenetic techniques are limited because they require metaphase stage chromosomes, but only one third of all embryos analyzed show good quality metaphases. Of these, only one quarter will have all their cells analyzed, or less than 8% overall.2,3 This means that mosaicism can be severely underestimated; furthermore, while arrested, embryos cannot be analyzed. Fluorescence in situ hybridization (FISH) has been used with much higher efficiencies (90%) to study the chromosomal constitution of cleavage stage human embryos, arrested or not.4–11 FISH with multiple probes can differentiate polyploidy from aneuploidy; also haploidy from monosomy, and when most or all cells of an embryo are analyzed, mosaicism can be differentiated from FISH or fixation failure, as well as from aneuploidy.12,13 But FISH supplies information only about a limited number of chromosomes for which the probes are specific. Other approaches such as comparative genome hybridization (CGH),14 quantitative polymerase chain reaction (PCR),15 or chip DNA technology cannot as yet perform single cell analysis with enough accuracy to be applied clinically, but show potential for the future.16 Finally, spectral

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karyotyping (SKY) based on FISH technology, has been able to karyotype poor quality metaphases of polar bodies, oocytes and blastomeres, but still requires those cells to be at metaphase stage and those metaphases to be well spread.17

CHROMOSOME ABNORMALITIES IN EMBRYOS DERIVED FROM ZYGOTES WITH ABNORMAL PRONUCLEAR NUMBER OR MORPHOLOGY The centrosome inheritance in human embryos needs to be understood to comprehend the different patterns of chromosome abnormalities produced by different numbers and parental origin of pronuclei. Centrosome inheritance, unipronuclear, tripronuclear, apronuclear zygotes, zygotes with uneven or distant pronuclei, distribution and morphology of nucleolar precursor bodies, and shape and alignment of first and second polar bodies will be discussed here. CENTROSOME INHERITANCE IN HUMAN ZYGOTES The centrosome is a center of organization involved in forming the mitotic spindle. During interphase it reproduces and doubles in anticipation of cell division. In most cells it consists of two morphologically distinct objects called centrioles: a pair of cylinders in a complex and asymmetrical arrangement and the pericentriolar material from which spindle microtubules are generated. These organelles reproduce by forming daughter copies, which then segregate. In the development of fertilized animal eggs, specific mechanisms must exist at the gamete or zygote level to control centrosome inheritance. If centrosomes from both gametes were retained and remained functional, the zygote would enter first mitosis with two doubled sets of centrosomes and four centrioles; resulting first in multipolar or extra spindles and then in mosaicism.18 Three types of centrosomal inheritance have been described, being under paternal or maternal control or controlled by both gametes. Wilson and Matthews discovered a century ago that in sea urchins centrosomal inheritance is paternal.19 Similar mechanisms exist for all mammals studied with the exception of the mouse, which apparently displays no distinct centrosomal complex.20 The presence of sperm centrioles in fertilized monospermic and dispermic human embryos was shown by Sathananthan et al using transmission electron microscopy.21 Tripolar spindles were found in tripronuclear zygotes with two centrioles. Analysis of the incidence and onset of mosaicism in human digynic, dispermic, and enucleated embryos provides evidence that the sperm centrosome controls the first mitotic division after fertilization, although the use of maternal centrosomal material cannot be excluded. Results prove that dispermic zygotes usually

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do not have bipolar spindles and become mosaic. This situation is determined by an extranuclear factor, since removal of a single pronucleus does not correct the abnormal division pattern.22 In contrast, monospermic digynic embryos become triploid, indicating that the three pronuclei are organized in a single spindle at syngamy. Removal of one pronucleus from such zygotes restores the embryo to a normal diploid state. Both the incidence of mosaicism and the onset of the anomaly in digynic embryos are similar to those found in normally developing monospermic embryos.23 SINGLE PRONUCLEATE OR UNIPRONUCLEAR ZYGOTES Single pronucleate zygotes can be obtained after IVF and intracytoplasmic sperm injection (ICSI) at frequencies ranging from 2% to 5%. Single pronucleate embryos occasionally (3%) develop following insemination in our laboratory, but they occur more frequently (9%) after ICSI. CHROMOSOME STUDIES IN 1PN AFTER IVF Karyotype studies indicate that 46–80% of 1PN embryos are in fact diploid.24,25 However, those studies could not differentiate between diploidy produced by pronuclear fusion or fertilization by parthenogenetic activation and subsequent diploidization by asynchronous pronuclei inflation. To differentiate between parthenogenetic activation and true fertilization (resulting in asynchronous pronuclear inflation or fusion) sexing of these embryos is necessary since the presence of a Y chromosome will indicate the occurrence of fertilization. FISH studies, summarized in Table 25.1, have shown that when embryos are diploid most of them are fertilized.26,27 However, true parthenogenesis may also be present since haploid embryos were also detected. These FISH studies suggest that most diploid

Table 25.1. FISH studies in 1PN embryos from IVF. N Mosaic (%) 3n (%) 2n (%) Y (%) n (%) (26) 21 19 0 66 48 14 (27) 115 37 1 49 45 13 N: analyzed, 3n: triploid, 2n: diploid, n: haploid, Y: chromosome Y detected. 1PN embryos develop from fertilized oocytes. But two mechanisms could be responsible, asynchronous appearance of the two pronuclei or formation of a single fertilization or zygote pronucleus (fused pronuclei). Levron et al demonstrated that single pronucleate zygotes from IVF are usually formed after fusion of the male and female pronuclei.28 They partitioned the pronucleus (karyoplast) from the rest of the egg

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(cytoplast), and analyzed them by FISH. Of the diploid pronuclei, 66% contained XY chromosomes and 33% contained XX chromosomes. The corresponding cytoplasts were DNA free, indicating that asynchronous inflation of pronuclei is rare in such zygotes or does not occur at all. CHROMOSOME ANALYSIS OF 1PN EMBRYOS AFTER ICSI Single pronucleate zygotes from ICSI have also been analyzed by FISH in two studies.26,27 The results, summarized in Table 25.2, indicate that single pronucleate ICSI zygotes are usually activated, not fertilized, but able to cleave. Such embryos are not being replaced into the patient. TRIPRONUCLEAR ZYGOTES DISPERMY (3 PN AFTER IVF) Dispermy is the most common fertilization anomaly in the human.29 Most of these embryos will cleave but arrest prior to differentiation. Cleavage patterns are highly irregular; most dispermic zygotes immediately divide into three or four cells after the first division, but only a minority divides into two cells.29 Although several karyotype studies have been performed on 3 PN embryos30 they suffer from the same problems of most karyotype studies and also cannot distinguish between 3 PN-2 PB (dispermic) from 3 PN-1 PB zygotes (digynic). Multiprobe FISH studies have found that while most of the embryos are mosaics, very few are pure triploids (Table 25.3).22,27 The different number of diploids between centers, ranging from 3% to 21%, could be due to differences in recording vacuoles and pronuclei. Staessen and Van Steirteghem found a ratio of XXX:XXY:XYY similar to 25%:50%:25% which would be expected from a dispermic origin.27 Some of these embryos could still be digynic since polar bodies are occasionally fragmented, and it is difficult to distinguish if there is one fragmented PB or two PBs. They also observed that three PN zygotes after IVF cleaved more rapidly than two PN oocytes, with more 3 cell embryos resulting from three PN (20%) than two PN (12%) embryos. Zygotes dividing into 3 cell embryos had less uniformly triploid cells than those dividing into two or four cells. In human zygotes, size appears to be variable and sperm tail remnants can almost never be identified by light microscopic observation.31 To ascertain the presence of the male pronuclei, the X/Y ratio needs to be determined. The X/Y ratio is determined by the

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Table 25.2. FISH studies in 1PN embryos from ICSI. N Mosaic (%) 3n (%) 2n (%) n (%) Y (%) (26) 21 39 0 14 47 19 (27) 61 41 0 28 31 31 N: analyzed, 3n: triploid, 2n: diploid, n: haploid, Y: chromosome Y detected. Table 25.3. FISH studies of 3PN embryos from IVF, with and without enucleation. N Mosaic (%) 3n (%) 2n (%) n (%) Intact: (22) 36 89 3 3 0 Staessen, Van Steirteghem 1997 71 63 13 21 3 Enucleated: (22) 15 100 0 0 0 N: analyzed, 3n: triploid, 2n: diploid, n: haploid, Y: chromosome Y detected. fact that the female pronucleus always contains an X-chromosome, whereas male pronuclei can have X as well as Y chromosomes. The X/Y ratio in dispermic zygotes and their daughter blastomeres should be 1:3. Removal of a male pronucleus should render a normal X/Y distribution of 1:1 if the distal pronucleus (the one furthest from the polar body) is indeed of paternal origin. A total of 51 dispermic embryos were biopsied between the 2 to 11 cell stage, of which 15 were previously enucleated. The proportion of dispermic and enucleated embryos with X- as well as Ychromosomes was similar to that of the theoretical value; 72% and 53% respectively.32 However, although the sex ratio was corrected, the resulting embryos continue to be mosaics (Table 25.3).22 Analyses of the types of mosaicism and the frequency of abnormal cells have indicated that the anomaly occurs at syngamy.12 Nevertheless, mosaicism in monospermic embryos usually occurs after the embryo has divided in two cells.12 The mosaicism probably occurs as a consequence of abnormal tripolar division in some embryos.24 DIGYNIC ZYGOTES (3PN AFTER ICSI) Four per cent of eggs injected with single sperm cells have three pronuclei and a single polar body. We consider these embryos to be monospermic digynic, and their genetic status can be analyzed by performing FISH in blastomeres.23,27 Most of these embryos were found to be perfect triploids (Table 25.4), and the differences in the percentage of diploid embryos could be caused by the presence of a misleading vacuole. None of the studies found XYY embryos, and equal number of XXX and XXY were

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found, again indicating that the mechanism was digyny. In addition, significantly fewer mosaics were found in three PN after ICSI than three PN after IVF, and mosaicism occurred mostly at the second division after ICSI compared with the first division after IVF. This, and the fact that many perfectly triploid embryos were found, indicates that the chromosomes of the three pronuclei had organized into a single bipolar spindle at syngamy, and suggests that only one centrosome is active in such zygotes. Three of the four monospermic and digynic embryos from which the pronucleus next to the first polar body was removed, cleaved, properly and became normally diploid, also indicating that a single centrosome is active in such embryos. The fourth one was a diploid mosaic embryo. APRONUCLEAR (oPN) ZYGOTES About ≤1% of zygotes with two polar bodies do not show pronuclei. Using FISH with XY,18 and 13/21 probes, Manor et al have found that 57% of them are normal diploid, 30% polyploid and or mosaic, and 13% aneuploid.33 They recommend the transfer of these embryos when not enough dipronucleated embryos are available. It is likely that pronuclei are hidden owing to granularity or missed because of an abnormal development speed. UNEVEN OR DISTANT PRONUCLEI Preimplantation development and chromosomal contents were evaluated in individual embryos derived from monospermic zygotes with uneven pronuclei. The average pronuclear sizes were 12.5µm and 22.3µm. Less than 2% of the studied PN zygotes (n=4527) had this dysmorphism, but they were found in 14% of patients. Chromosomal status was assessed in 15 of these zygotes and 87% were considered abnormal, mostly

Table 25.4. FISH studies of 3PN embryos from ICSI. N Mosaic (%) 3n (%) 2n (%) (23) 7 14 86 0 (27) 71 28 56 13 N: analyzed, 3n: triploid, 2n: diploid, n: haploid.

n (%) 0 0

mosaics.34 More studies are needed to identify the origin of each pronucleus. In the meantime transfer of such embryos is not recommended, especially since the data were derived by analyzing a restricted number of chromosome pairs. Recently, Tesarik and Greco noticed that when one pronucleus was at least twofold larger than the other, the embryo invariably arrested.35 They found that this occurred in 1.3% of zygotes (n=446 analyzed). These

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zygotes are often observed after fertilization with immature sperm cells,36,37 and may be related to incomplete nuclear protein transition in the male gamete.35 Tesarik and Greco35 also noticed that when equal-sized pronuclei were not contiguous to each other but were separated by certain distance they invariably arrested development. They found that this occurred in 0.9% of zygotes (n=446 analyzed). Lack of pronuclear apposition is probably the cause of malfunction of the paternally derived centriole.38 DISTRIBUTIONS AND MORPHOLOGY OF NUCLEOLAR PRECURSOR BODIES An early phase of nucleogenesis consists of the assembly, growth and fusion of nucleolar precursor bodies (NPB). Tesarik and Greco have described six patterns of size and distribution of NPB, some of which correlate with an increased frequency of embryonic arrest and reduced implantation.35 Pattern 0, which represents 28% of zygotes, is characterized by (a) having a similar number of NPBs between both pronuclei (±3), but never fewer than three; (b) when there were seven or less NPBs, they were always polarized towards the other pronucleus, but if they were more than seven per pronucleus, they were always scattered throughout both pronuclei; (c) the number of NPBs in a pronucleus was never fewer than three; (d) the distribution of NPBs was either polarized or non-polarized in both pronuclei but never polarized in one and non-polarized in the other. Other patterns differed in one or more of the above characteristics. When these patterns were compared with embryo arrest, pattern 0 arrested only 8.5% compared with 20–30% for all the other patterns. Less clear was the fact that pattern-0 embryos showed less multinucleation and better embryo morphology than other patterns. This resulted in a high pregnancy rate (50%) in transfer with at least one pattern 0 embryo, compared with only 9% transfers with no pattern 0 embryos transferred.35 Nucleolar polarization represents an important step in the establishment of embryonic axes.39 However, this is a dynamic process, and polarization of NPB is not evident from the very beginning of pronuclear development. The two morphologies described for pattern 0, one not polarized with many NPBs, and another polarized, with fewer but larger structures, represent earlier and latter polarization steps, both normal. The morphology patterns 1–5 presumably correspond to deviations from the normal pattern (0) and are characterized basically by asynchrony. No correlation with chromosome abnormalities has been made yet.

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SHAPE AND ALIGNMENT OF FIRST AND SECOND POLAR BODIES According to Ebner et al the selection of oocytes based on the morphology of the first PB (the best are round, ovoid, intact) three hours after retrieval permits a significant increase in pregnancy rates than when the selection was based on day 2 morphology.40

ANALYSIS OF CLEAVAGE-STAGE HUMAN EMBRYOS KARYOTYPE ANALYSIS OF MORPHOLOGICALLY NORMAL EMBRYOS Some reports have been published that give a rate of chromosome abnormalities between 20% and 40%. The disparity of these results may in fact be caused by the small number of embryos and the low number of cells analyzed. Observations are also complicated since the follicular stimulation protocols were different as well as the etiology of patients and culture systems. From data collected at several programs it is

Table 25.5. Difference in chromosome abnormalities found by karyotyping when one or more cells were analyzed. good quality 2 PN poor quality 2 PN (a) (b) (a) (b) No processed 1574 163 686 178 No analyzed 32% 72% 39% 49% Diploidy 63% 78% 13% 38% Aneuploidy 15% 3% 30% 10% Mosaicism 10% 15% 13% 40% other abnormal 12% 5% 49% 11% (a) One or more cells analyzed. Review by Pellestor30; (b) two or more cells analyzed. Data from Almeida and Bolton.42 becoming apparent that many factors determine chromosomal aneuploidy rates.41 For example, when only embryo for which at least two cells were analyzed is included, lower rates of aneuploidy and higher rates of mosaicism were reported42 than when only one or more cells were analyzed.30 The results are summarized in Table 25.5. The difference between these studies is remarkable and indicates that a sizable part of aneuploidy detected before Almeida and Bolton42 may not have been true aneuploidy but mosaicism. These results underline the need for different

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and/or improved techniques, such as FISH and SKY, to study chromosomal abnormalities in cleavagestage embryos. MORPHOLOGICAL TRAITS AND CHROMOSOME ABNORMALITIES Certain types of dysmorphisms have been studied in correlation to chromosome abnormalities: fragmentation, multinucleation, giant eggs and dominant blastomere embryos. As summarized in Table 25.6, some of these morphological abnormalities are very well correlated with chromosomal abnormalities but others are not. A review of each dysmorphism and its association (or lack of) with chromosomal abnormalities follows.

Table 25.6. Summary of morphological abnormalities and its relation to chromosomal abnormalities. Embryo morphology FISH analysis Reference Normal morphology 20 to 34 years old 16% abnormal 7 35 to 39 years old 37% abnormal 7 40 to 45 years old 53% abnormal 7 Dysmorphic zygotes: 3 PN after regular IVF 80–100% abnormal 22, 27 3 PN after ICSI 100% abnormal 22, 27 cleaving 1 PN after regular IVF 34–50% abnormal 27, 26 cleaving 1 PN after ICSI 70–85% abnormal 27, 26 uneven PN 87% abnormal 34 cleaving o PN 43% abnormal 33 Dysmorphic embryos: giant embryos (>220mm) triploid 13 dominant single blastomere polyploid 13 >20% fragments, normal devel 56% abnormal Munné, unpublished multinucleated embryos 74% abnormal 52 Table 25.7. Chromosomal abnormalities detected by FISH and fragmentation rate. Fragments analyzed aneuploid other abnormal* 0 to 5% 502 21.9% 29.7% 6 to 15% 420 19.3% 32.4% 16 to 25% 260 19.2% 34.2% 26 to 35% 161 11.8% 41.0%

normal 48.4% 48.3% 46.5% 47.2%

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11.7% P<0.01

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54.0% P<0.001

34.4% P<0.005

*mosaics, polyploid, haploid. Munné et al, unpublished data up to 8/24/99. FRAGMENTATION Fragmentation percentage has been associated with chromosome abnormalities.43,44 We have found that the percentage of fragmentation is strongly correlated with mosaicism,1 whereas aneuploidy did not appear to predict fragmentation. Our most updated results (Table 25.7) show again a very significant increase in mosaicism, polyploidy and haploidy with increasing fragmentation, in particular when fragmentation is 35% or higher. There is, however, a puzzling decrease of aneuploidy with fragmentation. Aneuploidy increases with maternal age but maternal age is not linked to an increase in embryo fragmentation.45 Such decrease of aneuploidy with increasing dysmorphism was already shown in previous studies7 and could be due to the difficulty of identifying aneuploidy when the embryo is mosaic. Alikani et al reported that the microsurgical removal of fragments in embryos with 6–35% of fragmentation implant with a similar frequency as those without fragments.45 The beneficial effect of fragment removal can be explained through two non-exclusive hypotheses. The first is that removal of extracellular fragments restores the spatial relationship of cells within the embryo, facilitating cell to cell contact, compaction, cavitation and blastocyst formation. The second hypothesis is that the removal of fragments prevents the degeneration of adjacent cells to those fragments. The observation that the removal of fragments in embryos with more than 35% fragmentation does not improve implantation may be explained by the fact that these embryos have higher rates of chromosomal abnormalities (only 34% are normal). In addition, different patterns of fragmentation have been observed,45 some of them associated with programmed cell death (Warner et al, in press). One of this fragment types (type IV), which is characterized by having large fragments, produces lower pregnancy rates than other types of fragments. However, there was no clear correlation between chromosome abnormalities and fragmentation type (Munné et al, unpublished). MULTINUCLEATION FREQUENCY Multinucleated blastomeres (MNBs) occur at any time between the first cleavage division and blastocyst stage, but were found more often in 2 cell

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than 4 cell or later stage embryos. This last observation may be less precise since nuclear observations are hindered once embryos contain more cells and fragments. The occurrence of multinucleated blastomeres (MNB) has been studied by two large studies, including 1885 embryos studied on day 346 and 3557 embryos, studied on day 2 of development.47 They found 44% and 74% of patients possessed embryos with at least one MNB, and 15% and 31% of embryos contained MNBs, respectively. MNBs have been associated dysmorphism and fragmentation.7,47 In addition most studies show a decreased developmental potential of MNBs. The impairment in development is probably due to the arrest of the MNB cells, because Hardy et al reported that binucleated cells are usually arrested.48 This is translated in a significantly lower cell count in MNB embryos,47 or the arrest of the whole embryo in 57% of embryos, while only 14% reached expanded blastocyst stage.46 According to Balakier and Cadesky there was no correlation with maternal age,46 but Jackson et al indicated that patients with MNBs tend to be younger (P<0.01).47 Jackson et al47 also found that cycles containing MNBs have a double the concentration of E2 on the day of HCG (2401 v. 1270 pg/ml, P<0.001), double numbers of oocytes collected (22 v. 10, P<0.001), and need fewer ampoules of gonadotropins (P<0.001) than cycles without MNBs. Previous studies have shown that high E2 concentrations49 or high numbers of retrieved oocytes50 are associated with lower implantation rates. The observation that, in repeated cycles, MNB was not observed as a recurrent phenomenon,47 indicates that culture conditions or stimulation regimes are more probably causes of multinucleation than sperm or oocyte factors. CHROMOSOME ABNORMALITIES FISH studies on MNBs showed that the chromosomal content of each MNB nucleus was not always the same as the chromosomal content of the nuclei of the sibling blastomere MNBs.6,51 Two patterns of multinucleation have been suggested, one occurring at 2 cell stage and the other occurring at 8–16 cell stage. Regarding MNB occurring at the 2 cell stage, Kligman et al found that the presence of multinucleated cells in 22 non-arrested day 2 or day 3 human embryos is indicative in 74% of the cases of extensive mosaicism and/or polyploidy when those embryos were analyzed on day 4 with five probes.52 Using only two probes and analyzing on day two or three Staessen and Van Steirteghem found 72% of the 101 embryos analyzed to be chromosomally abnormal.53 However, when only 2 cell stage embryos with both cells MNB were studied at the 2 cell stage only 55% were chromosomally abnormal. In a smaller series also with three chromosomes studied, Laverge et al found 44% of chaotic mosaics and other abnormalities in 27 MNB embryos.10 The differences

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between these studies are a result of the use of different probes and the longer time in culture in the Kligman et al study to produce arrest and polyploidization in culture.52 Nevertheless, this pattern of high rates of chromosome abnormalities in MNBs detected on day 2 differs from previous analyses in which MNBs were detected on day 4. In those studies, morphologically normal embryos with a single or few MNBs were mostly chromosomally normal.6,51 This suggests two patterns of multinucleation. One occurs at the 2 cell stage, which normally involves multinucleation and produces mostly chromosomally abnormal embryos. The other pattern occurs at the 4 to 16 cell stage and usually involves binucleation probably produced by cytokinesis failure, with each of the two nuclei being chromosomally normal and the cell arrested.48 CLINICAL IMPLICATIONS Usually it is not recommended to transfer embryos containing MNBs at the 2 cell stage, whereas embryos with binucleated cells at the 8 cell stage may be transferred if no other morphologically normal embryos are available. MNB embryos can implant,54 and healthy babies have been born from the transfer of only MNB embryos,46,47,55 but according to Jackson et al47 implantation sites from cycles containing MNB embryos are six times more likely to abort (19% v. 3%, P=0.006). However, the clinical pregnancy rate from mixed transfers containing MNBs was significantly reduced, from 29% to 17% (128 cycles mixed with MNBs, P<0.01) in one study55 and from 29% to 8% in mixed transfers and to 4% in transfers with only MNB embryos replaced (P<0.05) in another one.47 Unpublished data from our center also indicates a step decrease in implantation from 43% per nonmultinucleated embryo to 13% per multinucleated embryo (P<0.001). GIANT EGGS AND DOMINANT BLASTOMERE EMBRYOS We have detected two instances in which monospermic embryos with a particular morphological abnormality were chromosomally unique.13 The first were embryos with only one large dominant cell surrounded by smaller blastomere-sized extracellular fragments. These embryos were polyploid and frequently polyploid mosaic, and the single cell was normally multinucleated. The second group developed from giant oocytes with diameters of 220µm or more, and zygotes displaying two polar bodies and two pronuclei. These embryos are invariably triploid or triploid mosaics, with XXX or XXY gonosome constitutions, which suggests a higher contribution of maternal genomes. We have seen also giant GV stage eggs containing two nuclei, indicating that giant GV stage oocytes originate from cytokinetic failure or from fusion of two GVs. These embryos should not be transferred.

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EMBRYO DEVELOPMENT AND CHROMOSOMAL ABNORMALITIES Significant differences in the total amount of chromosome abnormalities are found between arrested, slowly developing and normally developing embryos when studied by FISH.7 These differences have been later confirmed by karyotyping, although only slow and normally developing embryos could be studied (55% abnormal v. 27%, P<0.00142). In total we have studied over 1255 human embryos with probes for at least chromosomes X, Y, 13, 18, 21 and part of them also with chromosome 16 (Marquez et al, unpublished).7 Embryos were divided into three groups according to development competence: (1) group A— embryos that had reached the 6 to 8 cell stage by day 3, had cleaved in the previous 24 hours, had less than 20% fragmentation, and did not present multinucleated blastomeres. These embryos were not frozen and donated for research; (2) group B—slow embryos, which had cleaved in the previous 24 hours but had more than 20% fragmentation, had multinucleate blastomeres, or had not reached 6 cells by day 3 of development; (3) group C—embryos that had not cleaved during a 24 hour period and were considered arrested. We found the highest rate of polyploidy (32%) in group C embryos while the other two groups had less than 10% polyploidy (Table 25.8) (Marquez et al. unpublished).7 High rates of polyploidy in arrested embryos have been also described by Laverge et al.56 Extensive diploid mosaicism, that is those embryos with a diploid average of chromosomes per cell and with more than 3/8 abnormal cells, increased with decreasing competence, from 15% in group A to 25% in group C (P<0.001) (Table 25.8). It seems therefore that those chromosome abnormalities that mostly originate during or after fertilization (polyploidy and mosaicism) increase with decreasing embryo

Table 25.8. Chromosome abnormalities in day 3 and day 4 of development according to development competence of the embryo. Development groups Group A Group B Group C Embryos analyzed Day 3 254 363 114 Day 4 188 154 182 total 442 517 296 Extensive diploid mosaicism Day 3 15.7 a 21.5 a 28.1 a Day 4 13.3 21.4 20.9 total 14.7 a 21.5 a 25.1 a Polyploidy, polyploid mosaics Day 3 6.3 a 7.7 a 22.2 a,c

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Day 4 2.1 d 13.0 d 44.4 c,d total 4.5 d 9.9 d 31.5 d Haploidy, haploid, mosaics Day 3 4.3 4.4 5.3 Day 4 3.7 1.9 2.7 total 4.0 3.4 4.2 Aneuploidy XY, 18 Day 3 2.8 3.3 2.6 Day 4 5.3 5.8 4.9 total 3.8 4.3 3.6 Aneuploidy 13, 21 Day 3 10.6 e 8.3 e 2.6 e Day 4 18.4 f 11.2 f 2.2 f total 13.9 9.5 2.4 Aneuploidy 16 Day 3 0.9 4.3 3.4 TOTAL Day 3 40.6 49.5 64.2 Day 4 42.8 53.3 75.1 total 41.5 50.6 67.8 a Significant differences between morphological groups (P<0.01, GLM analysis) d,e,f Significant differences between morphological groups (P<0.001, GLM analysis) c Significant differences between both both series (P<0.001, X2 test) The mean values have been averaged over the other factor ‘age group’ competence, with haploidy being constant in the three groups at around 4% (Table 25.8). In contrast aneuploidy, which originates mostly during meiosis I, was more common in groups A and B (18–19%) than C (9%) (Table 25.8). We have argued before that a decrease in aneuploidy in arrested (group C) embryos could be an underestimation of aneuploidy in arrested embryos that in addition are often polyploid and/or mosaic.7 There is another group of embryos, those with accelerated development on day 3, which we have not thoroughly studied. According to Harper et al these embryos are usually mosaics.57 They could be polyspermic, but the extra pronucleus may have been missed during zygote observation. The few embryos that we have analyzed are also chromosomally abnormal and usually show poor cell adhesion. In total, chromosome abnormalities were found in 41% of group A embryos and increased with decreasing development competence being 68% of group C embryos. Therefore it seems that chromosome abnormalities do not impair development, at least up to the 8 cell stage, because even group A embryos have high rates of chromosome abnormalities. Such lack of negative effect has been explained by the observation that genome activation does not start until the 4 to 8 cell stage,58 and therefore chromosomal abnormalities cannot produce embryonic arrest until later stages.

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POLYPLOIDY The origin of polyploidy is arrested (group C) embryos was discussed previously,6 and polyspermic fertilization was considered unlikely since zygotes were observed under high magnification with inverted scopes and only embryos developing from two PN zygotes were included in the study. Although cellular division stops in arrested embryos, DNA synthesis has been observed to continue,59 explaining the increase in polyploidy in arrested embryos. A further evidence of that is the observation that embryos analyzed on day 47 had more polyploidy than embryos studied on day 3 (Marquez et al, in preparation) (Table 25.8). Arrested embryos on day 4 had an extra day to replicate their DNA without cleaving, thus becoming polyploid at a higher rate than arrested embryos on day 3. Oocyte quality or culture conditions may be the cause of their arrest. The fact that the most compromised embryos tend to arrest in culture and become polyploid has repercussions at the time of deciding if to place embryos on extended culture or not. For example, while blastocyst culture will prevent the transfer of a sizable part of polyploid embryos it may also produce the arrest of compromised embryos that otherwise may implant if transferred earlier. Preliminary data comparing embryos replaced on day 3 and day 5 indicates that day 3 transfers may allow some developmentally and/or morphologically compromised embryos to implant, while those same embryos will arrest if left in extended culture (Alikani et al, unpublished). MOSAICISM IN MONOSPERMIC HUMAN EMBRYOS High rates of mosaicism have been reported by using FISH1,6,7,12,59–61 or karyotype analysis of at least two blastomeres.42 In karyotype studies, the rate of mosaicism detected has been underestimation based on the analysis of only one or two cells per embryo while aneuploidy was overestimated.42 For mosaicism studies to be accurate, most cells from each embryo should be analyzed. TYPES OF MOSAICS The types of mosaics observed in cleavage stage embryos are more diverse than those observed in spontaneous abortions, indicating that some of them are incompatible with later stages of embryo development. We have classified mosaics according to their overall ploidy (haploid, diploid, or polyploid mosaics), and then subdivided the diploid mosaics according to mechanism of formation (mitotic non-disjunction, anaphase lag, endorreduplication, chaotic mosaics, diploid/polyploid (Table 25.9). Some groups59,62 do not consider chaotic embryos as mosaics, but here they are considered as such. Table 25.9 shows recent data from our

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laboratory for the different types of mosaics found and their frequency (and published data).1 The most frequent types of diploid mosaics are chaotic (47%), followed by diploid/polyploid embryos (28%), diploid/aneuploid embryos (19%), and

Table 25.9. Types of mosaics, % of abnormal cells and onset of mosaicism. Onset of mosaicism % abnormal cells 1st division 2nd division 3rd division total 80–100% 79–40% >39% All diploid mosaics 41% 34% 24% 649 chaotic 68% 24% 7% 305 2n/pol 7% 40% 52% 181 2n/ane 32% 43% 24% 123 anaphase lag 19% 53% 28% 36 endoreduplication 0% 0% 100% 4 polyploid mosaic 73% 22% 4% 49 haploid mosaic 80% 8% 12% 25 dispermic 69% 30% 0% 36 anaphase lag (6%), with endoreduplication being very rare. Endoreduplication mosaics usually have just one cell abnormal this having many copies of the same chromosome. Although we previously indicated that some types of diploid mosaics seem to be more common in arrested and poorly developing embryos,1 now with a larger sample these differences are not present anymore, and most mosaic types seem to be evenly distributed among arrested, slow and normally developing embryos (unpublished data). While aneuploidy is found to increase significantly with maternal age mitotic non-disjunction is not. ONSET OF MOSAICISM The onset of mosaicism in cleavage stage embryos can be determined by assessing the number of blastomeres of each type. This can only be accomplished when most cells in a given embryo are analyzed. All blastomeres of monospermic embryos are abnormal when the chromosome abnormality occurs during the first embryonic division. When one half or one fourth of the blastomeres are abnormal, mosaicism arises at the second and third division, respectively.12 We previously reported that the onset of mosaicism in polyploid and haploid mosaic embryos is usually at the first division, whereas monospermic diploid mosaics were usually generated at the second or later divisions.12 However, that study was performed analyzing only two chromosome pairs. With up to eight chromosome pairs analyzed per cell, more mosaics

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can be detected. Our latest data (Table 25.9, unpublished) show that 40% of diploid mosaics occur at the first mitotic division, compared to polyploid mosaics (73%) and haploid mosaics (80%). However, the type of diploid mosaicism is also important. Chaotic mosaics are mostly generated during the first mitotic division (68%) compared to the other diploid mosaics (7–32%), which are mostly produced during the second or third division (Table 25.9). High rates of first mitotic mosaicism may indicate either an abnormal number of male centrioles (haploids none, polyspermics two), or suboptimal centriole function. In both cases the first mitotic spindle will not be formed properly creating two different chromosomally abnormal cells. FATE OF MOSAIC CELLS

(a)

(b)

(c)

(d)

It has been repeatedly suggested that abnormal cells within mosaic embryos are self correcting, that they have less developmental potential than normal cells, and/or end up in the trophectoderm.24,29,63 Nevertheless, studies in several laboratories show a different outcome for abnormal cells, depending on the type of mosaicism. Diploid/polyploid mosaics—They apparently represent a normal developmental process leading to trophoblast formation. Polyploid nuclei have been described in most mammals studied including human blastocysts.64 Experimental production of tetraploid or diploid mouse embryos suggests that a mechanism exists to exclude chromosomally abnormal cells from the primitive ectoderm lineage since at 12 days no tetraploid cells can be detected in the fetus.63 The cells derived from the tetraploid part appear mostly on trophectoderm derivative tissues or to amnion, yolk sac mesoderm, or primitive endoderm (tissues derivative of the inner cell-mass other than the fetus). This means that although some tetraploid cells can become incorporated in the inner cell mass, they do not contribute, at least in the mouse, to form the fetus. On the other hand, the fact that they appear as early as the cleavage stage has been suggested to indicate cytokinesis breakdown due to unsuitable culture conditions.65 In that case, their fate is probably cellular arrest. Binucleated blastomeres—The fate of binucleated blastomeres found in cleavage stage embryos has been determined by measuring blastomere size. Using this approach, Hardy et al concluded that binucleated blastomeres are mostly arrested.48 It is unlikely that mural trophectoderm giant cells, which are polyploid, could originate from binucleated cells because these giant cells are formed at the blastocyst stage while most binucleated cells originate and arrest at the third cleavage division, before trophectoderm differentiation.48 Aneuploid cells—Unlike polyploid and multinucleated cells, aneuploid cells experimentally combined with normal diploid cells to form mosaic mammalian embryos are demonstrably able to participate in embryogenesis and contribute to postimplantation stages and viable offspring.66,67 Preliminary results from our lab indicate that human diploid mosaics with an aneuploid cell line can reach blastocyst stage.68 Chaotic embryos—Most or all cells of these embryos are chromosomally abnormal,

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and, in most cases, each cell has a different chromosome complement. Surprisingly, we have detected chaotic embryos reaching blastocyst stage, but in those cases, the embryo never has more than 60 cells.68 FACTORS INDUCING MOSAICISM The suspected factors contributing to the formation of mosaicism are diverse and are by no means fully investigated. Factors have been identified in the gametes and also in culture conditions. (a) Male factors—Centrosomes are the microtubule organizing centers of the cell, therefore abnormal microtubule configurations may lead to mosaicism. Mosaicism may be caused by an abnormal number of centrioles.23 For example, excessive duplication of centrosomes or ectopic assembly of microtubule nucleating proteins could lead to the formation of spindles with multiple poles. In cancer, an abnormal number of centrioles leads to mosaicism. Recently, a human serine/theonine kinase named STK15, associated with centrosomes, which is amplified in many cancers known to be aneuploid has been found.69 Cells overexpressing STK15 had amplified number of centrosomes and chromosomes were missegregated in these cells, becoming aneuploid cells and producing mosaic tissues. Presumably, an increase in kinase concentrations causes centrosome dysfunction, leading to the assembly of aberrant spindles, the improper segregation of chromosomes, resulting in loss or gain of chromosomes and either cell death or malignant transformation.70 Abnormal centrioles may be the explanation for male originated IVF failure in some patients. We have recently identified a couple which produced only mosaic embryos when autologous sperm was used but not when the sperm was donated.71 (b) Female factors—Although neither maternal age nor elevated levels of basal FSH had been linked to mosaicism, there still may be a female contribution to mosaicism. For example, van Blerkom et al found a significant correlation between embryos with at least one MNB at the 2 cell stage and follicular underoxygenation.72 As shown before, MNBs are usually mosaics. Another factor that may contribute to mosaicism are the use of different superstimulation protocols. As we showed in a previous study downregulation produced significantly fewer mosaic embryos than stimulation with clomiphene citrate.41 (c) Culture conditions—Embryos with only a fraction of their cells abnormal may be produced by culture conditions more often than by abnormal gametes. For example, a drop in temperature may affect cytokinesis, resulting in diploid/polyploid embryos or embryos with MNBs. Spindle microtubles are highly thermosensitive, and even a small change in temperature can disturb the spindle structure of the oocytes. We have demonstrated that embryos from different laboratories cultured under different conditions and stimulation protocols have very diverse rates of mosaicism.41 Unsuitability of water and air has been recently linked to sudden decreases in implantation rate.73,74 However, no chromosomal analysis has been performed on those embryos. Furthermore, while some chemicals have been suggested as the culprits for lower than expected implantation

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rates, the damage that they might produce is at the gene level but probably not the chromosome level. ANEUPLOIDY Genetic analysis of abortuses and live offspring has shown that older women are at a higher risk of delivering trisomic fetuses. Most of this aneuploidy is the result of non-disjunction in maternal meioisis I,75–77 and even trisomy 18, which occurs in similar frequencies in maternal meiosis I and II78 may also originate mostly in meiosis I but be classified by molecular techniques as originated at meiosis II.79 FISH studies of cleavage stage embryos and oocytes have also found a significant increase in aneuploidy with maternal age.7,80 For chromosomes 13, 18, and 21, aneuploidy in clinically recognized pregnancies increases from 1.3% in 35–39 year-old group to 4.3% in the 40–45 group.81 However, in morphologically and developmentally normal cleavage stage embryos, we found that aneuploidy rates for these chromosomes increased from 4% in the 20–34 year old group, to 37% in the 40–45 year old group. The difference between aneuploidy rates at cleavage stage and recognized pregnancies is probably a result of the low survival rate of some aneuploidies. For example, preliminary data from our lab indicate that monosomies, with the exception of monosomy 21 and X seldom reach blastocysts stage.68 Similarly, some trisomies are commonly detected in cleavage stage embryos and seldom or never detected in spontaneous abortions, as is the case with trisomy 1.10,82 Because the chromosome abnormalities detected at cleavage stage are not necessarily the same found in first trimester conceptions, it is imperative to know which are the chromosomes most involved in aneuploidy at cleavage stage if PGD is to be performed with the purpose to increase implantation rates. For example, in a study by Bahçe et al,83 abnormalities for chromosomes 1, 4, 6, 7, 14, 15, 17, 18, and 22 were studied in cleavage stage embryos. That data in combination with previous works7 and recent data (Bahçe et al, unpublished data) indicate that of the chromosomes studied 1, 7, 15, 16, 21, and 22 are those with the highest aneuploidy rate (>3%) in cleavage stage embryos, while chromosomes X, Y, 4, 6, 14, and 18 have frequencies below 2% (Table 25.10). Of these chromosomes, we could demonstrate a significant increase in aneuploidy with maternal age effect for 4, 6, 7, 15, 16, and 21 (Table 25.10).

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Table 25.10. Chromosome specific aneuploidy rates in human cleavage-stage embryos. Ave Maternal Ave. maternal Chromosome # analyzed age analyzed aneuploid age aneuploid embryos embryos ±STD (%)ε embryos ±STD 1 190 36.8±4.4 9 (4.7%) 34.7±4.8 4 211 37.3±4.4 4 (1.9%) 39.7±0.9 (++) 6 190 36.8±4.4 2 (1.1%) 41.7±0.6 (++) 7 215 37.3±4.3 7 (3.2%) 40.2±2.2 (++) 13 882 37.0±4.1 21 (2.4%) 38.2±4.0 14 277 37.2±4.1 3 (1.1%) 37.3±3.4 15 302 37.5±4.1 15 (5.0%) 39.7±2.8 (+) 16 520 37.2±4.2 27 (5.2%) 39.7±2.8 (++) 17 190 36.8±4.4 5 (2.6%) 39.0±3.8 18 999 36.9±4.1 17 (1.7%) 38.8±4.3 21 882 37.0±4.1 38 (4.3%) 39.4±3.2 (++) 22 302 37.5±4.1 17 (5.6%) 38.3±3.9 XY 999 36.9±4.1 12 (1.2%) 38.7±3.9 ε Double aneuploidies counted twice, once for each chromosome. + (p<0.05 and ++ (p<0.01) were the result of comparing the maternal ages of aneuploid embryos versus non-aneuploid embryos, at specified chromosomes. CHROMOSOME ABNORMALITIES AND DEVELOPMENT TO BLASTOCYST STAGE The advantages of culturing embryos to blastocyst stage in an IVF lab are being discussed. The results so far seem promising because those embryos reaching blastocyst stage implant at very high rates.84 CHROMOSOME ANALYSIS OF BLASTOCYSTS Data about the genetic composition of the embryos that reach blastocyst stage have been reported but the interpretation is still unclear.85–88 In all those studies, mosaicism is a common feature. The types of mosaics found are classified according to the overall ploidy (haploid, diploid, polyploid mosaics) and to the mechanism (non-disjunction, endoreduplication, chaotic mosaics, diploid/other ploidy).7 Evsikov and Verlinsky reported that mosaicism in blastocysts was found only in 10.5% of the embryos, with an average of five aneuploid cells per blastocyst.11 Clouston et al described the karyotype analysis of human blastocyst and,

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although just few cells were analyzed, they found 23% of diploid/tetraploid mosaics and two thirds of embryos with aneuploid cells were diploid/aneuploid mosaics.86 Veiga et al reported that 88% of blastocyst were mosaics with an average of 17% of abnormal cells.88 Harper et al found that all of the embryos that had reached the blastocyst stage were chromosomally mosaic, with an average percentage of abnormal cells of 27%.86 Most of the mosaicism found in those blastocysts is due to the presence of tetraploid and polyploid cells, but also aneuploid and chaotic cell lines have been found. Polyploid nuclei have been described in many mammalian blastocysts including human blastocysts.64 They have been found mostly in the trophoblasts in day-10 pig embryos90 and only in the trophectoderm bovine blastocysts,91 and could be the precursors of giant trophectodermic cells.92 The presence of polyploid cells in blastocysts probably represent a normal developmental process leading to trophoblast formation and is considered a normal feature of human embryo development.11,64,65,93 Although aneuploid cells seem detrimental for embryo development, high levels of mosaicism and chaotics can still be detected in blastocyst stage.11,68

FATE OF ABNORMAL CELLS IN MOSAIC EMBRYOS It has been suggested that abnormal cells within mosaic embryos are self correcting, have less development potential, and/or locate ultimately in the trophectoderm.24,29,63 The evidence does not support either theory. For example, James and West63 created tetraploid/diploid mouse embryos and at 12½ days of development no tetraploid cells were detected in the fetus and those cells rarely contributed to other derivative of the primary ectoderm and trophectoderm lineages. In contrast, aneuploid/diploid mouse chimeras show aneuploid cells participating in embryogenesis and contributing to post-implantation stages and viable offspring.66,67 Mottla et al found that each blastomere of the early cleavage stage human embryo can participate in both trophectoderm (TE) and inner cell mass (ICM) formation, and therefore genetically abnormal blastomeres should have the same chance of contributing to the ICM.94 Similarly, Evsikov and Verlinksy reported that there is probably no selection for euploid ICM, on the basis of the fact that the average degree of aneuploidy in the ICM was similar to the overall blastocyst mosaicism.11 Delhanty and Handyside also concluded that aneuploid cells are not necessarily diverted to TE on the basis of the fact that a substantial proportion of trisomic fetuses are due to postzygotic mitotic error.95

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SELECTION OF CHROMOSOMALLY ABNORMAL EMBRYOS Impaired development of some embryos during extended culture could be due, at least in part, to chromosome abnormalities. Significant differences in the total amount of chromosome abnormalities have been found between arrested, slow embryos and normal developing ones in cleavage stages.7 Jany and Menezo argued that selection against chromosome abnormalities may occur during extended culture because many embryos arrest during morula stage.96 Evsikov and Verlinsky suggested that cavitation initiates a negative selection against aneuploid cells, and therefore if the aneuploid cells at morula stage reach some threshold level, this would lead to the self elimination of the whole embryo.11 Although Evsikov and Verlinksy do not show evidence of selection against any specifically chromosome abnormality,11 our results suggest that there is a strong selection against all monosomies, with the exception of monosomy X and 21, as well as for haploidy.68 The fact that just these two types of monosomies are found in blastocyst stage agrees with prenatal diagnosis data, were not other monosomies are detected in first trimester abortions.97 Menezo et al reported in vitro selection of unbalanced translocated embryos during extended culture.98 Triploid and tetraploid blastocyst are able to develop to blastocyst stage,11,68,86 which is in concordance with first trimester conceptuses data. Embryos with high frequency of mosaicism can occasionally develop to blastocyst. However, we found that chaotic mosaics reaching blastocyst stage were probably developmentally jeopardized, since they never had more than 60 cells, compared with an average of 114 in other blastocysts.68 In summary, a strong selection against chromosomally abnormal embryos has been observed, but most chromosome abnormalities can still be detected at blastocyst stage. It is still unknown if there is a link between blastocyst morphology and chromosomal status, which would allow for the selection of normal embryos. Also, more studies on the impact of mosaicism and cell allocation are needed.

CHROMOSOME ABNORMALITIES AND TYPES OF PATIENTS RECURRENT MISCARRIAGES Recurrent miscarriages (RM) have been defined as three or more consecutive spontaneous abortions of less than 20 weeks’ gestation, excluding any spontaneous abortion with documented aneuploidy by karyotype analysis.99 0.3% of couples of reproductive age should have a

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history of three consecutive losses, but epidemiological studies estimate that this history occurs more frequently, in the range of 1% to 2%.100 In addition, couples without an antecedent live birth and three or more consecutive abortions have a 50% risk of having a subsequent spontaneous abortion.101 The causes of multiple abortion are: 43% unexplained, 20% auto-immune, 20% endocrine, 16% anatomical, 3– 3.5% genetic, and 0.5% infections.99,102,103 Of the cases resulting from genetic factors Stephenson found that they were caused because the parents were carriers of translocations (n=5), insertions (n=1), or inversion (n=1).99 Another study reported that up to 9.2% of fertile couples experiencing three or more consecutive first trimester abortions are carriers of translocations.104 While some studies have reported a higher frequency of chromosome abnormalities in spontaneous abortions, others have not.105–107 Kiefer et al reported that oligoasthenozoospermia could be another factor.108 Oligoasthenospermic patients (those with less than 107 motile sperm per ml) had a significantly higher rate of spontaneous abortions (40%) than patients with normal semen (12%), even though both populations had similar maternal ages. No results on chromosome analysis of SPA were reported. Simon et al and Pellicer et al have detected significantly more chromosome abnormalities in embryos from women with recurrent miscarriages than in their respective control groups.109,110 Pellicer et al, for instance, found 58.5% chromosomally abnormal embryos in nine women with RM, compared with 17% in 10 females without previous miscarriages and similar ages.110 They gave a probability of 0.001 but comparing embryos instead of patients, which would had been more accurate. However, the use of PGD in those patients did not improve implantation rates.

Table 25.11. Chromosome abnormalities in recurrent miscarriage patients. Groups N maternal normal aneuploid (%) other abnormalities age (%) (%) PGD current 4 <35 57 20 22 aborters 16 35–39 53 21 26 other PGDs 24 <35* 51 23 26 36 35–39 50 25 25 *why PGD?: 5 because X-linked, 5 repeated IVF failure, 3 prev trisomy, 3 had one previous loss, 8 unk Data from SBMC 3/00, unpublished. In contrast, a more extensive group of PGD patients from our center (Munné et al, unpublished data) did not show any difference in

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chromosome abnormalities when comparing PGD patients with or without previous history of RM (Table 25.11). Our study reanalyzed the nontransferred embryos, in comparison to the study by Pellicer et al,110 which did not. The major difference between these two studies is that by Pellicer et al included only patients 35 or younger, while most of our patients were 35 and older, and the cause of RM in our population could be a result of maternal age. REPEATED IVF FAILURES In 27 cycles that had failed three or more IVF cycles it was found that in several of them all the embryos were chromosomally abnormal, and the rest had a high percentage of chromosome abnormalities (54%, n=74 embryos) for an average maternal age of only 32 years. However, when they underwent PGD for chromosome abnormalities and their implantation rates were compared, the PGD group showed a 17% implantation rate, not statistically higher than the 10% found in the control group.111 OVERRESPONDERS TO HORMONAL STIMULATION Previous studies have shown that high E2 levels on the day of human chorionic gonadotrophin (HCG)49 or high numbers of retrieved oocytes50 are associated with lower implantation rates. Recently, Jackson et al also found that cycles containing MNBs have double the level of E2 on the day of HCG (2401 v. 1270pg/ml, P<0.001), double numbers of oocytes collected (22 v. 10, P<0.001), and need fewer ampules of gonadotropins (P<0.001) than cycles without MNBs.47 As reviewed before, most MNB embryos are chromosomally abnormal,52 which may be related to the lower implantation rates observed in these cycles. PATIENTS WITH HIGH BASAL FSH Although maternal age is a well established risk factor for aneuploidy, it is not well known if the chronological age of the mother or the physiological age of the ovary is more important. High concentrations of FSH112,113 and lower concentrations of estrogen114 are characteristic of advanced maternal age, but are also found in women with unilateral oophorectomy (ULO) and younger women with premature menopause. Warburton suggested that if aneuploidy was caused by oocyte depletion, women who have had a trisomic conception at a young age might exhibit signs of early oocyte depletion, such as premature menopause.115 Brook et al observed that mice with ULO had premature menopause and elevated risk of aneuploidy.116 They concluded that the risk for aneuploidy is determined by the distance in time from the

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menopause rather than the chronological age of the mother and that the number of follicles limits the reproductive life span. Recently Freeman et al have reported a significant increase in Down’s syndrome in babies from young women with reduced ovarian complement,117 whereas other reports have found that women who have had an aneuploid conceptus exhibited elevated serum concentrations of FSH.118,119

Table 25.12. Chromosome abnormalities found in ICSI and IVF embryos developing from bipronucleated zygotes. Age ≤39 years Age >39 years All ages Chromosome abnormalities IVF ICSI IVF ICSI IVF ICSI Number of embryos analyzed 135 102 110 34 245 136 Normal (%) 39 48 28 24 34 42 Aneuploid (%)* 10 6 12 18 11 9 Gonosomal (%) 2 1 Haploid (%; mosaic or not) 2 5 2 6 2 5 Polyploid (%; mosaic or not) 10 4 13 12 11 6 Extensive 2N mosaicism (%) 28 20 38 38 33 24 Low 2N mosaicism (%) 10 18 7 3 9 14 *4 monosomies 13; 2 nullisomy 13; 6 monosomies 21; 2 monosomies 18; 6 monosomy 16; 2 trisomy 13; 3 trisomy 16; 7 trisomy 21; 1 trisomy 18; 3 monosomy X; 1 disomy Y; 1 trisomy XXY; 1 trisomy XYY. These data are consistent with Warburton’s “limited oocyte pool hypothesis”,115 which states that in a limited pool of oocytes, an oocyte in a suboptimal state of development could become the dominant follicle. These results also suggest that women with elevated FSH and/or ULO should be offered prenatal testing. Preliminary results in our center indicate also an elevated frequency of aneuploidy in patients with elevated concentrations of FSH. We found a significant increase in the rate of aneuploidy in women aged 35–39 years when they had a basal FSH concentration of >10 (P<0.02) (Table 25.12). The prevalence of genetic abnormalities was uniformly high in embryos from women over the age of 40, independent of their basal FSH status. Aneuploidy rates in women 35–39 with elevated FSH levels (>10) were equivalent to those over age 40 (Table 25.12). The finding of uniformly high chromosome abnormality rates in women over the age of 40 independent of their FSH status demonstrates that an elevated FSH level is a marker, which becomes abnormal late in the process of depleting ovarian reserve and is consistent with lower pregnancy rates seen in this population.

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MALE FACTOR We have compared the chromosomal abnormalities found in cleavage stage embryos produced by ICSI and IVF, and after controlling for morphological development and maternal age we found similar rates of chromosome abnormalities in both groups,120 thus indicating that ICSI is not a teratogenic method. The rate of serious malformations in babies born after ICSI was found to be similar to the general population.121 However, some studies show an increased risk of chromosome abnormalities, mostly gonosomal chromosome, in babies born from ICSI procedures.122–125 In another study, Kiefer et al reported that oligoasthenozoospermic patients (<07 motile sperm/ml) had more spontaneous abortions (40%) than patients with normal semen (12%).108 These chromosome abnormalities may have several different sources: MALE CHROMOSOME CONSTITUTION One hypothesis for this high rate of sex chromosome abnormalities may be due to gonosomal mosaicism.127 Although 47XYY males usually produce normal sperm,127–128 mosaic 46XY/47XXY130–133 and true 47XXY (Esgop et al, in preparation) produced high rates of 24,XY sperm. MEIOTIC DISRUPTION Infertile men have an increased frequency of pairing disruptions resulting in meiotic arrest.133 The sex chromosome bivalent is particularly susceptible to pairing abnormalities since there is generally only one crossover in the pseudoautosomal region. Thus, it is quite possible that infertile men have decreased recombination and pairing leading to both meiotic arrest (oligospermia) and non-disjunction of the sex chromosomes.134 These chromosome abnormalities will not be found in peripheral blood. ABNORMAL CENTRIOLE As previously discussed in the section on factors inducing mosaicism, there is some evidence that male factor patients may produce also high rates of mosaic and chaotic embryos, while the patient is chromosomally normal.71 As discussed in that section this may indicate an abnormal centriole function or number. Recently, we have compared the chromosome constitution of embryos obtained by ICSI and those from patients requiring surgical production of sperm, and we found that while aneuploidy was similar in both groups, post-meiotic abnormalities (mostly

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mosaicism) were significantly (P<0.001) more prevalent in those from surgical treatments than from regular ICSI.135

EMBRYO SELECTION: FINAL REMARKS Successful embryo selection is one of the best tools used in embryology for achieving high implantation rates. There are three types of selection, which are not exclusive. One is by selecting against morphologically and developmentally abnormal embryos on the basis of observations up to day three of development. On the basis of previous data on chromosome abnormalities, dysmorphism, abnormal development, and survival rates, it is possible to select embryos with higher implanting potential and lower risk of carrying chromosome abnormalities than others. According to that selection approach an ideal embryo for transfer would be one that develops from a regular size oocyte, resulting in a bipronucleated zygote with NPB distributed as pattern 0. During the first three days of development such embryo should not display multinucleation, but must cleave to about four cells by day 2 and eight cells by day 3, and do so without displaying type IV fragments or more than 15% fragmentation. Even with this selection, a third to a half of these embryos, depending on the maternal age, will still be chromosomally abnormal.7 A different selection approach involves culturing embryos up to six days in vitro. In doing so, it is assumed that many chromosomally and morphologically abnormal embryos will arrest at morula stage. Our preliminary data indicate that monosomies and some mosaics may arrest before reaching blastocyst, but trisomies, polyploidies, and some mosaics continue developing.68 Thus, negative selection of trisomic and other abnormal embryos can only be done through preimplantation genetic diagnosis (PGD). We proposed that PGD has the potential to increase implantation rates in women of advanced maternal age.5 So far more than 1000 cases of PGD of aneuploidy have been performed, either using embryo biopsy or polar body biopsy.5,7,112,137–140 Up to eight chromosome pairs (XY, 13, 14, 15, 16, 18, 21, and 22) can now be simultaneously analyzed in single cells with an efficiency of 90%.140 Recently different groups have reported a significant twofold increase in implantation rate111 and a 2.5-fold decrease in spontaneous abortions accompanied by an increase in the take-home baby rate.136 Thus, morphological and developmental criteria combined with PGD could produce significant increases in implantation rates and a reduction in spontaneous abortions. Now that most morphologic, developmental, and chromosomally abnormal types of embryos have been identified, the next step should be to ascertain, and if possible prevent, the action of agents causing chromosomal abnormalities, as well as embryonic dysmorphism and arrest. This may imply changes in hormonal stimulation to produce better

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matured oocytes; prevention of molecular organic and inorganic contamination using better air and water filters, as well as purer culture ingredients and products; and better selection of spermatozoa to avoid damaged centrioles producing mosaicism. Although most of these agents are not known or the anomalies cannot be identified without destroying the embryo, new methods are being proposed to prevent these processes in the gamete or embryo, such as cytoplasm donation.140

REFERENCES 1 Munné S, Cohen J. Chromosome abnormalities in human embryos. Hum Reprod Update (1998); 4:842–55. 2 Pellestor F, Dufour MC, Arnal F, Humeau C. Direct assessment of the rate of chromosomal abnormalities in grade IV human embryos produced by in-vitro fertilization procedure. Hum Reprod (1994); 9:293–302. 3 Santaló J, Veiga A, Calafell JM, et al. Evaluation of cytogenetic analysis for clinical preimplantation diagnosis. Fertil Steril (1995); 64:44–50. 4 Griffin DK, Wilton LJ, Handyside AH, Wiston RML, Delhanty JDA. Dual fluorescent in-situ hybridization for simultaneous detection of X and Y chromosome-specific probes for the sexing of human preimplantation embryonic nuclei. Hum Genet (1992); 89:18–22. 5 Munné S, Lee A, Rosenwaks Z, Grifo J, Cohen J. Diagnosis of major chromosome aneuploidies in human preimplantation embryos. Hum Reprod (1993); 8:2185–91. 6 Munné S, Grifo J, Cohen J, Weier HUG. Chromosome abnormalities in arrested human preimplantation embryos: a multiple probe fluorescence in-situ hybridization (FISH) study. Am J Hum Genet (1994); 55, 1:150–9. 7 Munné S, Alikani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmental rates and maternal age are correlated with chromosome abnormalities. Fertil Steril (1995); 64:382–91. 8 Munné S, Weier U. Simultaneous enumeration of chromosomes 13, 18, 21, X and Y in interphase cells for preimplantation genetic diagnosis of aneuploidy. Cytogenet Cell Genet (1996); 75:263–70. 9 Harper JC, Coonen E, Ramaekers FCS, 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. 10 Laverge H, De Sutter P, Verschraegen-Spae MR, De Paepe A, Dhont M. Triple colour fluorescent in-situ hybridization for chromosomes X, Y and 1 on spare human embryos. Hum Reprod (1997); 12:809–14. 11 Evsikov S, Verlinsky Y. Mosaicism in the inner cell mass of human blastocysts. Hum Reprod 11:3151–5.

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12 Munné S, Weier HUG, Grifo J, Cohen J. Chromosome mosaicism in human embryos. Biol Reprod (1994); 51:373–9. 13 Munné S, Alikani M, Grifo J, Cohen J. Monospermic polyploidy and atypical embryo morphology. Hum Reprod (1994); 9:506–10. 14 Kallioniemi A, Kallioniemi OP, Sudar D, et al. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science (1992); 258:818–21. 15 Von Eggeling F, Freytag M, Fashold R, Horsthemke B, Claussen U. Rapid detection of trisomy 21 by quantitative PCR. Hum Genet (1993); 1:567–70. 16 Wells D, Sherlock JK, Handyside AH, Delhanty DA. Detailed chromosomal and moleular genetic analysis of single cells by whole genome amplification and comparative genome hybridization. Nucleic Acid Res (1999); 27:1214–8. 17 Marquez C, Cohen J, Munné S. Chromosome identification on human oocytes and polar bodies by spectral karyotyping. Cytogenet Cell Genet (1998); 81:254–8. 18 Sluder G, Miller FJ, Lewis K, Davison ED, Rieder CL. Centrosome inheritance in starfish zygotes: Selective loss of the maternal centrosome after fertilization. Dev Biol (1989); 131:567–79. 19 Wilson EB, Mathews A. Maturation, fertilization and polarity in the echtoderm egg. J Morphol (1895) 10:319–42. 20 Schatten H, Schatten G, Mazia D, Balczon R, Simerly C. Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs. Proc Natl Acad Sci USA (1986); 83:105–9. 21 Sathananthan H, Kola I, Osborn J, Trounson A, Ng SC, Bongso A, Ratman SC. Centrioles in the beginning of human development. Proc Natl Acad Sci USA (1991); 88:4806–10. 22 Cohen J, Levron J, Palermo G, et al. Atypical activation and fertilization patterns in humans. Theriogenology (1995); 43:129–40. 23 Palermo G, Munné S, Cohen J. The human zygote inherits its mitotic potential from the male gamete. Hum Reprod (1994); 9:1220–5. 24 Plachot M. Chromosome analysis of oocytes and embryos. In: Verlinsky Y, Kuliev A, eds. Preimplantation genetics New York: Plenum Press (1991); 103–112. 25 Staessen C, Janssenwillen C, Devroey P, Van Steirteghem AC. Cytogenetic and morphological observations of Single pronucleated human oocytes after in-vitro fertilization. Hum Reprod (1993); 8:221– 3. 26 Sultan KM, Munné S, Palermo GD, Alikani M, Cohen J. Ploidy assessment of embryos derived from singlepronucleated human zygotes obtained by regular IVF and intra-cytoplasmic sperm injection (ICSI). Hum Reprod (1995); 10:132–6. 27 Staeessen C, Van Steirteghem AC. The chromosomal constitution of embryos developing from abnormally fertilized oocytes after

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intracytoplasmic sperm injection and conventional in-vitro fertilization . Hum Reprod (1997); 12:321–7. 28 Levron J, Munné S, Willadsen S, Rosenwaks Z, Cohen J. Male and female genomes associated in a single pronucleus in human zygotes. Biol Reprod (1995); 52:653–7. 29 Kola I, Trounson A, Dawson G, Rogers P. Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos. Biol Reprod (1987); 37:395–401. 30 Pellestor F. The cytogenetic analysis of human zygotes and preimplantation embryos. Hum Reprod Update (1995); 1:581–5. 31 Malter H, Cohen J. Embryonic development following microsurgical repair of polyspermic human zygotes. Fertil Steril (1989); 52:373–80. 32 Tang YX, Munné S, Reign A, et al. The parental origin of the distal pronucleus in dispermic human zygotes. Zygote (1994); 2:79–85. 33 Manor D, Kol S, Lewit N, et al. Undocumented embyros: do not trash them, FISH them. Hum Reprod (1996); 11:2502–6. 34 Sadowy S, Tomkin G, Munné S, Ferra-Congedo T, Cohen J. Impaired development of zygotes with uneven pronuclear size. Zygote (1998); 6:137–41. 35 Tesarik J, Greico E. The probability of abnormal preimplantation development can be predicted by a single static observation pronuclear stage morphology. Hum Reprod (1999); 14:1318–23. 36 Ogura A, Matsuda J, Yaganimachi R. Birth of normal young after electrofusion of mouse oocytes with round spermatids. Proc Natl Acad Sci USA (1994); 91:7460–2. 37 Tesarik J, Mendoza C. Spermatid injection into human oocytes. I. Laboratory techniques and special features of zygote development. Hum Reprod (1986); 11:772–9. 38 Schatten G. The centrosome and its mode of inheritance: the reduction of the centrosome during gametogenesis and its restoration during fertilization. Dev Biol (1994); 165:299–335. 39 Edwards RG, Beard HK. Oocyte polarity and cell determination in early mammalian embryos. Mol Hum Reprod (1997); 3:863–905. 40 Ebner T, Moser M, Yaman C, Feichtinger O, Hartl J, Tews G. 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. 41 Munné S, Magli C, Adler A, et al. Treatment-related chromosome abnormalities in human embryos. Hum Reprod (1997); 780–4. 42 Almeida PA, Bolton VN. The relationship between chromosomal abnormality in the human preimplantation embryo and development invitro. Reprod Fertil Devel (1996); 8:235–41. 43 Plachot M, Junca AM, Mandelbaum J, De Grouchy J, Salat-Baroux J, Cohen J. Chromosome investigations in early life II. Human preimplantation embryos. Hum Reprod (1987); 1:29–35.

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44 Pellestor F, Sele B. Assessment of aneuploidy in the human female by using Cytogenetics of IVF failure. Am J Hum Genet (1988); 42:274– 83. 45 Alikani M, Cohen J, Tomkin G, Garrisi J, Mack C, Scott RT. Human embryo fragmentation in vitro and its implications for pregnancy and implantation. Fertil Steril (1999); 71:836–42. 46 Balakier H, Cadesky K. The frequency and developmental capability of human embryos containing multinucleated blastomeres. Hum Reprod (1997); 12:800–4. 47 Jackson KV, Ginsburg ES, Hornstein D, Rein MS, Clarke RN. Multinucleation in normally fertilized embryos is associated with an accelerated ovulation induction response and lower implantation and pregnancy rates in in vitro fertilization-embryo transfer cycles. Fertil Steril (1998); 70:60–6. 48 Hardy K, Winston RML, Handyside AH. Binucleate blastomeres in preimplantation human embryos in vitro: failure of cytokinesis during early cleavage. J Reprod Fertil (1993); 98:549–58. 49 Forman R, Fries N, Testard J, Belaish-Allart J, Hazout A, Frydman R. Evidence for an adverse effect of elevated serum estradiol concentrations on embryo implantation. Fertil Steril (1988); 49:118– 22. 50 Pellicer A, Ruiz A, Castellvi RM, et al. Is the retrieval of high numbers of oocytes desirable in patients treated with gonadotropin-releasing hormone analogues (GnRHa) and gonadotropins? Hum Reprod (1989); 4:536–40. 51 Munné S, Cohen J. Unsuitability of multinucleated human blastomeres for preimplantation genetic diagnosis. Hum Reprod (1993); 8,7:1120– 5. 52 Kligman I, Benadiva C, Alikani M, Munné S. The presence of multinucleated blastomeres in human embryos correlates with chromosomal abnormalities. Hum Reprod (1996); 11:1492–8. 53 Staessen C, Van Steirteghem AC. The genetic constitution of multinucleated blastomeres and their derivative daughter blastomeres. Hum Reprod (1998); 13:1625–31. 54 Mohr LR, Trouson AO, Leeton JR, et al. Evaluation of normal and abnormal human embryo development during procedures in vitro. Beier HM, Lindner HR, eds. Fertilization of the human egg in vitro. Heidelberg: Springer Verlag (1983):211–22. 55 Pelinck MJ, De Vos M, Dekens M, Van der Elst J, De Sutter P, Dhont M. Embryos cultured in vitro with multinucleated blastomeres have poor implantation potential in human in-vitro fertilization and intracytoplasmic sperm injection. Hum Reprod (1998); 13:960–3. 56 Laverge H, Van der Elst J, De Sutter P, Verschraegen-Spae MR, De Paepe A, Dhont M. Fluorescent in-situ hybridization on human embryos showing cleavage arrest after freezing and thawing. Hum Reprod (1998); 13:425–9.

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57 Harper JC, Robinson F, Duffy S, et al. Detection of fertilization in embryos with accelerated cleavage by fluorescence in-situ hybridization (FISH). Hum Reprod (1994); 9:1733–7. 58 Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature (1988); 333:459–61. 59 Artley JK, Braude PR, Johnson MH. Gene activity and cleavage arrest in human pre-embryos. Hum Reprod (1992); 7:1014–21. 60 Delhanty JDA, Harper JC, Ao A, Handyside AH, Winston RML. Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients. Hum Genetics (1997); 99:755–60. 61 Harper JC, Coonen E, Handyside AH, Winston RML, Hopman AHN, Delhanty JDA. Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos. Prenatal Diagn (1995); 15:41–9. 62 Harper JC, Delhanty JDA. Detection of chromosomal abnormalities in human preimplantation embryos using FISH. JARG (1996); 13:137–9. 63 James RM, West JD. A chimeric animal model for confined placental mosaicism. Hum Genet (1994); 93:603–4. 64 Benkhalifa M, Janny L, Vye P, Malet P, Boucher D, Menezo Y. Assessment of polyploidy in human morulae and blastocysts using coculture and fluorescent in-situ hybridization. Hum Reprod (1993); 8:895–902. 65 Angell RR, Sumner AT, West JD, Thatcher SS, Glasier AF, Baird DT. Post-fertilization polyploidy in human preimplantation embryos fertilized in vitro. Hum Reprod (1987); 2:721–7. 66 Epstein CJ, Smith S, Cox DR. Production and properties of mouse trisomy 15 ← → diploid chimeras. Devel Genet (1984); 4:159–65. 67 Dyban AP, Baranov VS. Functional activity of Chromosomes and control mechanisms of early embryonic development. Cytogenetics of mammalian embryonic development. Oxford: Clarendon Press (1987):267–94. 68 Sandalinas M, Sadowy S, Calderon G, et al. Abstracts of the 16th Annual Meeting of the European Society of Human Reproduction, Bologna, Italy (in press). 69 Zhou H, Kuang J, Zhong L, et al. Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation. Nature Genet (1998); 20:189–93. 70 Doxsey S. The centrosome-a tiny organelle with big potential. Nature Genet (1998); 20:104–6. 71 Obasaju M, Kadam A, Sultan K, Fateh M, Munné S. Evidence that sperm quality may adversely affect the chromosome constitution of embryos resulting from ICSI. Fertil Steril (1999); 72:1113–50. 72 Van Blerkom J, Antezak J, Schrader R. The developmental potential of the human oocyte is related to the dissolved oxygen content of

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follicular fluid: association with vascular endothelial growth factor levels and perifollicular blood flow characteristics. Hum Reprod (1997); 12:1047–55. 73 Cohen J, Gilligan A, Esposito W, Schimmel T, Dale B. Ambient air and its potential effects on conception. Hum Reprod (1997); 12:1742– 9. 74 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. 75 Hassold T, Chiu D. Maternal age-specific rates of numerical chromosome abnormalities with special reference to trisomy. Hum Genet (1985); 70:11–7. 76 Warburton D, Kline J, Stein Z, Strobino B. Cytogenetic abnormalities in spontaneous abortions of recognized conceptions. In: Porter IH, Willey A, eds. Perinatal genetics: diagnosis and treatment. New York: Academic Press (1986):133–48. 77 Antonorakis SE, Lewis JG, Adelsberg PA, et al. Parental origin of the extra chromosome in trisomy 21 revisited: DNA polymorphism analysis suggests maternal origin in 95% of cases. N Engl J Med (1991); 324:872–6. 78 Fisher JM, Harvey JF, Morton NE, Jacobs PA. Trisomy 18: Studies of the parent and cell division of origin and the effect of aberrant recombination on nondisjunction. Am J Hum Genet (1995); 56:669–75. 79 Lamb N, Freeman S, Savage-Austin A, et al. Susceptible chiasmate configurations of chromosome 21 predispose to nondisjunction in both maternal meiosis I and meiosis II. Nat Genet (1996); 14:400–5. 80 Dailey T, Dale B, Cohen J, Munné S. Association between nondisjunction and maternal age in meiosis-II human oocytes detected by FISH analysis. Am J Hum Genet (1996); 59:176–84. 81 Snijders RJM, Holzgreve W, Cuckle H, Nicolaides KH. Maternal agespecific risks for trisomies at 9–14 weeks’ gestation. Prenat Diagn (1994); 14:543–52. 82 Watt JL, Templeton AA, Messinis I, Bell L, Cunningham P, Duncan RO. Trisomy 1 in an eight cell human preembryo. J Med Genet (1987); 24:60–4. 83 Bahçe M, Cohen J, Munne S. PGD of aneuploidy: were we looking at the wrong chromosomes? J Assist Reprod Genet (1999); 16:176–81. 84 Gardner D, Schoolcraft W, Wagley L, Schlenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in vitro fertilization. Hum Reprod (1998); 13:3434–40. 85 Clouston H, Fenwick J, Webb A, Herbert M, Murdoch A, Wolstenhome J. Detection of mosaic chromosome abnormalities in 6 to 8 day-old human blastocysts. Hum Genet (1997); 101:30–6. 86 Harper J, Ruangvutilert P, Marchat S, Delhanty J. High levels of mosaicism persist to the blastocyst stage. In: Abstracts of the 11th

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World Congress on In vitro fertilization and Human Reproductive Genetics, 9–14 May 1999. Sydney, Australia, 0–002, p. 62. 87 Bergers-Jansen JM, Derhaag JG, Ignoul-Vanvuchelen RCM, van Wissen LCP, Coonen E, Dumoulin JCM, Evers JLH. Inventory of chromosome abnormalities in human blastocysts using multiple probes and repeated fluorescence in-situ hybridization. Abstracts of the 15th Annual meeting of the ESHRE, Tours, France (1999). 88 Veiga A, Gil Y, Boada M, et al. Confirmation of diagnosis in preimplantation genetic diagnosis (PGD) through blastocyst culture: preliminary experience. Prenat Diagn (1999); 19:1242–7. 90 Long SE, Williams CV. A comparison of the chromosome complement of inner cell mass and trophoblast cells in day-10 pig embryos. J Reprod Fertil (1982); 66:645–8. 91 Iwasaki S, Hamano S, Kuwayama M, et al. Developmental changes in the incidence of chromosome anomalies of bovine embryos fertilized in vitro. J Exp Zool (1992); 261:79–85. 92 Barlow DH, Sherman SL. The biochemistry of differentiation of mouse trophoblast: studies on polyploidy. J Embryol Exp Morphol Apr (1972); 27:447–65. 93 Wilmut I, Schinieke AE, Mcwhir J, Kind AJ, Campbell KHS. Viable offsprings derived from fetal and adult mammalian cells. J Womens Health (1998); 475–6. 94 Mottla GL, Adelman MR, Hall JL, Gindoff PR, Stillman RJ, Johnson KE. Lineage tracing demonstrates that blastomeres of early cleavagestage human pre-embryos contribute to both trophectoderm and inner cell mass. Hum Reprod (1995); 10:384–91. 95 Delhanty JDA, Handyside AH. The origin of genetics defects in the human and their detection in the preimplantation embryo. Hum Reprod Update (1995); 1:201–15. 96 Janny L, Menezo YJ. Maternal age effect on early human embryonic development and blastocyst formation. Mol Reprod Dev (1996); 45:31–7. 97 Eiben B, Bartels I, Bahr-Porsch S, Borgmann S, et al. Cytogenetic analysis of 750 spontaneous abortions with the direct-preparation method of chorionic villi and its implications for studying genetic causes of pregnancy wastage. Am J Hum Genet (1990); 46:656–63. 98 Menezo YJ, Bellec V, Zaroukian A, Benkhalifa M. Embryo selection by IVF, co-culture and transfer at the blastocyst stage in case of translocation. Hum Reprod (1997); 12:2802–3. 99 Stephenson MD. Frequency of factors associated with habitual abortion in 197 couples. Fertil Steril (1996); 66:24–9. 100 Roman E. Fetal loss rates and their relation to pregnancy order. J Epidemiol Community Health (1984); 38:29–35. 101 Poland BJ, Miller JR, Jones DC, Trimple BK. Reproductive counselling in patients who have a spontaneous abortion. Am J Obstet Gynecol (1977); 127:685–91.

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102 Stray-Pedersen B, Stray-Pedersen S. Etiological factors and subsequent reproductive performance in 195 couples with a prior history of habitual abortion. Am J Obstet Gynecol (1984); 148:140–6. 103 Tulppala M, Palosuo T, Ramsay T, Miettinen A, Salonen R, Ylikorkala O. A prospective study of 63 couples with a history of recurrent spontaneous abortions: contributing factors and outcome of subsequent pregnancies. Hum Reprod (1993); 8:764–77. 104 Stern C, Pertile M, Norris H, Hale L, Baker HWG. Chromosome translocations in couples with in-vitro fertilization implantation failure. Hum Reprod (1999); 14:2097–101. 105 Strobino B, Fox HE, Kline J, Stein Z, Susser M, Warburton D. Characteristics of women with recurrent spontaneous abortion and women with favourable reproductive histories. Am J Public Health (1986); 76:986–91. 106 De Braekeleer M, Dao TN. Cytogenetic studies in couples experiencing repeated pregnancy losses. Hum Reprod (1990); 5:519– 28. 107 Daniely M, Aviram-Goldring A, Barkai G, Goldman B. Detection of chromosomal aberrations in fetuses arising from recurrent spontaneous abortions by comparative genome hybridization. Hum Reprod (1998); 13:805–9. 108 Kiefer D, Check JH, Katsoff D. Evidence that oligoasthenozoospermia may be an etiologic factor for spontaneous abortion after in vitro fertilization-embryo transfer. Fertil Steril (1997); 68:545–8. 109 Simon C, Rubio C, Vidal F, Moreno C, Parrilla JJ, Pellicer A. Increased chromosome abnormalities in human preimplantation embryos after in-vitro fertilization in patients with recurrent miscarriages. Reprod Fertil Devel (1998); 10:987–92. 110 Pellicer A, Rubio C, Vidal F, et al. In vitro fertilization plus preimplantation genetic diagnosis in patients with recurrent miscarriages: an analysis of chromosome abnormalities in human preimplantation embryos. Fertil Steril (1999); 71:1033–9. 111 Gianaroli L, Magli C, Ferraretti AP, Munne 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. 112 Khalifa E, Toner JP, Muasher SJ, Acosta AA. Significance of basal follicle-stimulating hormone levels in women with one ovary in a program of in vitro fertilization. Fertil Steril (1992); 57:835–9. 113 Backer LC, Rubin CS, Marcus M, Kieszak SM, Schober SE. Serum follicle-stimulating hormone and luteinizing hormone levels in women aged 35–60 in the US population: the third national health and nutrition examination survey (NHANES III, 1988–1994). Menopause (1999); 6 (1):29–35.

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114 Lass A. The fertility potential of women with a single ovary. Hum Reprod Updates (1999); 5:546–50. 115 Warburton D. The effect of maternal age on the frequency of trisomy: Change in meiosis or in utero selection? Prog Clin Biol Res (1989); 311:165–81. 116 Brook JD, Gosden RG, Chandley AC. Maternal ageing and aneuploid embryos: Evidence from the mouse that biological and not chronological age is the important influence. Hum Genet (1984); 66:41–5. 117 Freeman SB, Yang Q, Allran K, Taft LF, Sherman SL. Women with a reduced ovarian complement may have an increased risk for a child with Down syndrome. Am J Hum Genet (2000); 66:1680–3. 118 Van Montfrans JM, Dorland M, Oosterhuis GJE, Van Vugt JGM, Rekers-Mombarg LTM, Lambalk CB. Increased concentrations of follicle-stimulating hormone in mothers of children with Down’s syndrome. Lancet (1999); 353:1853–4. 119 Nasseri A, Mukherjee T, Grifo JA, Noyes N, Krey L, Copperman AB. Elevated day 3 serum follicle stimulating hormone and/or estradiol may predict fetal aneuploidy. Fertil Steril (1999); 71:715–8. 120 Munné S, Marquez C, Reing A, Garrisi J, Alikani M. Chromosome abnormalities in embryos obtained following conventional IVF and ICSI. Fertil Steril (1998); 69:904–8. 121 Bonduelle M, Legein J, Buysse A, et al. Prospective follow-up study of 423 children born after intracytoplasmic sperm injection . Hum Reprod (1996); 11:1558–64. 122 In’t Veld P, Brandenburg H, Verhoeff A, Dhont M, Los F. Sex chromosomal abnormalities and intracytoplasmatic sperm injection. Lancet (1995); 346:773. 123 Bonduelle M, Legein J, Derde MP, et al. Comparative follow up study of 130 children born after intracytoplasmic sperm injection and 130 children born after in-vitro fertilization. Hum Reprod (1985); 10:3227–331. 124 Tournaye H, Liu J, Nagy Z, et al. Intracytoplasmic sperm injection (ICSI): The Brussels experience. Reprod Fertil Dev (1995); 7:269–79. 125 Liebaers I, Bonduelle M, Van Assche E, Devroey P, Van Steirteghem A. Sex chromosome abnormalities after intracytoplasmic sperm injection. Lancet (1995); 346:1095. 126 Persson JW, Peters GB, Saunders DM. Genetic consequences of ICSI. Is ICSI associated with risks of genetic disease? Implications for counselling, practice and research. Hum Reprod (1996); 11:921–32. 127 Benet J, Martin R. Sperm chromosome complements in a 47XYY man. Hum Genet (1988); 78:313–15. 128 Han T, Ford J, Flaherty S, et al. A fluorescent in situ hybridization analysis of the chromosome constitution of ejaculated sperm in a 47,XYY male. Clin Genet (1994); 45:67–70.

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129 Cozzi J, Chevret E, Rousseaux S, et al. Achievement of meiosis in XXY germ cells: study of 543 sperm karyotypes from an XY/XXY mosaic patient. Hum Genet (1994); 93 (1):32–4. 130 Blanco J, Rubio C, Simon C, et al. A fluorescent in-situ hybridization study of the spermatogenic process and sperm production in a 47XYY and a 47XXY/46XY male: implications for ICSI. Hum Reprod (1996); 11:159–60. 131 Chevret E, Rousseaux R, Monteil R, et al. Increased incidence of hyperhaploid 24,XY spermatozoa detected by three-colour FISH in a 46,XY/47,XXY male. Hum Genet (1996); 93:32–4. 132 Martini E, Geraedts JPM, Liebaers I, et al. Constitution of semen samples from XYY and XXY males as analysed by in-situ hybridization. Hum Reprod (1996); 11:1638–43. 133 Egozcue J, Templado C, Vidal F, Navarro J, Morer-Fargas F, Marina S. Meiotic studies in a series of 1100 infertile and sterile males. Hum Genet (1983); 65:185–8. 134 Martin R. The risk of chromosomal abnormalities following ICSI. Hum Reprod (1996); 11:924–5. 135 Munné S, Magli C, Wamsley R, Ferrareti AP, Sadowy S, Gianaroli L. Chromosome analysis of embryos from TESA and MESA. Fertil Steril (2000); Suppl. (abstract in press.) 136 Munné S, Magli C, Cohen J. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum Reprod (1999); 14:2191–9. 137 Verlinsky Y, Cieslak J, Frieidine M, et al. Pregnancies following preconception diagnosis of common aneuploidies by fluorescence in-situ hybridization. Hum Reprod (1995); 10:1923–7. 138 Verlinsky Y, Cieslak J, Ivakhnenko V, Lifchez A, Strom C, Kuliev A, 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. 139 Verlinsky Y, Kuliev A. Polar body preimplantation diagnosis. Proc. of the 15th annual meeting of ESHRE. Precongress course embryology and genetics. Tours, France. 47–54. 140 Munné S, Magli C, Bahçe 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. Prenatal Diagn (1998); 18:1459–66. 141 Cohen J, Scott R, Alikani M, et al. Ooplasmic transfer in mature human oocytes. Molecular Hum Reprod (1998); 4:269–80.

26 Genetic analysis of the embryo Yuval Yaron, Ronni Gamzu, Mira Malcov

INTRODUCTION For couples at risk of transmitting a genetic defect, preimplantation genetic diagnosis (PGD) and transfer of only 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 PCR to amplify Y-chromosome specific sequences, and only embryos determined to be females were transferred.1 During the last decade, the range of genetic abnormalities that can be detected by PGD has increased enormously. It is expected that with the completion of the Human Genome Project, this number will increase even further. PGD is still riddled with technical difficulties including the limiting amounts of genetic material, the inherent pitfalls including amplification failure, allele dropout (ADO), and contamination. There is also a rather narrow window of opportunity to perform diagnosis within hours to enable embryo transfer without jeopardizing pregnancy rates. To overcome these difficulties, new strategies have been developed, as will be outlined in this chapter.

BASIC PRINCIPLES OF PGD POLYMERASE CHAIN REACTION Single cell molecular analysis for PGD was made possible by the polymerase chain reaction (PCR), first introduced in the mid-80s. 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

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nucleotides (dNTPs) are added in succession to recreate a double stranded 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 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 a result of 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. Owing to these limitations, when the number of initial DNA template molecules is limited, as in a single cell PGD analysis, the quantity of amplified DNA may be insufficient for a complete molecular analysis. The two-step, 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 since the nested primers anneal to sites that have not been eroded. This technique also decreases the rate of non-specific 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.2,3 There seems to be an association

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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.4,5 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. 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 (ADO) 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.6 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 amplifications, or none of the cells.5,7–12 The causes of ADO are still not fully understood. Current hypotheses include imperfect denaturing temperature, incomplete cell lysis, and DNA degradation prior to PCR. Ray and Handyside showed that an increase in denaturing temperature from 90°C to 96°C during PCR may be associated with a fourfold reduction in ADO at the cystic fibrosis locus and an 11fold reduction at the beta-globin locus.8 The use of alkaline lysis buffer or lysis buffer containing proteinase K and detergent have also been suggested to reduce ADO.8,13 Degenerated and apoptotic cells show increased ADO probably as a result of partial degradation of the DNA strands. It has been suggested that ADO is higher in blastomeres than in other cell types.10 This may be explained at least partly, by the higher rate of haploidity of blastomeres.14 In cases of diagnosis of dominant disorders or recessive diseases when both parents 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

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minimally amplified allele, resulting in ADO.7–11–15 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.16 In the setting of PGD, there may be three main sources for possible contamination. First, paternal genome contamination may rise 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 from either 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.

ADVANCED MOLECULAR METHODS FOR PGD MULTIPLE PCR Multiplex PCR refers to the simultaneous amplification of more than one fragment in the same PCR reaction by using more than one pair of unrelated primers.7,10,11,17 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 more than four different loci within the same multiplex PCR reaction have been reported in single cells.7,11 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

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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.17 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.9,10,18,19 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.19,20 The probability of ADO affecting both mutation site and the linked polymorphic site are very low and thus the mutant allele is more likely to be detected. Such strategies have been reported for cystic fibrosis,10,21 beta-thalassemia,19 and others. 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 comparatively similar size nor 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 called 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, digitally amplified, and analyzed by a computer. With this technique, it is possible to separate, detect and analyze the fluorescent labeled PCR products with sensitivity 1000 times greater than that achieved using conventional methods.22 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 primers 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.7,11,15 In addition, since the detection efficiency is several magnitudes higher, fewer PCR cycles are required, thereby reducing the risk of contamination playing a significant role. Moreover, since fewer cycles are needed, less time is required for the complete analysis. Using this approach, Sermon et al have successfully reduced the rate of ADO by a factor of four in the

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diagnosis of myotonic dystrophy,15 and Findlay et al reported an accurate diagnosis in as much as 97% of the cases.7 QUANTITATIVE FLUORESCENT PCR Quantitative fluorescent (OF) PCR provides information on the ploidity of the cell.23 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 different sized alleles. During the initial exponential phase of PCR amplification, the amount of DNA product is proportional to the original number of repeats.24 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).23 This method has been successfully used in prenatal diagnosis of aneuploidy.25 In PGD however, OF PCR is only reliable in identifying triallelic trisomies, since the interpretation of di-allelic trisomies is problematic because of the possibility of preferential amplification.11 REVERSE TRANSCRIPTION (RT) PCR The high number of mRNA transcripts of a single allele in a single cell may also provide a theoretical solution for ADO. By using reverse transcription of mRNA molecules present in the cell it is possible to obtain numerous cDNA copies of a specific allele. Thus, RT PCR followed by mutation analysis has been suggested to reduce both amplification failures and ADO.26 However, since the embryonic genome is activated only at the 4–8 blastomere stage, this method may be applied only for genes that are already expressed prior to the biopsy. Additionally, genomic imprinting and residual transcripts derived from the oocyte may also result in misdiagnosis. WHOLE GENOME AMPLIFICATION (WGA) 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—whole genome amplification (WGA)—have been developed.16,27 These techniques amplify a great 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.

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PRIMER EXTENSION PREAMPLIFICATION (PEP) Primer extension preamplification (PEP) is a WGA method designed mainly for single cells. Using random sequence primers of 15 bp it has been claimed to amplify at least 70% of the genome in more than 30 copies.27 This however, is likely to be a rather conservative estimate since Paunio et al reported that PEP yields at least 1000 copies of the genome, and Wells et al have suggested that more than of 90% of genomic sequences are represented in PEP amplification products.16,28 One of the drawbacks of PEP is the time required, which is usually more than 12 hours. Sermon et al have successfully adopted a modified protocol that requires less than six hours, and Tsai et al have improved the efficiency by further technical modifications.29,30 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 and familial adenomatous polyposis coli.9,31,32 DEGENERATE OLIGONUCLEOTIDE PRIMED PCR (DOP-PCR) A second form of WGA called degenerate oligonucleotide primed PCR (DOP-PCR), has been recently applied to PGD.16,33 DOP-PCR amplifies a similar proportion of the genome as does PEP, but reportedly to a greater extent, providing sufficient DNA for over 100 subsequent PCR amplifications,16 or for other analytical procedures such as comparative genomic hybridization (CGH). It has been shown that using a combination of DOP-PCR, CGH, and OF PCR it is possible to determine the copy number of each chromosome and conduct various molecular studies on single cells and blastomeres.16,34 Some of the inherent problems with PGD are not overcome by WGA, and ADO rates after PEP or DOP-PCR do not differ significantly from methods using direct amplification of single cells. Yet, whole genome amplification using PEP or DOP-PCR seems to be promising in light of the recent development of CGH and DNA microarrays (chips). Whole genome amplification would enable the generation of sufficient DNA to conduct complete chromosomal analysis using by CGH and “whole genome mutation screen” by DNA microarrays on a single cell.35 MICROSATELLITES AND OTHER POLYMORPHIC MARKERS Multiplex PCR and whole genome amplification allow both the analysis of the tested gene for mutation, as well as analyzing polymorphic genetic markers such as short tandem repeats (STRs), also known as microsatellites, in a process referred to as “DNA fingerprinting”. This technique is useful for ruling out contamination from various sources

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described earlier, and thus improve reliability of the diagnosis. The amplification of one or more highly polymorphic STRs allows the determination of the source of DNA amplified.36 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.7,11,36 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 PGD37 and Duchenne muscular dystrophy. In the latter, only 60% of patients exhibit detectable large scale deletions in the dystrophin gene. Since it is the largest known human gene, spanning more than 2 million bp, it is often impossible to detect small deletions or point mutations.38 Linkage analysis has also been suggested for the diagnosis of disease with large tri-nucleotide repeat expansions, such as fragile X and myotonic dystrophy.15,39 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.40 CELL RECYCLING In the cell recycling method, single cells are fixed to a slide or “dipstick”, and subjected to sequential PCR or fluorescent in situ hybridization (FISH) analysis.41 This method allows sequential analysis of specific genes of interest and STRs as discussed earlier. Both molecular and cytogenetic results may be successfully obtained from 65–85% of cells.41,42 However, ADO rates are significantly higher, and accuracy is lower than in other methods.42,43 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 spinal muscular atrophy, the actual PCR amplification reaction is sufficient for making a diagnosis since it is based on the lack of

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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, denaturant gradient gel electrophoresis and others. These methods are based on the fact the normal DNA strands, mutant DNA strands, and various combinations thereof, often have varying electrophoretic migratory properties under different conditions, allowing to distinguish between them. These techniques often help in scanning for a mutation in diseases that are caused by numerous different mutations. Although PGD employing these techniques has been reported in conditions such as beta-thalassemia,44–46 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 provide bona fide evidence of the mutation, and also facilitates the development of direct diagnostic techniques such as restriction endonuclease digestion of DNA or amplification refractory mutation system. RESTRICTION ENDONUCLEASE DIGESTION Alteration in the DNA sequence caused by mutations, may often lead to a creation or abolition of specific restriction endonucleases 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. After 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 Haelll. This allows sex determination to be performed more accurately than on the basis of the presence or lack of amplification of the Y-chromosome specific SRY gene.

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AMPLIFICATION REFRACTORY MUTATION SYSTEM The amplification refractory mutation system employs three primers in the PCR reaction: a common primer that anneals upstream of the mutation site and two other primers, which differ slightly, each specific for either the normal or mutant alleles. The site specific primers may be designed to vary in length, to contain a restriction site, or are tagged by different fluorescent markers.11 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. By 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.47 DNA MICROARRAY TECHNOLOGY DNA microarrays or “chips” allow the simultaneous detection of up to thousands of different polymorphism or mutations in defined genes. Numerous oligonucleotide probes (usually 20–25bp) are arrayed in microscopic predefined regions on a solid surface such as a thumbnail sized glass slide. The probes are complementary to known mutations in defined genes or single nucleotide polymorphisms throughout the genome. The microarray is hybridized with a fluorescent labeled 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.

LABORATORY TECHNIQUES IN PGD 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, and 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 will be 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

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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, lab technicians should wear disposable outer clothings, caps, masks, shoe covers, and powder free gloves, that 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 and DNAse-free, sterilized by autoclaving, filtered through a 0.22µ filter and by UV irradiation. All reagents should be prepared in a 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 pipetting and sampling 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, intracytoplasmic sperm injection (ICSI) is employed. THE 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 owing to ADO, it is possible to biopsy and analyze two blastomeres from the same embryo.48 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 PBS. The suspension is centrifuged at 7.5g for five 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.49,50,51

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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 pulled glass micropipette under a stereo-microscope.28 Lymphocytes may be isolated from peripheral blood by the FicollPaque method, washed three times in PBS, resuspended, and diluted in culture medium on a glass slide. Individual cells are then selected using pulled glass micropipette under an inverted microscope, washed three times in PCR buffer (50mM KCl, 10mM Tris-HCl pH8.3) 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 20µl of concentrated lymphocytes are added to 40µl of fetal calf serum, 120µl of RPMI medium and 20µl of dimethyl sulphoxide (DMSO) and kept in liquid nitrogen until required. Cells can be stored for up to a year. Thawing is performed by several washes with culture medium.52 A lymphoblast cell-line carrying the known mutation is probably the best choice, since its establishment provide 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.53 Once the cell line is established, single cells may be aspirated and transferred to 1.5ml Eppendorf tubes, washed three times with PBS, resuspended in 50µl PBS and kept at 4°C until use.54 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 (50mM KCl, 10mM TrisHCl pH 8.3) supplemented with 0.01% polyvinylpyrrolidone (PVP) or 4mg/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.7,8,39,52,55–57 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, efficiency and reliability of PGD.13 Among the several

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options, the 3 most commonly used lysis solutions are water, alkaline lysis buffer, and proteinase K/SDS buffer. There is 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 60µl of biotechnology grade water. An aliquot from the last washing droplet is added to a PCR tube containing 60µl 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.7,58 Proteinase K/SDS: Single blastomeres are washed three times in PBS or PCR buffers supplemented with PVP or BSA and transferred individually to PCR tubes containing K/SDS buffer: 17µM sodium dudecyl sulphate (SDS) and 125ng/µl proteinase K. Cells are incubated at37°C for 1 hour followed by denaturation at 99°C for 15 minutes to inactivate the enzyme.44,50,51 A modification of this procedure, includes raising the proteinase K concentration to 400 ng/µl, elevating the proteolysis temperature to 50°C, and using 0.5% Tween-20 instead of SDS.63 Lysates can be stored at −80°C until used. Alkaline lysis buffer: Single cells are transferred as above to PCR tubes containing 5µl alkaline lysis buffer (ALB- 200mM KOH, 50mM 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.52,57,59,60 After lysis, 5µl neutralization buffer (300mM KCl, 900mM Tris-HCl pH8.3, 200mM HCl) is added. Lysates are centrifuged briefly and placed on ice for immediate use or stored at −20°C until use.61 PRIMARY AND NESTED PCR CONDITIONS For the primary PCR reaction, the following are mixed with the biopsied cell lysate to a final volume of 50µl: PCR buffer (10mM Tris-HCl, 50mM potassium chloride and 2.5mM MgCl2 pH 8.3), 0.3mM dNTP, 1–2 U Taq polymerase, and 0.5mM outer primers. It is recommended to perform optimization of the reaction by using 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)SO4 or detergent, and by the choice of a mixture of Taq and proof-reading polymerases. 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.59 This is followed by 30 cycles of denaturation at 94°C for 1 minute, annealing at 52–65°C (according to the primers melting temperature) for 1 minute, and extension at 72°C for 1

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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, 2–5µl of the primary PCR product serves as a 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 requested for this step. Other reagents and PCR conditions may be similar to those used in the primary PCR reaction.60,62 MULTIPLEX PCR According to the standard protocol, each 50µl reaction includes 1–1.5 units of Taq polymerase, 0.3mM for each dNTP, 0.5–2.5mM MgCl2 and 0.1–0.5mM of each primer. The reaction 10×PCR buffer is usually composed of 500mM KCl, 100mM Tris-HCl pH 8.3 but at least one of the following ingredients is usually added: glycerol, gelatin, betain, DMSO, (NH4)SO4 and detergent. The PCR thermocycler program begins with a prolonged initial denaturation at 96°C for 5 minutes (ensuring appropriate accessibility to the DNA strands). This is followed by a 30 cycle of 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 2–5µl aliquot of the product serves as a DNA template for a nested-PCR reaction. PRIMER EXTENSION PREAMPLIFICATION (PEP) 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 60µl includes: 33mM random primers, 10×PCR buffer (100mM Tris-HCl pH 8.3, 25mM MgCl2, 1mg/ml gelatin and 500mM KCl), 0.1mM 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 sec/°C until 55°C, and extension at 55°C for 4 minutes.27,32 Improvement of amplification can be achieved by raising the denaturation temperature, elongating the denaturation period, raising the pH buffer from 8.3 to 8.8, modifying the MgCl2 and gelatin concentrations, reducing the KCl concentration, using a more thermostable DNA polymerase, and one that has minimal exonuclease activity. Addition of glycerol, betain, BSA, detergents, spermidine and (NH4)SO4 may also improve the products yield. Primers should be dissolved in Tris-HCl 5–10mM pH8.3 and not in TE buffer to prevent the chelation of Mg++ ions by EDTA. The PEP-PCR product should produce an even smear on ethidium bromide gel

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electrophoresis. A 2–10µl 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.28,35,63 DEGENERATED OLIGONUCLEOTIDE PRIMED PCR (DOP-PCR) DOP-PCR is based on multiple rounds of extensions using a universal primer containing a 6bp degenerate region representing all possible nucleotide combinations, flanked with a GC-rich short sequence to improve hybridization to genomic DNA. DOP-PCR reaction mixture in a final volume of 100µl contains 2.0mM degenerated primers, 10×PCR buffer (100mM Tris-HCl pH8.3, 25mM MgCl2, and 500mM KCl). However, the buffer should be K+ free if the cell was lysed by alkaline lysis buffer), 0.2mM dNTPs and 2.5 U of Taq polymerase.35 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.35 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. FLUORESCENT PCR Fluorescent PCR is performed in a final volume of 25µl of 10×PCR buffer containing 15mM MgCl2, 0.2mM of each dNTP, and fluorescent-tagged primers at a final concentration of 0.05mM. After a “hot start,” 0.6–1.5U 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 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.11,47,64 RESTRICTION ENZYME DIGESTION For each different restriction enzyme different conditions, such as buffer, temperature and time, 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 law 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, resolved by electrophoresis on agarose or acrylamide gels.

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PRODUCTS DETECTION Ethidium bromide gel electrophoresis: An aliquot of the PCR products is applied to 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 not sensitive nor accurate because it does not detect PCR products if the amplification yield is low, nor does it allow distinguishing between alleles differing in length by a few bp. GeneScan: following fluorescent PCR, size separation is performed on an acrylamide gel or by 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 1–2bp. ACKNOWLEDGEMENT We would like to thank Dr Dalit Ben-Yosef for her assistance in the preparation of this chapter.

REFERENCES 1 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. 2 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); 10:2214–7. 3 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. 4 Cui KH, Matthews CD. Nuclear structural conditions and PCR amplification in human preimplantation diagnosis. Mol Hum Reprod (1996); 2:63–71. 5 Ray PF, Ao A, Taylor DM, Winston RM, Handyside AH. Assessment of the reliability of single blastomere analysis for preimplantation diagnosis of the delta F508 deletion causing cystic fibrosis in clinical practice. Prenat Diagn (1998); 18:1402–12.

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6 Grifo JA, Tang YX, Munne S, Alikani M, Cohen J, Rosenwaks Z. Healthy deliveries from biopsied human embryos. Hum Reprod (1994); 9:912–6. 7 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. 8 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). 9 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. 10 Rechitsky S, Strom C, Verlinsky O, et al. Allele dropout in polar bodies and blastomeres. J Assist Reprod Genet (1998); 15:253–7. 11 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. 12 Dreesen JC, Bras M, de Die-Smulders C, et al. Preimplantation genetic diagnosis of spinal muscular atrophy. Mol Hum Reprod (1998); 4:881– 5. 13 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. 14 Harper JC, Coonen E, Handyside AH, Winston RM, Hopman AH, Delhanty JD. Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos. Prenat Diagn (1995); 15:41–9. 15 Sermon K, De Vos A, Van de Velde II, 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. 16 Wells D, Sherlock JK. Strategies for preimplantation genetic diagnosis of single gene disorders by DNA amplification. Prenat Diagn (1998); 18:1389–401. 17 Eggerding FA. A one-step coupled amplification and oligonucleotide ligation procedure for multiplex genetic typing. PCR Methods Appl (1995); 4:337–45. 18 Blake D, Tan SL, Ao A. Assessment of multiplex fluorescent PCR for screening single cells for trisomy 21 and single gene defects. Mol Hum Reprod (1999); 5:1166–75. 19 Kuliev A, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis of thalassemias. J Assist Reprod Genet (1998); 15:219–25.

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20 Xu K, Shi ZM, Veeck LL, Hughes MR, Rosenwaks Z. First unaffected pregnancy using preimplantation genetic diagnosis for sickle cell anemia. JAMA (1999); 281:1701–6. 21 Dreesen JC, Jacobs LJ, Bras M, et al. Multiplex PCR of polymorphic markers flanking the CFTR gene; a general approach for preimplantation genetic diagnosis of cystic fibrosis. Mol Hum Reprod (2000); 6:391–6. 22 Hattori M, Yoshioka K, Sakaki Y. High-sensitive fluorescent DNA sequencing and its application for detection and mass-screening of point mutations. Electrophoresis (1992); 13:560–5. 23 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. 24 Ferre F. Quantitative or semi-quantitative PCR: reality versus myth. PCR Methods Appl (1992); 2:1–9. 25 Verma L, Macdonald F, Leedham P, McConachie M, Dhanjal S, Hulten M. Rapid and simple prenatal DNA diagnosis of Down’s syndrome. Lancet (1998); 352:9–12. 26 Eldadah ZA, Grifo JA, Dietz HC. Marfan syndrome as a paradigm for transcript-targeted preimplantation diagnosis of heterozygous mutations. Nat Med (1995); 1:798–803. 27 Zhang I, Cui X, Schmitt K, Hubert R, Navidi W, Arnheim N. Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci USA (1992); 89:5847–51. 28 Paunio T, Reima I, Syvanen AC. Preimplantation diagnosis by wholegenome amplification, PCR amplification, and solid-phase minisequencing of blastomere DNA. Clin Chem (1996); 42:1382–90. 29 Sermon K, Lissens W, Joris H, Van Steirteghem A, Liebaers I. Adaptation of the primer extension preamplification (PEP) reaction for preimplantation diagnosis: single blastomere analysis using short PEP protocols. Mol Hum Reprod (1996); 2:209–12. 30 Tsai YH. Cost-effective one-step PCR amplification of cystic fibrosis delta F508 fragment in a single cell for preimplantation genetic diagnosis. Prenat Diagn (1999); 19:1048–51. 31 Snabes MC, Chong SS, Subramanian SB, Kristjansson K, DiSepio D, Hughes MR. Preimplantation single-cell analysis of multiple genetic loci by whole-genome amplification. Proc Natl Acad Sci USA (1994); 91:6181–5. 32 Kristjansson K, Chong SS, Van den Veyver IB, Subramanian S, Snabes MC, Hughes MR. Preimplantation single cell analyses of dystrophin gene deletions using whole genome amplification. Nat Genet (1994); 6:19–23. 33 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.

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34 Voullaire L, Wilton L, Slater H, Williamson R. Detection of aneuploidy in single cells using comparative genomic hybridization. Prenat Diagn (1999); 19:846–51. 35 Wells D, Sherlock JK, Handyside AH, Delhanty JD. Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genomic hybridization . Nucleic Acids Res (1999); 27:1214–8. 36 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. 37 Harton GL, Tsipouras P, Sisson ME, et al. Preimplantation genetic testing for Marfan Syndrome. Mol Hum Reprod (1996); 2:713–5. 38 Lee SH, Kwak IP, Cha KE, Park SE, Kim NK, Cha KY. Preimplantation diagnosis of non-deletion Duchenne muscular dystrophy (DMD) by linkage polymerase chain reaction analysis. Mol Hum Reprod (1998); 4:345–9. 39 Sermon K, Lissens W, Joris H, et al. Clinical application of preimplantation diagnosis for myotonic dystrophy. Prenat Diagn (1997); 17:925–32. 40 Daniels R, Holding C, Kontogianni E, Monk M. Single-cell analysis of unstable genes. J Assist Reprod Genet (1996); 13:163–9. 41 Thornhill AR, Monk M. Cell recycling of a single human cell for preimplantation diagnosis of X-linked disease and dual sex determination. Mol Hum Reprod (1996); 2:285–9. 42 Rechitsky S, Freidine M, Verlinsky Y, Strom CM. Allele dropout in sequential PCR and FISH analysis of single cells (cell recycling). J Assist Reprod Genet (1996); 13:115–24. 43 He ZY, Liu HC, Mele CA. Veeck LL, Davis O, Rosenwaks Z. Recycling of a single human blastomere fixed on a microscopic slide for sexing and diagnosis of specific mutations by various types of polymerase chain reaction. Fertil Steril (1999); 72:341–8. 44 El-Hashemite N, Wells D, Delhanty JD. Single cell detection of betathalassaemia mutations using silver stained SSCP analysis: an application for preimplantation diagnosis. Mol Hum Reprod (1997); 3:693–8. 45 Vrettou C, Palmer G, Kanavakis E, et al. A widely applicable strategy for single cell genotyping of betathalassaemia mutations using DGGE analysis: application to preimplantation genetic diagnosis. Prenat Diagn (1999); 19:1209–16. 46 Kanavakis E, Vrettou C, Palmer G, Tzetis M, Mastrominas M, Traeger-Synodinos J. Preimplantation genetic diagnosis in 10 couples at risk for transmitting beta-thalassaemia major: clinical experience including the initiation of six singleton pregnancies. Prenat Diagn (1999); 19:1217–22.

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47 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. 48 Findlay I, Quirke P. Fluorescent polymerase chain reaction. Part I. A new method allowing genetic diagnosis and DNA fingerprinting of single cells. Human Reprod Update (1996); 2:137–52. 49 Findlay I, Lilford R. Sources and detection of contamination in preimplantation diagnosis. Proceedings of the XII Annual Scientific Meeting of the Fertility Society of Australia (1994): (abstract 101). 50 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); 30:903–09. 51 loulianos A, Dagan W, Harper JC and Delhanty JDA. A successful strategy for preimplantation diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. Prenat Diagn (2000); 20:593–98. 52 Hussey ND, Dongguil H, Froiland DAH, 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. 53 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. 54 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. 55 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. 56 Ao A, Handyside AH. Cleavage stage human embryo biopsy. Hum Reprod Update (1995); 1:3. 57 De Vos A, Sermon K, Van de Velde H, et al. Pregnancy after preimplantation genetic diagnosis for Charcot-MarieTooth disease type 1A. Mol Hum Reprod (1998); 4:978–84. 58 Gibbons WE, Gitlin SA, Lanzendorf SE, Kaufmann RA, Slotnick RN, Hodgen GDH. Preimplantation genetic diagnosis for Tay-Sachs disease: successful pregnancy after pre-embryo biopsy and gene amplification by polymerase chain reaction. Fertil Steril (1995); 63:723–28. 59 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. 60 Van den Veyver IB, Chong SS, Cota J, et al. Single-cell analysis of the RhD blood type for use in preimplantation diagnosis in the prevention

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of severe hemolytic disease of the newborn. Am J Obstet Gynecol (1995); 172:533–40. 61 Cui XF, Li HH, Goradia TM, et al. Single-sperm typing: determination of genetic distance between the G gammaglobin and parathyroid hormone loci by using the polymerase chain reaction and allelespecific oligomers. Proc Natl Acad Sci USA (1989); 80:9389–93. 62 Cui KH, Haan EA, Wang LJ, Matthews CD. Optimal polymerase chain reaction amplification for preamplification diagnosis in cystic fibrosis ( F508). BMJ (1995); 311:536–40. 63 Hahn S, Zhong XY, Troeger C, Burgemeister R, Gloning K, Holzgreve W. Current application of single-cell PCR. Cell Mul Life Sci (2000); 57:96–105. 64 Findlay I, Quirke P, Hall J, Rutherford A. Fluorescent PCR: A new technique for PGD of sex and single-gene defects. J Assist Reprod Genetics (1996); 13:96–103.

27 Polar body biopsy Yury Verlinsky, Anver Kuliev

INTRODUCTION Polar bodies (PB) are the byproducts of female meiosis, extruded during maturation and fertilization of oocytes. The first PB (PB1) is extruded as a result of the first meiotic division, while the second PB (PB2) is the outcome of the second meiotic division. Because neither PB1 nor PB2 have biological significance in pre-implantation and postimplantation development they may be removed and analyzed to investigate the genetic normality of the corresponding oocytes. It is, of course, possible to obtain the genotype of an oocyte by direct analysis, but this analysis will destroy the oocyte. Although the information about genotypes may also be inferred from the materials secreted into culture media, so far such attempts for non-invasive testing of oocytes or embryos before transfer have not proved successful. A number of ovarian markers have been found to be useful for predicting developmental potential of oocytes, including perifollicular vascularity, vascular endothelial growth factors, expression of some enzymes by granulosa cells in vitro, adhesion and proliferation, and steroidogenic activity of cumulus cells.1,2 However, the relation of these factors with the genotype of the oocyte and their clinical relevance for the preselection of oocytes for fertilization and transfer needs to be further evaluated before considering clinical application in assisted reproduction practices. Therefore, removing PB1 and PB2, and performing genetic analysis of PB DNA is the only way to evaluate the genetic quality of oocytes. Modlinsky and McLaren attempted to visualize PB2 chromosomes a long time ago, but the method was not sufficiently accurate to be acceptable for clinical application.3 There was also other experimental work by Monk and Holding to use the PB approach for testing the possibility of amplification of beta-globin sequences in the mouse model.4 We introduced the PB approach 10 years ago for testing oocytes for preconception diagnosis in couples at risk of having a child with autosomal recessive condition.5 Initially, PB1 was only tested on the basis of the fact that, in the absence of crossing over, PB1 will be homozygous for the allele not contained in the oocyte and PB2. However, the PB1 approach was not applicable for predicting the eventual genotype of the

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oocytes if crossing over occurred, because the primary oocyte in this case will be heterozygous for the abnormal gene. As the frequency of crossing over varies with the distance between the locus and the centromere, approaching as much as 50% for telomeric genes, the PB1 approach appeared to be of a limited value, unless the oocytes can be tested further on. So, the analysis of PB2 has been introduced to detect hemizygous normal oocytes resulting after the second meiotic division.6 The technique presently involves a two step oocyte analysis, which requires a sequential testing of PB1 and PB2. Although this is no longer a preconception diagnosis, as it has originally been designed, it is still possible to freeze the oocytes at the pronuclear stage, following exposure to sperm or introcytoplasmic sperm injection (ICSI), so that only mutation free oocytes are allowed to develop into embryo and be transferred. Therefore, the PB approach might still be useful in those ethnic groups, where not only termination of pregnancy, but also any manipulation with embryo is not allowed after fertilization. Together with other available methods for prenatal and preimplantation diagnosis, the PB approach will make it possible to offer the whole range of options for couples at risk of having children with genetic and chromosomal disorders.

POLAR BODY REMOVAL Polar body removal (PBR) is performed after stimulation and oocyte retrieval by using a standard in vitro fertilization (IVF) protocol, as described in detail elsewhere.7 After aspiration, oocytes are checked for extrusion of PB1, and then, by using the standard micromanipulation set up, the zona pellucida is opened mechanically by a microneedle, at the position away from PB1, to avoid possible damage to the meiotic spindle. Through this zona opening the aspirating blunt micropipette is passed to PB1 to aspirate it by gentle suction. The oocyte is then washed in IVF medium, inseminated with motile sperm or by using ICSI, examined for the presence of pronuclei and extrusion of PB2, which is removed in the same way as PB1. This two step procedure of sequential removal of PB1 and PB2 is performed for preimplantation genetic diagnosis (PGD) for single gene disorders, although it is also possible to sample simultaneously both PB1 and PB2, to avoid additional invasive procedure. The latter technique may be applied only for PGD of chromosomal aneuploidies, because both PB1 and PB2 may be fixed and analyzed on the same slide by fluorescent in situ hybridization analysis (FISH), without mixture of the results of each PB. The biopsied oocytes are then returned to culture, checked for cleavage and currently, also for blastocyst formation, and transferred, depending on the genotype of the corresponding oocytes. The embryos resulting from unaffected oocytes are transferred back to the uterus within the implantation window or in the next cycle, if oocytes were frozen at the pronulear stage, while those

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predicted to be affected are used for follow up analysis by polymerase chain reaction (PCR) or FISH analysis to confirm the PB diagnosis. Although, as mentioned, PB1 and PB2 are not expected to have any biological role in the development of embryo, direct observations on the subject were not available. We have explored a possible detrimental effect of PB1 and PB2 removal by following up and evaluating the resulting micromanipulated oocytes at different stages of development.7–8 We did not observe any significant decrease in fertilization rate for oocytes, or cleavage of the resulting embryos, after PB1 removal. The percentage of embryos entering cleavage was similar in biopsied and non-biopsied oocytes. There was no increase in the percentage of polyspermic embryos either. Data on long term effects of the procedure, inferred from culturing the embryos to the blastocyst stage, showed that the proportion of embryos reaching the blastocyst stage was similar to that known for nonmicromanipulated oocytes. A follow up study of the viability of the sampled oocytes through implantation and postimplantation development of the resulting embryos also suggested no detrimental effect. As will be described below, the procedure of PB1 has already been applied in about 5000 oocytes in the process of PGD of age related aneuploidies and single gene disorders, showing no effect of PB1 removal on fertilization, preimplantation, and, possibly, postimplantation development. No deleterious effect was also observed in the follow up study of more than one hundred children born following PB sampling.9 Similar data were obtained in a study of a possible effect of PB2 sampling.10 The viability and the developmental potential of the resulting embryos were analyzed by using mouse oocytes, which were compared with the control for the percentage of embryos reaching the blastocyst stage. No difference was observed between the embryos resulting from biopsied and non-biopsied oocytes in forming morphologically normal blastocysts. No differences were found in the cell count of blastocysts obtained from biopsied and control groups either, suggesting no detectable effect of PB2 sampling on preimplantation development. Finally, similar to PB1 removal, there were also no long term effects of the procedure, as is shown by the follow up study of children born after PGD for genetic and chromosomal disorders, performed by PB1 and PB2 biopsy.9

POLAR BODY DIAGNOSIS OF MENDELIAN DISORDERS As mentioned PB biopsy was first applied to PGD for autosomal recessive disease ten years ago.5 Since then PB diagnosis has been performed for more than two dozen different single gene disorders (Table 27.1), and, as will be described below, has been found to be accurate and reliable method for PGD of single gene disorders. Despite the

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Table 27.1. Preimplantation diagnosis by polar body biopsy: list of conditions. Cystic fibrosis Alpha-1-antitrypsin deficiency Thalassemia Achondroplasia Sickle cell disease Adenosine deaminase deficiency Tay-Sachs disease Multiple epiphyseal dysplasia Phenylketonuria X-linked hydrocephalus Hemophilia A and B Neufibromatosis I & II Gaucher disease Myotonic dystrophy Long-chain 3-hydroxyacyl-CoA dehydrogenase Fragile X syndrome deficiency Epidermolis bullosa Fancony anemia Retinitis pigmentosa P53 oncogene mutations Ornithine-transcarbamylase deficiency Chromosomal abnormalities Alport disease well known limitations of DNA analysis in single cells, the currently available experience of genetic analysis of more than 1000 oocytes, shows that it is possible to overcome these limitations and avoid a possible misdiagnosis in PGD.11–13 In fact, DNA analysis in PB seemed to be more accurate and reliable than DNA analysis in single blastomeres. DNA analysis in different types of single cells showed that the possibility of allele specific amplification failure, or allele drop out (ADO), in PB1 is at least half that in blastomeres.11 In addition, a considerable proportion of ADO is detectable by sequential analysis of PB1 and PB2. PCR analysis of 16 alleles in both PB1 and PB2 obtained from hundreds of oocytes showed that more than half of all ADOs can be detected simply by sequential analysis of PB1 and PB2.12–13 This may allow avoiding misdiagnosis owing to ADO even in those cases when no informative polymorphic markers are available, as in cases of PGD in some isolated populations and ethnic groups, such as in Cyprus PGD program for thalassemias.14–15 However, if two or more linked polymorphic markers are available for the gene under study, almost all ADOs are detected, making the PB diagnosis a completely reliable procedure. The other recent development, which will make possible to improve detection of potential misdiagnoses in PB PGD, is the application of fluorescence PCR (F-PCR). Because preferential amplification (PA) like ADO may be represented by a lack of one of the alleles in a single heterozygous cell analyzed by conventional PCR analysis, most ADO may be actually the result of PA. With the introduction of F-PCR, it has become possible to distinguish ADO from PA, allowing the definite

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diagnosis of oocytes with PA manifested as ADO by conventional methods. We studied this phenomenon by testing single cells heterozygous for cystic fibrosis (CF) Delta F-508 mutation with both conventional and F-PCR and showed that PA has a minor contribution to misdiagnosis (unpublished data). Fifty PGD cycles for couples at high risk for having children with single gene disorders have been performed by using PB biopsy (Table 27.2). These included 20 cycles for CF, 18 for

Table 27.2. Preimplantation diagnosis by polar body biopsy: clinical experience. Disorders Cycles Oocytes Resulting normal Transfers Pregnancies Babies studied embryos transferred Single gene 50 636 126 47 20 19 Chromosomal 777 4720 1816 717 159 116 Total 827 5356 1942 764 179 135 beta-globin gene mutations, two for Gaucher disease, two for LCHAD, one for hemophilia B, and one for phenylketonuria (PKU). The couples underwent one or more IVF protocols, providing the possibility to obtain sufficient number of oocytes for preselection of unaffected embryos for transfer.13–15 The design of primers used for detection of the above mutations, including the optimal conditions for PCR reaction, were described for each condition elsewhere.11–15 The general principle of genetic analysis was multiplex nested PCR analysis. The genes and polymorphic markers were first amplified in the same PCR reaction (first round PCR), using the mixture of all outside primers involved. The PCR product obtained in the first round was then amplified separately with inside primers for each gene and corresponding marker. We used one or more linked markers for each gene, representing short tandem repeats (STRs) to exclude the possibility of ADO, which may lead to misdiagnosis, as mentioned. In addition, STRs on other chromosomes were also used to trace and exclude a possible DNA contamination and to monitor the origin of the DNA studied. Mutations and linked STRs studied were described earlier.11–15 The strategy for preselection of mutation free oocytes is based on detection of both the mutant and the normal alleles in PB1, together with the presence of the mutant one in PB2, in agreement with the pattern of the linked STRs, corresponding to these genes. Fluorescent F-PCR was also used, either in parallel or following conventional PCR, in an attempt to improve ADO detection rate, as well as to distinguish ADO from PA, as mentioned.13 Overall, 636 oocytes were available for study from 50 PGD cycles, ranging from two to 28 oocytes per each cycle. The average number of

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oocytes with both PB1 and PB2 data was 8.2, allowing preselection and transfer of one to three unaffected embryos in almost all the clinical cycles. More than half of oocytes (65%) were heterozygous, on the basis of the analysis of corresponding PB1, with variation from 58% in LCHAD to 72% in thalassemia, the only exception being the case of PGD for Gaucher disease.13 In all cycles, priority in transfer was given to the embryos resulting from the heterozygous oocytes following the first meiotic division as detected by PB1 analysis, because in the absence of DNA contamination, this indicates the absence of ADO from either the normal or the mutant alleles. Identification of the mutant gene in the sequential PB2 analysis of these heterozygous oocytes allowed preselection of mutation free oocytes for transfer. Unfortunately, PB2 analysis was available only in 67% heterozygous oocytes, which, however, allowed detection of 126 normal embryos for transfer, based on the presence of the mutant gene in the corresponding PB2. Although most of the transferred embryos were preselected using this particular strategy, 22 (17.5%) embryos preselected for transfer still originated from homozygous normal oocytes, inferred from homozygous mutant status of PB1 and hemizygous normal status of PB2. Such embryos were accepted for transfer only in cases in which the possibility of ADO may have been excluded by using linked polymorphic marker analysis. Otherwise, such embryos were excluded from transfer and exposed to follow up study to investigate the accuracy of the method. In fact, the follow up study showed, that 15 oocytes (5.4%), initially inferred as normal from finding of homozygous mutant PB1 and hemizygous normal PB2, appeared to be affected following the analysis of linked markers, indicating to ADO of a normal alleles in an apparently heterozygous PB1 in these cases. The presence of ADO was also obvious in 18 cases (6.5%), on the basis of contradictory results from PB1 and PB2, showing the identical homozygous genotypes, owing to undetected ADO of one of the PB1 alleles, opposite to one detected in PB2. This approach, in combination with multiplex PCR analysis of the genes and linked polymorphic markers, made it possible to detect ADO in 33 oocytes (10.9%), which could have contributed to misdiagnosis should they be used for transfer. The follow up analysis of the embryos excluded from transfer either because they were affected, or there were insufficient information to preselect them for transfer, has provided valuable data for evaluating the proportion of undetected ADO. The data indicate to 97% accuracy, which may be quite acceptable for clinical use of PB PGD. This work has resulted in preselection and transfer of mutation free embryos in almost all cycles, with 20 established pregnancies, resulting in the birth of 19 healthy children (Table 29.2). These include healthy children born after PGD for several CF mutations, sickle cell disease, and the most common thalassemia mutations.13–16 PGD has also provided for couples with one homozygous affected partner, including thalassemia and PKU, resulting in an unaffected pregnancy and birth of healthy children in

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the latter case.13 Overall, the data show that the availability of approximately 6–7 oocytes per cycle with data for both PB1 and PB2 makes it possible to preselect at least two to three mutation free oocytes for transfer per cycle, resulting in an acceptable pregnancy rate. The method, may therefore be acceptable for clinical practice, as it allows a preselection of a sufficient number of unaffected embryos for transfer and provides the possibility to minimize misdiagnosis owing to ADO. Further optimization of the method may allow to avoid completely the risk for misdiagnosis owing to ADO and other limitations of single cell PCR analysis in PGD for single gene disorders.

POLAR BODY DIAGNOSIS OF CHROMOSOMAL DISORDERS PB1 and PB2 are of special interest for cytogenetic analysis, representing a direct approach for testing the outcomes of the first and second meiotic divisions. Because PB1 is the byproduct of the first meiotic division and represented by metaphase chromosomes, PB1 was extremely useful for preimplantation testing for maternally derived translations.17 It also makes it possible to apply the recent spectral karotyping (SKY) technique to attempt complete karyotyping of oocytes.18 The method for karyotyping PB2 has been recently also developed and may be used in combination with PB1 cytogenetic analysis for PGD of translocation and aneuploidies.19 PGD for aneuploidy was done by PB1 and interphase PB2 FISH analysis, by using commercial probes specific for a three (chromosomes 13, 18, and 21) and currently for five chromosomes (chromosomes 13, 16, 18, 21 and 22) (Vysis).20–22 Overall, 777 such cycles have been performed for patients of advanced maternal age.20–22 Of 4255 oocytes obtained and subjected to PB sampling and FISH analysis, results were available in 82.1% of oocytes (5 oocytes per cycles). As much as 42.9% of oocytes with FISH results were predicted to be aneuploid, leaving the rest (3.1 per cycle) for possible transfer. More than two thirds (71.1%) of these oocytes had data for both PB1 and PB2, 16% with only PB1, and 12.9% with only PB2 data. As expected most errors were observed in PB1 (34.9%), although a considerable number of abnormalities were also seen in PB2 (26.4%). The types of abnormalities in PB1 were represented by missing chromatids in 51.9%, extra chromatids in 16.6%, missing chromosomes in 8.0%, extra chromosomes in 0.6%, and complex abnormalities, involving different types of abnormalities, in 21.9%. The proportion of abnormal oocytes with missing and extra chromatids in their PB2 was 40.6% and 45.8%, respectively. PB2 of the rest of the oocytes were with complex abnormalities, involving missing and extra chromatids of the different chromosomes. Of a total of abnormal oocytes, 48.3% were with meiosis I errors, 29.3% with meiosis II errors, and 22.4% with both meiotic errors.

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The same chromosome was involved in more than half of these oocytes, resulting in a balance status in 37.6% of them. As many as 43.8% of the complex abnormalities contained errors of different chromosomes. The study of chromosome specific patterns of errors in the first and second meiotic divisions was possible in 2419 oocytes for chromosomes 18 and 21, and 1682 oocytes for chromosome 13. Of 352 oocytes with chromosome 21 errors, 46.3% originated from meiosis I, 32.7% from meiosis II, and 21.0% from both meiotic divisions. A similar pattern was observed for chromosome 13 errors, with 48.4% originating from meiosis I, 35.2% from meiosis II, and 16.4% from both. Although the chromosome 18 errors also occurred predominantly in meiosis I (59%), this is opposite to the data derived from postnatal cases of trisomy 18, shown to originate predominantly in the meiosis II.23 Collection of further data may allow studying the biological significance of these differences. As many as 37.4% of abnormal oocytes had complex errors, involving the same chromosome in both meiotic divisions in 31.2% of them, and more than one chromosome in 68.8%, of which 88% were with abnormalities of two, and 12% with abnormalities of three chromosomes studied. This further supports earlier reports suggesting a possible increase of mitotic spindle formation errors with age.24 The other observation supporting this hypothesis is a non-significant increase of aneuploidy rate with the application of an additional chromosome specific probe for PGD of age related aneuploidies. For example, the aneuploidy rate was 39.8% when we applied two chromosomes specific probes (chromosomes 18 and 21) for analysis of 2839 oocytes, and has increased to only 42.9% with the addition of the third chromosome specific probe (chromosome 13), involving the analysis of further 1500 oocytes. It may be predicted that the application of additional chromosome specific probes will probably affect the proportion of abnormal oocytes with complex errors, rather than the overall incidence of aneuploid oocytes. Of 2212 detected aneuploidy free oocytes, 1816 (2.5 per cycle) were transferred in 717 treatment cycles, resulting in 159 (22.2%) clinical pregnancies and 116 healthy children born after confirmation of PB diagnosis (Table 29.2). In addition to avoiding the transfer of embryos resulting from 1665 aneuploid oocytes (2.1 per cycle), contributing to prevention of the birth of children with common aneuploidies, PB testing of oocytes could have also improved the chances of IVF patients to become pregnant. Of 4720 oocytes obtained in 777 IVF patients of advanced maternal age (six per cycle), only half (three per cycle) would have been selected in a routine IVF. In the absence of genetic preselection, this number may have incidentally included at least one or two aneuploid embryos, which would have led to reproductive failures. In addition, because the number of embryos transferred is limited to only three, selection of aneuploid embryos might have caused euploid embryos to be not transferred. Therefore, this should have contributed to 22.2% pregnancy rate in our group of patients whose average maternal age was

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approximately 39 years. However, more data will be needed to investigate the impact of preselection on aneuploidy free oocytes on the efficiency of IVF. The application of additional probes to the analysis of the oocytes will be of great clinical value, because the errors of most of autosomes may contribute significantly to implantation rate. Five color FISH analysis is currently applied routinely, which involves, in addition to chromosomes 13, 18, and 21, the analysis of chromosomes 16 and 22, known to contribute significantly to fetal loss (data not shown). Re-hybridization with additional probes specific for chromosomes 1 and 17 is also being initiated to collect data on the impact of errors of these chromosomes on implantation. The ideal, of course, might be the development of the methods for full karyotyping of PB1 and PB2. However, immediately after extrusion, PB1 chromosomes are uncountable if analyzed. But after two to three hours of in vitro culture and during the next two to three hours, the individual PB1 chromosomes become recognizable and countable, with following degeneration six to seven hours after extrusion.7 This makes PB1 particularly useful for SKY, or application of whole chromosome specific fluorescence probes or other chromosome segment specific probes for testing of chromosomal translocations. Munne et al used this method for PGD of translocations of maternal origin in more than two dozen clinical cycles.17 Although the method resulted in a significant reduction of spontaneous abortions in the PGD cycles and led to the birth of more than a dozen unaffected children, it is sensitive to malsegregation of chromatids and recombination between chromatids. So the visualization of PB2 chromosomes, in combination with PB1 FISH analysis, will ideally be required for accurate PGD of chromosomal abnormalities. However, it is known that PB2 forms a nucleus and never transforms into metaphase. To visualize PB2 chromosomes, Modlinsky and McLaren transplanted the mouse PB2 into fertilized eggs, which in some cases transformed the PB2 nucleus in a presumably haploid group of mitotic chromosomes, but the success rate was very low, and even when the chromosomes were visualized they were unsuitable for karyotyping.3 Various techniques have recently been developed for converting PB2 into the metaphase stage.19,25,26 One of these approaches involved electrofusion of the mouse PB2 with intact and/or the enucleated mouse zygotes and resulted in PB2 nuclei transformation into the metaphase plate in 34% of cases. The same results were obtained by electrofusion of PB2 with foreign 1 cell mouse embryo, with the proportion of metaphase plates reaching 65% when the recipient 1 cell stage mouse embryo was enucleated.25 The other approach involved the treatment of the 1 cell stage mouse embryos with okadaic acid (a specific inhibitor of phosphatase 1 and 2A) leading to visualization of PB2 chromosomes in up to 80% of cases.26 The visualized PB2 chromosomes were unichromatid G1 premature condensed chromosomes of good quality, suitable for differential staining. However, because, in contrast to the mouse data, the

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okadaic acid treatment of human PB2 leads to further condensation of already picnotic PB2 nuclei, PB2 has to be first decondensed by its introduction into the mouse cytoplast.19 By then using electrofusion in conjunction with inhibition of protein synthesis by ocadaic acid, PB2 chromosomes of a good quality were obtained suitable for differential staining and FISH. Analyzable chromosomes were obtained in 66% of cases, and the method has already been applied to PGD of maternally derived translocations in clinical cycles (unpublished data).

CONCLUSION A method for genetic testing of oocytes by PB1 and PB2 biopsy was introduced, which has been shown to be accurate, reliable, and safe. The method has been applied in 827 clinical cycles for PGD for single gene and chromosomal disorders. Overall, the work involved genetic testing by PCR or FISH of more than 5000 oocytes, from which, about 2000 were pre-selection and transferred in 764 cycles, resulting in 179 clinical pregnancies and the birth of 135 healthy children (Table 29.2). An average pregnancy rate of 23.4% in the whole series seems quite acceptable, taking into consideration that most patients were 35 years and older, which represents the major indication for PGD. In the group of patients with the latter indication, the average age was about 39 years, which suggests that the aneuploidy testing may not only allow avoiding the birth of children with age related aneuploidies, but can also improve the pregnancy rate in IVF patients of advanced maternal age. Although more data are clearly needed to come to that conclusion, there is no doubt that preselection of aneuploid oocytes will reduce at least to some extent the transfer of potentially non-viable oocytes. Although the intrinsic genetic variables 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 fertilization and transfer could be avoided. Together with other predictive factors, such as different clinical and epigenetic characteristics, preselection of euploid oocytes by PB1 and PB2 sampling may allow in the future to distinguish those few oocytes with the maximum potential to result in clinical pregnancy and the birth of a healthy child. POLAR BODY BIOPSY PROTOCOL An oocyte is transferred to a micromanipulation dish with a drop containing sucrose. Then the oocyte is secured by the holding pipette and oriented using the microneedle to visualize PB1 at the 6 o’clock position. By using the microneedle the opening is made in the zona pellucida at the 4–5 o’clock position, by rubbing the microneedle against the holding pipette. The aspirating blunt micropipette is then passed through the opening to PB1 and gentle suction is applied to aspirate PB1 into the micropipette Pressure from the

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hydraulic system is equilibrated before withdrawing the aspirating micropipette to avoid damage to the oocyte. The same procedure is applied for PB2 removal except there is no need for partial zona dissection, which has already been created in the zona pellucida prior to PB1 removal. The oocyte is rotated to position the opening at 3 o’clock, with PB2 its focus, and the micropipette is advanced to PB2. By using gentle suction PB2 is aspirated into the micropipette and the procedure continues as for PB1 removal. Oocytes are returned to culture dishes and observed for cleavage next morning. Contrary to PGD for single gene disorders when PB1 and PB2 are removed separately in sequence, PB1 and PB2 are removed simultaneously for the purpose of PGD for chromosomal abnormalities. The procedure of simultaneous PB1 and PB2 biopsy is similar to those performed for PB1 removal, except it does not require precautions for DNA contamination.

REFERENCES 1 Van Blerkom J. Epigenetic influences on oocyte developmental competence: perifollicular vascularity and intrafollicular oxigen. J Assist Reprod Genet (1998); 15:226–34. 2 Gregory L. Ovarian markers of implantation potential in assisted reproduction, Hum Reprod (1998); 13:117–32. 3 Modlinsky J, McLaren A. A method for visualizating the chromosomes of the second polar body of the mouse egg. J Embryol Exp Morphol (1980); 60:93–7. 4 Monk M, Holding C. Amplification of beta-haemoglobin sequence in individual human oocytes and polar bodies. Lancet (1990); 335:985. 5 Verlinsky Y, Ginsberg N, Lifchez A, et al. Analysis of the first polar body: preconception genetic diagnosis. Hum Reprod (1990); 5:826–9. 6 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 Molec Med (1997); 62:182–7. 7 Verlinsky Y, Kuliev A. Preimplantation diagnosis of genetic diseases: a new technique for assisted reproduction. New York: Wiley-Liss (1993). 8 Verlinsky Y, Milayeva S, Evsikov S; et al. Preconception and preimplantation diagnosis for cystic fibrosis. Prenat Diagn (1992); 12:103–10. 9 Strom C, Levin R, White M, et al. Neonatal outcome of preimplantation diagnosis by polar body removal: the first 100 babies. Pediatr (in press). 10 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); 12:747–9.

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11 Rechitsky S, Strom C, Verlinsky O, et al. Allele drop out polar bodies and blastomeres. J Assist Reprod Genet (1998); 15:253–7. 12 Rechitsky S, Strom C, Verlinsky O, et al. Accuracy of preimplantation diagnosis of single-gene disorders by polar body analysis of oocytes. J Assist Reprod Genet (1999); 16:169–75. 13 Verlinsky Y, Rechitsky S, Verlinsky O, et al. Prepregnancy testing of single-gene disorders by polar body analysis. Genet Testing (1999); 3:185–90. 14 Kuliev A, Rechitsky S, Verlinsky O, et al. Preimplantation diagnosis for thalassemias. J Assist Reprod Genet (1998); 15:219–25. 15 Kuliev A, Rechitsky S, Verlinsky O, et al. Birth of healthy children after preimplantation diagnosis of thalassemias. J Assist Reprod Genet (1998); 16:207–11. 16 Kuliev A, Rechitsky S, Ivakhnenko V, et al. Preembryonic diagnosis for sickle cell disease. Proceedings of the III International Symposium on Preimplantation Genetics, Bologna, 22–23 June 2000. 17 Munne 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. 18 Marquez C, Cohen J, Munne S. Chromosome identification of human oocytes and polar bodies by spectral karyotyping. Cytogenet Cell Genet (1998); 81:254–8. 19 Verlinsky Y, Evsikov S. Karyotyping of human oocytes by chromosomal analysis of the second polar body. Molec Hum Reprod (1999); 5:89–95. 20 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. 21 Verlinsky Y, Cieslak J, Ivakhnenko V, et al. Prepregnancy genetic testing for common age-related aneuploidies by polar body analysis. Genet Testing (1998); 1:231–5. 22 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. 23 Nicolaides P, Peterson M. Origin and mechanisms of non-disjunction in human autosomal trisomies. Hum Reprod (1998); 13:313–9. 24 Battaglia DE, Goodwin P, Klein NA, Soules MR. Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum Reprod (1966); 11:2217–22. 25 Verlinsky Y, Dozortzev D, Evsikov S. Visualization and cytogenetic analysis of second polar body chromosomes following its fusion with one-cell mouse embryo, J Assist Reprod Genet (1996); 11:123–31. 26 Dyban A, De Sutter P, Verlinsky Y. Okadaic acid induces premature chromosome condensation reflecting the cell cycle progression in onecell stage mouse embryos. Mol Reprod Dev (1993); 34:403–15.

28 Embryonic regulation in the process of implantation Jose Luis de Pablo, Marcos Meseguer, Pedro Caballero-Campo, Antonio Pellicer, Carlos Simón

INTRODUCTION Embryonic implantation in humans is a progressive and a complex process, in which the embryo has to appose, then has to adhere to the maternal endometrial epithelium (EE), and finally has to invade it. The existence of a receptive phase or “window” for embryo implantation is required to make possible maternal-embryo interaction. The window process lasts approximately four days in women, from days 20–24 of the cycle.1 Timing from the luteinizing hormone (LH) peak, which precedes ovulation by about 36 hours, gives a window lasting from approximately day LH+7 to LH+11.2 The blastocyst in the apposition period faces the specific site, generally in the posterior wall of the uterus. In the next phase of implantation, the adhesion phase, the trophoectoderm of the blastocyst contacts directly with the EE. It occurs between day 6 and 7 after ovulation. This concept is not obvious and more researchers are starting to believe that epithelial penetration without adhesion of the blastocyst to the luminal surface is feasible.3 The last phase is the invasion of the embryonic trophoblast, traversing adjacent cells of the epithelial lining, and invasion into the endometrial stroma up to the uterine vessels. The blastocyst-induced apoptosis on EE seems to be a plausible mechanism to allow the embryo to traverse the epithelial barrier before it can invade deep into the stroma.4 Implantation mechanisms vary among species, nevertheless, interaction between the apical surface of luminal uterine epithelium and the external surface of embryonic trophoectoderm, which are immunologically and genetically different, is a shared event in which the molecular protagonists are still being defined. To understand the molecular mechanism involved in this process, several aspects have to be considered separately—endometrial receptivity, the presence of a chromosomally and functionally competent human embryo, and the impact of the implanting blastocyst to the endometrial epithelium.

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In this work, we present the results of research on embryonic regulation of endometrial epithelial molecules such as chemokines, adhesion molecules, and mucins.

EMBRYONIC REGULATION OF IMPLANTATION The embryo plays an important role in the receptive phase of implantation. The embryo further prepares the endometrium, modulating endometrial molecules and controlling the implantation process. In mammals an amazing “dialogue” exists between the embryo and the endometrium to establish the pregnancy, always helped by the corpus luteum (CL). It is well known that in rodents (mice), direct contact between the embryo and the endometrium is not necessary.5 The embryos send signals to the endometrial epithelium and stroma to favour epithelial receptivity. This crucial concept has also been demonstrated in the rabbit,6 and more recently, Fazleabas et al demonstrated in the nonhuman primate (Papio anubis) the modulation of uterine endometrium by CG during the period of uterine receptivity.7 In this study, the authors show a physiological effect of CG on the uterine endometrium in vivo, suggesting that the primate blastocyst signals modulate the uterine environment prior to and during implantation.7 Therefore, these studies strongly suggest that there is a third dimension to be considered in implantation studies: the effect of the embryo on the regulation of endometrial molecules during the apposition, adhesion and invasion phases. In humans, there is no direct evidence implicating the role of the human embryo in regulating endometrial receptivity, adhesion or invasion. Nevertheless, indirect data obtained from endometrial biopsies during a cycle of conception reinforce the concept of embryonic regulation of the implantation process.8 Endometrial biopsies obtained in two clinical series including 188 and 54 patients9 during a cycle of conception showed a histologic delay of the decidualization process lasting two days compared with the average cycle length of 25 days. In summary, we have focussed on relevant proteins dedicated to facilitate the endometrial-embryo communication during the adhesion phase, such as cytokines, integrins β3, α4 and α1 and the mucin MUC-1. In general, it has been stated that chemokines could act as a chemoattractant for resident leucocytes. Once the embryo adheres to endometrial epithelial cells (EEC) through the recognition of the adhesion molecules, such as integrins, it could induce the signalling transduction into cells. In contrast, MUC-1 is postulated to be an anti-adhesion protein that might be absent from the epithelial cells when the embryo attaches to it.

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MATERIALS AND METHODS COCULTURE Endometrial biopsies obtained in the luteal phase, were minced into small pieces to isolate EEC and endometrial stromal cells (ESC), so that an EEC monolayer can be obtained. Therefore, epithelial cells were cultured and grown to confluence in a steroiddepleted medium; 75% DMEM and 25% MCDB-105 containing antibiotics, supplemented with 10% charcoal dextran treated FBS and 5µg/ml insulin. The homogeneity of cultures was determined by morphological characteristics and was verified by immunocytochemical localization of cytokeratin, vimentin, and CD68 antigen. Confluence was reached in four to six days. Human embryos were cocultured in the autologous human endometrial epithelial cells (AEEC) monolayer with in vitro fertilization (IVF):CCM (1/1) during the first 24 hours and then CCM is used until the day of the transfer.10 IN VITRO MODEL FOR ENDOMETRIALEMBRYONIC INTERACTIONS IN THE APPOSITION AND ADHESION PHASE Based on our previous work,11 we have developed a coculture system with AEEC that retained many features of human endometrial epithelium. The model employed is based on a clinical IVF program in which single human embryos were cocultured with primary culture of AEEC until the blastocyst stage and then transferred back to the uterus.10 Embryos were obtained after ovarian superovulation and inseminated employing routine IVF procedures. EEC were isolated from endometrium of fertile patients and cultured until confluent as previously described.12,13 Individual human embryos were cocultured with AEEC for five days (from day 2 until day 6 of embryonic development). After embryo transfer, EEC wells were divided into three different groups according to the embryonic status reached: EEC with embryos that achieved the blastocyst stage, EEC with arrested embryos and EEC without embryos (Fig 28.1). Using this model we have studied the embryonic regulation of endometrial epithelial adhesion and antiadhesion molecules and chemokines during the apposition phase. Furthermore to seek specific paracrine human embryonic effect on the adhesion phase in vitro attachment assays could be done on threedimensional cultures (Fig 28.2). Spare blastocysts are cultured on these endometrial epithelial cells and are allowed to attach to the epithelial surface during 48–72 hours. (Fig 28.3).

Fig 28.1 Representative diagram of embryo coculture as an “in vitro” model. Biopsy is taken in the previous cycle in patients undergoing ART and immediately frozen. In the treatment cycle, the biopsy is thawed and processed to obtain the epithelial fraction, seeded and cultured until confluence. Embryos are cocultured under these conditions and blastocysts are transferred to the patients.10,11

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Fig 28.2 Model of the autocrine-paracrine regulation of the adhesion phase. We use three different monolayers: EEC, extracellular matrix (ECM) and ESC. It has been designed to create a culture similar to the human endometrium.

Fig 28.3 Human blastocysts attached to polarized EEC observed by a confocal microscopy. The field was captured by phase contrast in which the embryo appears as an opaque sphere. Epithelial cells could be discerned beneath the blastocyst. Magnification ×250. HORMONAL REPLACEMENT THERAPY PROTOCOL To study hormonal regulation in vivo, serum and endometrial samples for immunohistochemistry and Northern blot analysis were obtained in mock cycles from 10 patients (aged 23–29 years) undergoing oocyte donation and receiving hormone replacement therapy (HRT) as previously described.14 Their indications for oocyte donation were a low response to gonadotropin stimulation (4/10), poor quality of oocytes (4/10), and the menopause (2/10). Patients with ovarian function were desensitized with

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leuprolide acetate (LA, 1mg/day) in the secretory phase of the previous cycle. Hormonal replacement started on day 1 of the cycle with administration of E2- valerate 2mg/day on days 1 to 8; 4mg/day from days 9 to 11; and 6mg/day from days 12 to 20. Natural micronized progesterone (P), 800mg per day, was administered vaginally from day 16, and LA was discontinued. The regimen of 6mg/day E2-valerate and 800mg/day P was maintained until day 22. Serum samples (S) and uterine biopsies (B) were taken at days 13 (S1, B1), 18 (S2, B2) and 21 (S3, B3) from each patient. At the time that serum and biopsies were collected, patients were treated with 6mg/day of EV for two days and 6 mg/d E2valerate plus 100mg/d P for three and six days, respectively (Fig 28.4). Biopsies were dated histologically according to the method by Noyes et al.15 FIXATION AND SCANNING ELECTRON MICROSCOPY For the scanning electron microscopy (SEM), samples were fixed using 1ml 1% gluteraldehyde in PBS. Samples were stored in the fixative at 4°C for several days until they were processed. The specimens need to be dehydrated in a decrease grade series of alcohol and then dried according to the critical point method using CO2. Once the samples are dried, EEC monolayers were mounted on the specimen holder, sputter coated with gold (14nm thickness), and observed under accelerated voltage of 10.0kv at a short working distance in a Cambridge Stereoscan 360 scanning electron microscopy (Cambridge Instruments, Cambridge, MA). For measurements, the screen magnification was increased to 20000, and three representative areas of 4µm2 were examined for each specimen. All specimens were processed together. IMMUNOHISTOCHEMISTRY Endometrial samples for immunohistochemical experiments were fixed in formalin and embedded in paraffin, sectioned, and mounted on glass slides coated with Vectabond™ (Vector Laboratories, Burlingame, CA). Twelve serial sections (6µm) from each sample were prepared for immunohistochemistry and the first and last sections were stained with hematoxylin-eosin.

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Fig 28.4 Schematic representation of the hormonal replacement regimen used in patients undergoing oocyte donation. Treatment started on day 1 of the cycle. Representative diagram of embryo coculture as an “in vitro” model. Biopsy is taken in the previous cycle in patients undergoing ART and immediately frozen. Extravidin-peroxidase staining method or immunofluorescence were performed on human endometrium paraffin embedded sections for MUC1. Sections were previously deparaffinated and dehydrated.16 Samples were stained with primary antibodies (Abs) against molecules studied. Endogenous peroxidase was quenched with 3% (v/v) hydrogen peroxide (10 minutes at room temperature (RT), samples were rinsed three times for five minutes in PBS, and non-specific binding was blocked with non-fat milk dehydrated 50mg/ml in PBS. The samples were thereafter rinsed with PBS, pH 7.4, with 0.05% Tween-20 (PBS-T) three times and then incubated with the primary antibodies, each for 90 minutes at 37°C. After washing them four times with PBS-T sections were incubated with rabbit anti-mouse IgG (90 minutes, 1/300 dilution at 37°C). To amplify the signal, sections were rinsed four times with PBS-T and then incubated with extravidin-horse radish peroxidase conjugated (30 minutes, 1:40 dilution at RT, washed with PBS-T four times, and incubated 10 minutes with working substrate solution (0.2ml Stock AEC solution with 3.8ml of 0.05M acetate buffer, pH 5.0, and was added 20µl of 3% H2O2 immediately before use), reaction was terminated by rinsing the slides gently with distilled water. Finally, slides were counterstained with Mayer’s hematoxylin, rinsed with distilled water and mounted with glycerol gelatin and viewed with an Olympus BH2 microscope.

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Primary Ab detection by immunofluorescence was performed by using a double step amplification with biotinylated rabbit antimouse (IgG) and swine antirabbit F(ab') at dilution 1:100 in PBS and extravidin conjugated to fluorescein isothiocyanate (FITC) (1:75 in PBS) with an incubation of one hour at RT in the dark (Dako, High Wycombe, UK). Coverslips were mounted in Dako aqueous mounting media and viewed under a fluorescence microscope. When no signals were obtained after deparaffination, sections were boiled in citrate buffer (0.05M) in the microwave oven. Where indicated, sections were subjected to enzymatic digestion with sialidase, after deparaffination and rehydratation working dilution 1/100 in 0.1M sodium acetate and 1mM Ca Cl2 O/N at 37°C and rinsed for 15 minutes in PBS before the application of the HMFG-1 antibody. FLOW CYTOMETRY For flow cytometry experiments, EEC monolayers were detached by a cell scraper (Falcon 3085), retrieved with the coculture medium, or by digestion with collagenase, centrifuged (for studying the intracytoplasmic epitopes pellet was resuspended in ethanol 70% at −20°C for 20 minutes and centrifuged), and the cell pellet was blocked with 1% BSA in PBS for 120 minutes at 4°C. After washing with PBS-T (0.05%), cells were incubated with primary Ab directed against the molecules which expression will be studied (O/N at 4°C) according to providers instructions. EEC suspensions were washed and mixed for with FITCconjugated goat anti-mouse IgG whole molecule (150 minutes, 1:64 dilution at 4°C) or FITC-conjugated goat anti-rabbit IgG whole molecule (150 minutes, 1:160 dilution at 4°C). Cell suspensions which were not treated with ethanol (study of extracellular epitopes) were fixed with 1% paraformaldehyde for one hour at 4°C. Negative controls were included in each experiment by the delection of the primary antibody. Cells were resuspended in PBS, and analyzed in a Epics Elite flow cytometer (Coulter Cytometry, Hialeah, FL) using an argon-ion laser tuned at 488nm and 15mw. FITC fluorescence was collected by 575 DC+525BP filters. Data were collected in a four decade amplification. Debris was excluded by analysis of scatter properties. At least 10000 events per sample were stored in list mode files.17 Data were expressed as luminosity intensity FAU (fluorescence arbitrary units) or the percentage of positive cells for molecules studied. RT-PCR Total cellular RNA was extracted, purified, and reverse transcribed from secretory phase endometrial biopsies, endometrial epithelial cells, and adenoids by using Trizol reagent (Life Technologies, Gibco, Grand Island, NY) according to the manufacturer’s instructions. First strand cDNA was

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reverse transcribed from 1µg RNA employing an MMLV reverse transcriptase and advantage RT for PCR kit (60 mins/42°C) (Clontech, Palo Alto, CA). The cDNA solution was diluted to 100µl and stored at −20°C. The integrity of each cDNA preparation was analysed using βactin primers. The PCR amplification employed reagents supplied in a Taq DNA polymerase kit (BIOTAQ, Bioline, London, UK). Each reaction volume (25µl total) consisted of 67mM TrisHCl pH 8.8, 16mM (NH4)2SO4, 0.01% Tween-20, 2.5mM MgCl2, and 0.2mM dNTPs (Sigma, Saint Louis, MO), 0.2µM 3′ and 5′ primer and 2µl of cDNA, overlayed with 50µl mineral oil (Sigma. Taq DNA polymerase (0.5IU) was added at 94°C. PCR amplification was as follows: 5 minutes at 94°C, followed by multiple one minute cycles of denaturation at 94°C, annealing at 58–60°C, and extension at 72°C, followed by a final extension for 10 minutes at 71°C. The negative control included in each reaction consisted of H2O substituted for cDNA. PCR reaction products were analysed by gel electrophoresis in 2% agarose gel containing 0.5µg/ml ethidium bromide. Data were expressed as a percentage of the mean of all four samples and normalized for β-actin mRNA expression. The PCR assay was repeated at least three times on each cDNA sample.

HORMONAL AND EMBRYONIC REGULATION OF CHEMOKINES IL-8, MCP-1 AND RANTES IN THE HUMAN ENDOMETRIUM Cytokines and among them chemokines appear to be excellent candidates implicated in the early phases of human implantation. Chemokines are a family of cytokines with chemotactic activity for leukocytes. In reproductive biology these molecules and related cells are implicated in ovulation, menstruation, parturition, embryo implantation, and pathological processes such as preterm delivery, endometriosis, ovarian hyperstimulation syndrome, and HIV infection.16 Traditionally, based in the position of the first of two consecutive cysteine residues, they are classified into two groups, the α or CXC chemokines and the β or CC chemokines. Interleukin-8 (IL-8) (α-chemokines) is a potent chemoattractant and activator for neutrophils (Mukaida et al, 1994)18 and T-lymphocytes (Larsen et al, 1989).19 IL-8 is produced by a variety of cell types: monocytes, fibroblasts, lymphocytes, epithelial, and endothelial cells.20,21 It has been detected in the human reproductive tract, cervix,22 placenta,20 chorio-decidua23 and endometrium24–26 Monocyte chemoattractant protein (MCP-1), although belonging to the βchemokines subfamily, it is closely related to protein, and to a potent chemoattractant and activator for monocytes, macrophages, T-cells, basophils, mast cells, and also natural killers. MCP-1 is secreted by a number of cell types: endothelial cells,27 fibroblasts,28 monocytes,28 and

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lymphocytes.29 It has been detected in normal endometrium24,26 and endometriotic cells.30 The β-chemokine RANTES (regulated upon activation normal T cell expressed and secreted) is a chemoattractant for monocytes, eosinophils, and basophils. RANTES is located in eutopic endometrium and ectopic endometriosis implants.31 During implantation, leukocyte recruitment occurs in the endometrium, 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 driven by ovarian steroids32,33 and maybe seminal factors.34 Chemokines act on a range of leukocyte subsets, which in turn release a number of protease and other mediators that would facilitate embryo decidual invasion.23 In this study, we investigated the immunolocalization in vivo of chemokines IL-8, MCP1, and RANTES in the human endometrium and their hormonal regulation during the receptive phase of human implantation. Besides, embryonic regulation of the expression, production and secretion of these molecules in an in vitro model of EEC has been investigated by coculturing single human embryos with human EEC. To study the in vivo hormonal regulation of chemokines IL-8, MCP-1, and RANTES, endometrial biopsies were obtained in HRT cycles. Immunohistochemistry was carried out for protein localization. Flow cytometry was performed to study the effect in the intracellular produced chemokines by the EEC monolayers. Finally, Northern blot and RT-polymerase chain reaction (PCR) analysis were employed to study the chemokine mRNA expression by the EEC. This study shows that chemokines IL-8, MCP-1, and RANTES are produced in the human endometrium. RANTES was mainly detected in the stromal compartment and perivascular localization. Monolayers of endometrial epithelial cells do not appear to express RANTES,31 this is corroborated by the absence of RANTES mRNA in cultured normal endometrial epithelial cells (Fig 28.5). IL-8 and MCP-1 are localized in the glandular and luminal epithelium and vascular lining. Our results indicate that P, applied after a prior E2 priming, upregulates IL-8 and MCP-1 expression in the prereceptive (P+3; day 18) and in the receptive (P+6; day 21 of cycle), at the time when an embryo would be expected to implant. Other authors have reported similar findings employing a model of P withdrawal and maintenance in vivo.35 In experiments involving coculture of developing preimplantation embryos with confluent EEC monolayers, IL-8 mRNA and protein increase in the presence of blastocyst providing evidence for an embryonic regulation of this molecule. However, embryonic regulation of MCP-1 was not observed.36

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EMBRYONIC INDUCTION OF ADHESION MOLECULES IN HUMAN EEC During the adhesion phase of implantation different paracrine autocrine signals regulate molecular changes in endometrial cells. It has been reported that integrins, a class of cell adhesion molecules that participate in cell cell and cell substratum interactions and are present in all human cells, could act as mediators of embryo adhesion. Integrins are heterodimeric transmembrane proteins, composed of two subunits: α (130 to 210Kda) and β (90 to 200Kda).37 When the progesterone production begins and endometrial progesterone receptors (PR) are highest,38,39 the α1 and α4 subunits appear. In contrast, the production of the β3 is induced when the progesterone production is maximal and endometrial PR are lowest.38,39 To investigate the potential embryonic regulation of endometrial integrins we first localized the presence of immunoreactive β3, α4 and α1 in our in vitro human EEC model. Immunostaining for β3 was positive in plasma membrane of EEC, with increased staining intensity in wells from embryos that reached blastocyst stage compared to those EEC wells from arrested embryos. Flow cytometry showed a mean percentage of β3 stained cells of 24.1±5.7% in EEC cocultured with embryos that achieved the blastocyst stage (n=13) versus 9.5±1.6% (p<0.05) in those EEC cultured with arrested embryos (n=12). Immunostaining for

Fig 28.5 Agarose gel stained with ethidium bromide, showing the expression of IL-8 mRNA (N=3). Negative control (line 1). Next 9 lines (three patients); with the following order: control cells, EEC cocultured with embryos which arrest its development, and EEC cocultured with transferred embryos (blastocyst stage reached). In the lower

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panel the beta actin expression is showed as a housekeeping gene of the same samples. α1 and α4 integrins was negative in EEC monolayers studied, regardless of the presence or absence of embryos, and these findings were confirmed by flow cytometry.17 The possibility that the embryonic IL-1 system was involved in the endometrial β3 upregulation was investigated by neutralizing experiments showing a significant inhibition of β3 staining when EEC monolayers were cultured in the presence of EEC/blastocyst-conditioned media with (n=4) versus without (n=8) anti-human IL-1α+IL1β (1.65% versus 14.6%; p<0.05). SEM further showed that EEC cultured with conditioned media from cocultured blastocysts were healthy, and their shape was more rounded compared with the control EEC cultured without blastocyst conditioned media. In addition, retraction fibres (a sign of cell migration) and specifications of the plasma membrane, such as short stubby and long hairy microvilli, were much more abundant compared with the control. Strikingly, EEC in contact with conditioned media from cocultured blastocysts developed bulging of the membranes resembling pinopods, and this feature was not found in the control EEC. When the IL-1 system was blocked in those EEC cultured with conditioned media from cocultured blastocysts, SEM showed that the antibodies had clearly interrupted cell to plastic adhesion, but not cell to cell adhesion. EEC appeared generally similar to those cultured without antibodies, with the exception of retraction fibres.17 Dose response experiments further showed an upregulation of β3 positive cells when recombinant IL-1α+IL-1β were added to the medium at a concentration of 10pg/ml compared to control medium without IL1α+IL-1β (40% v. 20%, n=4). The functional relevance of the EEC β3 upregulation was tested using a mouse blastocyst adhesion assay. More mouse blastocysts attached to EEC previously in contact with human blastocyst (72.7%) compared with those EEC previously in contact with arrested embryos (40%).17 In conclusion, it has been shown that the embryonic IL-1 system is the mediator for individual human blastocysts to upregulate β3 integrin in cultured human EEC. These observations may imply an active role for the blastocyst in preparing the endometrium and regulating its own ability to implant.

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HORMONAL AND EMBRYONIC REGULATION OF ANTI-ADHESION MOLECULES IN HUMAN EEC The receptive status is the balance between the activation of adhesion molecules and the presence of a natural barrier that the implanting embryo may encounter in the epithelial glycocalix. Mucins are a family of highly glycosylated, high molecular weight (>250kDa) glycoproteins present on the surface of human epithelial cells including human EEC.40 MUC1 is an integral membrane glycoprotein with a large ectodomain containing a variable number (about 25–120) of 20 amino acid tandem repeats resulting in a large and highly extended structure, which is both immunogenic and extensively glycosylated.41 The short (56 residue) cytoplasmic region can be phosphorylated42 and seems to be associated with the cytoskeleton.43 There are two isoforms: a secreted form of MUC1, which lacks the cytoplasmic tail that may originate from a proteolytic cleavage site in the proximal extracellular domain or from a secreted variant arising from alternative splicing (MUC1/SEC)44,45 and MUC/Y (also originated by alternative splicing) which lacks the extracellular domain.42 MUC1/SEC efficiently binds and specifically interacts with the extracellular domain of MUC1/Y (Fig 28.6).46 It has been proposed that MUC1 functions as an anti-adhesion molecule and may inhibit the interaction between embryo and maternal apical epithelium during implantation, because of the possibility of creating an uterine barrier for implantation.47 This hypothesis is based on two lines of evidence: the unique structure of this molecule and the research performed in experimental animals. Evidences from the animal research support that MUC1 prevents embryo adhesion to endometrial epithelial cells in the mouse. First, selective enzymatic removal of mucins from the apical cell surface of uterine epithelia transform these cells from their normally non-receptive to a functionally receptive state. Second, uterine epithelium derived from

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Fig 28.6 Represented structure of a mature MUC1 complete isoform and its corresponding two isoforms originated by alternative splicing. The secreted isoform (MUC1/SEC) lacks the transmembrane domain and the cytoplasmic tail. The short transmembrane isoform (MUC/Y) (also originated by alternative splicing) lacks the extracellular domain consisting of a variable number of tendem repeats. MUC1/SEC efficiently binds and specifically interacts with the extracellular domain of MUC1/Y.43 MUC1 null mice are chronically receptive in vitro. Third, adhesion of cells that overexpress MUC1 to attachment competent mouse blastocysts is greatly reduced in comparison to their non-MUC1 expressing revertants. However, uterine control of MUC1 expression is speciesspecific and is not really predictable from a phylogenetic standpoint.48 MUC1 has been detected in the endometrium, and its expression varies within the menstrual cycle; moreover, variations are found among different species.41 In humans, MUC1 mRNA increases from the proliferative to secretory phase in endometrial tissue, decreasing in the late secretory phase.49 In patients who are having HRT no variation is observed with higher doses or time of exposure to E2; but when P is administered MUC1 increases in luminal and glandular epithelial cells, and this is corroborated with a higher expression of its mRNA.50,51

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Furthermore there is an increase in luminal epithelium and in mRNA expression of MUC1 comparing receptive endometrium in the implantation window with prereceptive endometrium before this period.51 In conclusion, MUC1 is highly expressed in the luminal and glandular epithelium when implantation is going to occur (Fig 28.7). Recently, we have observed that the complete MUC1 isoform protein and its mRNA is expressed in pre-adhesion blastocysts.52 MRNA expression increases the possibility that the embryo would be able, by alternative splicing, to synthesize the receptor binding protein isoforms. This expression could be an important mechanism of

Fig 28.7 Immunohistochemistry of paraffin embedded sections of endometrium from biopsy performed in the receptive phase of an HRT cycle with Mab HMFG-1 to the MUC1 mucin. Immunostaining appears intracellularly (a) and is also apically distributed (b). Signal appears also in gland secretions (c). No staining was observed in negative controls, (d) corresponds to the negative control. Binding of HMFG-1 to its MUC1 epitope is significantly enhanced after treatment with sialidase (b and c). Magnifications (a) and (c): ×400, (b) and (d): ×200.

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cross talk during the implantation phase. Implications of this interaction with morphological events makes it a candidate for the plasma membrane transformation that has been reported in preparation for blastocyst attachment like cytoskeleton disorganization.53 Paracrine downregulation of endometrial MUC-1 expression in the embryo has been reported in rabbits,54 a species in which the hormonal regulation of MUC1 is very similar to humans, suggesting that only healthy embryos have the ability to decrease MUC1 expression. In humans, using our in vitro model, by using flow cytometry we observed that MUC1 protein was increased by the presence of the embryo in the apposition phase. MUC1 mRNA expression was also increased in EEC with blastocysts.55 Therefore, although progesterone upregulates endometrial MUC-1 during the implantation window and the human blastocyst induces an increase of this molecule in human EEC, it is controversial as to whether this molecule is a true anti-adhesion molecule or serves as the first site of attachment for the embryo to the apical epithelial surface. One interesting possibility is that the healthy embryo may act to reinforce the maternal barrier to premature attachment. Similarly, expression of MUC1 in the blastocyst may prevent attachment at an anatomically inappropriate site such as the tubal epithelium. Nevertheless we can not exclude that embryo performs a local effect at the implantation site, cleaving or locally downregulating MUC1 presence as has been observed in rabbits.54

FUTURE PROSPECTS The bidirectional signals between the embryo and the endometrium are a prerequisite for normal embryonic implantation, and this new field constitutes a third dimension in the future studies on implantation. In humans, we hypothesize that normal hormonally regulated endometrium is the trigger of molecular events announcing the blastocyst that has to produce a new set of molecules to efficiently communicate and influence the endometrium. The human preadhesive blastocysts is able to upregulate endometrial epithelial adhesion molecules and anti-adhesion molecules as MUC1, endometrial epithelial chemokines such as IL-8 during the apposition phase (Fig 28.8). The embryonic molecules responsible for these endometrial effects require further investigation. Furthermore, more studies should be developed regarding the adhesion phase and the paracrine effect that implanting blastocysts could produce in these molecules. These findings have far reaching implications for assisted reproductive technology (ART) programs, where low implantation success rates remain a major problem. Further studies will clearly be required to address the mechanisms of synergistic communication between maternal and embryonic cells.

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Fig 28.8 Embryonic regulation of endometrial epithelial molecules in human implantation. Using our in vitro model we have observed an increase in plasma membrane associated molecules such as the integrin β3, the MUC1 and the IL-8 chemokine mediated by human blastocyst

REFERENCES 1 Bergh PA, Navot D. The impact of embryonic development and endometrial on the timing of implantation. Fertil Steril (1992); 58:537– 42. 2 Aplin JD. The cell biology of human implantation. Placenta (1996); 17:269–75. 3 Lopata A. Blastocyst-endometrial interaction: an appraisal of some old and new ideas. Mol Hum Reprod (1996); 7:519–25. 4 Galán A, O’Connor JE, Valbuena D, et al. The human blastocyst regulates endometrial epithelial apoptosis in embryonic adhesion. Biol Reprod (2000); (in press). 5 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. 6 Harper MJK, Kudolo GB, Alecozay AA, Jones MA. Platelet activating factor (PAF) and blastocyst-endometrial interactions. Prog Clin Biol Res (1989); 294:305–15. 7 Fazleabas A, Donnelly KM, Srinivasan S, Fortman JD, Miller JB. Modulation of the baboon (papio anubis) uterine endometrium by chorionic gonadotrophin during the period of uterine receptivity. Proc Natl Acad Sci (1999); 96:2543–8.

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8 Sulewski JM, Ward SP, McGaffic W. Endometrial biopsy during a cycle of conception. Fertil Steril (1980); 34:548–51. 9 Colston A, Herbert C, Maxson W, Hill G, Pittaway D. Cycle of conception endometrial biopsy. Fertil Steril (1986); 46:196–99. 10 Simón C, Mercader A, Garcia Velasco J, Remohí J, Pellicer A. Coculture of human embryos with autologous human endometrial epithelial cells in patients with repeated implantation failures. J Clin Endocrinol Metabol (1999); 84:2638–46. 11 De los Santos MJ, Mercader A, Frances A, et al. Immunoreactive human embryonic interleukin-1 system and endometrial factors regulating their secretion during embryonic development. Biol Reprod. 54:563–74. 12 Simón C, Piquette G, Frances A, Polan ML. Localization of interleukin-1 type I receptor and interleukin-1 Beta in human endometrium throughout the menstrual cycle. J Clin Endocrinol Metab (1993); 77:549–55. 13 Simón C, Piquette GN, Frances A, El-Danasouri I, Polan ML. The effect of interleukin-1 beta (IL-1b) on the regulation of IL-1 receptor type I and IL-1 beta messenger ribonucleic acid (mRNA) levels and protein expression in cultured human endometrial stromal and glandular cells. J Clin Endocrinol Metab (1994); 78:675–82. 14 Remohí J, Gartner B, Gallardo E, et al. Pregnancy and birth rates after oocyte donation. Fertil Steril (1997); 67:717–23. 15 Noyes RN, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril (1950); 1:3–25. 16 Simón C, Caballero-Campo P, Garcíia-Velasco JA, Pellicer A. Potential implications of chemokines in the reproductive function: an attractive idea. J Reprod Immunol (1998); 38:169–93. 17 Simón C, Gimeno MJ, Mercader A, et al. Embryonic regulation of integrins β3, α4 and α1 in human endometrial epithelial cells in vitro. J Clin Endocrinol Metabol (1997); 82:2607–16. 18 Mukaida N, Shiroo M, Matsushima K. Genomic structure of the human monocyte-derived neutrophil chemotactic factor IL-8. J Immunol (1989); 143:1366–71. 19 Larsen GG, Anderson AO, Apella A, Oppenheim YY, Hatsushima K. The neutrophil activating protein (NAP-1) is also chemotactic for lymphocytes. Science (1989); 243:1464–66. 20 Saito S, Kasahara T, Sakakura S, Mwekage H, Harada N, Ichijo M. Detection and localisation of interleukin-8 mRNA and protein in human placenta and decidual tissues, J Reprod Immunol (1994); 27:161–72. 21 Baggiolini M, Imbodem P, Detmers P. Neutrophil activation and the effects of interleukin-8-neutrophilactivating peptide-1 (IL-8/NAP-1). In: Baggiolini M, Sorg C. eds. Interleukin-8 (NAP-10) and Related Chemotactic Cytokines . Karger; Basel. (1992): 1–17.

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22 Barclay CG, Brennand JE, Kelly RW, Calder AA. Interleukin-8 production by the human cervix. Am J Obstet Gynecol (1993); 169:625–32. 23 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. 24 Arici A, Head JR, MacDonald PC, Casey ML. Regulation of interleukin-8 gene expression in human endometrial cells in culture. Mol Cell Endocrinol (1993); 94:195–204. 25 Critchley HOD, Kelly RW, Kooy J. Prerivascular localization of interleukin-8 in human endometrium: a preliminary report. Hum Reprod (1994); 8:1406–9. 26 Jones RL, Kelly RW, Critchley HOD. Chemokines and cyclooxygenase-2 expression in human endometrium coincides with leukocyte accmulation . Hum Reprod (1997); 12:1300–6. 27 Sica A, Wang JM, Colotta F, et al. Monocyte chemotactic and activating factor gene expression induced in endothelial cells by interleukin-1 (IL-1) and tumor necrosis factor (TNF-α). J Immunol (1990); 144:3034–8. 28 Yoshimura T, Leonard EJ. Secretion by human fibroblasts of monocyte chemoattractant protein-1, the product of the gene JE. J Immunol (1990); 144:2377–83. 29 Yoshimura T, Yuhki N, Moore S. Human monocyte chemoattractant protein 1 (MCP-1): full- length cDNA cloning, expression in mitogestimulated blood mononuclear leukocytes and sequence similarity to mouse competence gene JE.FEBS. Lett (1989); 244:487– 93. 30 Akoum A, Lemay A, McColl S, Turcot-Lemay L, Maheux R. Elevated concentration and biologic activity of monocyte chemotactic protein-1 in the fluid of patients with endometriosis. Fertil Steril (1996) 66:17– 23. 31 Hornung D, Ryan IP, Chao VA, Schriock DE, Taylor RN. Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells. J Clin Endocrinol (1997); 82:1621–8. 32 Robertson SA, Mayrhofer G, Seamark RF. Ovarian steroid hormones regulate granulocyte-macrophage colonystimulating factor synthesis by uterine epithelial cells in the mouse. Biol Reprod (1996); 54:265–77. 33 Wood GW, Hausmann X, Choudhuri R. Relative role of CSF-1, MCP1/JE, and RANTES in macrophage recruitment during successful pregnancy. Molecular Reprod Devel (1997); 46:62–70. 34 Robertson SA, Mau VJ, Tremellen KP, Seamark F. 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.

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35 Critchley HOD, Kelly RW, Lea RG, Drudy TA, Jones RL, Baird DT. Sex steroid regulation of leukocyte traffic in human decidua. Hum Reprod (1996); 11:2257–62. 36 Caballero-Campo P, Coloma J, Meseguer M, Simón C. Hormonal and embryonic regulation of chemokines IL-8, MCP-1 and RANTES in the human endometrium. J Clin Endocrinol Metab (2000); (submitted). 37 Lessey BA, Castelbaum AJ. Integrins in the endometrium. Reprod Med Rev (1995); 4:43–58. 38 Lessey BA, Yeh I, Castelbaum AJ, et al. Endometrial progesterone receptors and markers of uterine receptivity in the window of implantation. Fertil Steril (1996); 65:477–83. 39 Ingamells S, Campbell IG, Anthony FW, Thomas EJ. Endometrial progesterone receptor expression during the human menstrual cycle. J Reprod Fertil (1996); 106:33–8. 40 Devine P, McKenzie IFC. Mucins: structure, function and association with malignancy. Bioassays (1992); 14:619–25. 41 Meseguer M, Pellicer A, Simón C. MUC1 and endometrial receptivity. Molecular Hum Reprod (1998); 4:1089–98. 42 Zrihan-Licht S, Vos HL, Baruch A, et al. Characterization and molecular cloning of a novel MUC1 protein, devoid of tandem repeats, expressed in human breast cancer tissue. Eur J Biochem (1994); 224:787–95. 43 Boshell M, Lalani EN, Pemberton L, et al. The product of the human MUC1 gene when secreted by mouse cells transfected with the full length cDNA lacks the citoplasmic tail. Biochem Biphys Research Com (1992); 185:1–8. 44 Ligtenberg MJL, Kruishaar L, Buijs F, van Meijer M, Litvinov SM, Hilkens J. Cell associated epsialin is a complex containing two proteins derived for a common precursor. J Biol Chem (1992); 267:6171–7. 45 Aplin JD, Hey NA. MUC1, endometrium and embryo implantation. Bioch Soc Trans (1995); 23:826–31. 46 Baruch A, Hartmann M, Yoeli M, et al. The breast cancer-associated MUC1 gene generates both a receptor and its cognate binding protein. Can Res (1999); 59:1552–61. 47 Hey NA, Li TC, Devine PL, Graham RA, Aplin JD. MUC1 in secretory phase endometrium: expression in precisely dated biopsies and flushings from normal and recurrent miscarriage patients. Hum Reprod (1995); 10:2655–62. 48 Lagow E, DeSouza M, Carson DD. Mammalian reproductive tract mucins. Hum Reprod Upd (1999); 5:280–92. 49 Hey NA, Graham RA, Seif MW, Aplin JD. The polymorphic epithelial mucin MUC1 is regulated with maximal expression in the implantation phase. J Clin Endocrinol Metabol (1994); 78:337–42. 50 Meseguer M, Gaitán P, Mercader A, Caballero P, Remohí J, Pellicer A, Simón C. Hormonal regulation “in vivo” of MUC1 in mock cycles

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from patients undergoing oocyte donation. Abstract Book 1 Human Reproduction (1998); 13:121–22. (14th Annual Meeting of ESHRE). 51 Meseguer M, Caballero-Campo P, Martin JC, Galán A, Remohí J, Pellicer A, Simón C. Hormonal regulation “in vivo” of MUC1 in human endometrium. Fertil Steril (1999); 72:abstract. 52 Meseguer M, Caballero-Campo P, Aplin JD, Martin JC, Remohí J, Pellicer A, Simóon C. Hormonal and embryonic regulation of human endometrial MUC1. Biol Reprod 2000; submitted. 53 Murphey CR. The cytoskeleton of uterine epithelial cells: a new player in uterine receptivity and the plasma membrane transformation. Hum Reprod Update (1995); 1:567–80. 54 Hoffman LH, Olson GE, Carson DD, Chilton BS. Progesterone and implanting blastocyst regulate MUC1 expression in rabbit uterine epithelium. Endocrinology (1998); 139:266–71. 55 Meseguer M, Caballero-Campo P, O’Connor JE, Galán A, Martin JC, Remohí J, Pellicer A, Simón C. Supplement, J Soc Gyn Invest (1999); 6:143A.

29 The use of biomarkers for the assessment of uterine receptivity Bruce A Lessey

INTRODUCTION At the turn of the 20th century, we can look back proudly at the steady advances in the field of medicine. Despite the many successes, however, a surprisingly high number of couples remain who have difficulty in establishing or maintaining a pregnancy for reasons that defy detection. “Unexplained” infertility still accounts for up to 37% of cases.1 In 1995, it was estimated that one in six couples were infertile. The number of patients seeking care for infertility that year exceeded 9 million couples,2 up from 2.4 million reported in 1989.3 A large percentage of this increase is now thought to represent defects in implantation and a relative or absolute lack of uterine receptivity. As we enter the next century and new millennium, the study of implantation and such defects in uterine receptivity remain an area of rapid progress and great debate. A better understanding of potentially occult defects has recently been advanced by the use of endometrial biomarkers. The focus of this chapter will be on the use of endometrial biomarkers in implantation research, and how this area is developing to help us comprehend the implantation process and to improve diagnosis for disorders that affect fertility through interference with embryoendometrial interactions. In 1999, the National Institutes of Health (NIH) organized a symposium on the use of biomarkers for the diagnosis and treatment of a wide variety of medical conditions (http://www4.od.nih.gov/biomarkers/index.htm). Maximizing the utility of biomarkers in diagnosis and assessment infertility will require an extensive infrastructure of investigators. This infrastructure will need to be comprised of: an environment in which basic science can proceed to translational research; availability of technological resources; and clinical researchers, biostatisticians, and epidemiologists who design studies to validate the use of biomarkers. This chapter reviews the current status of uterine factors that have been considered as potentially useful biomarkers to evaluate the functional state of the endometrium. It is envisioned that the use of such markers will provide a means to understand the mechanism of implantation, identify women at risk for implantation

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failure, to study the efficacy of various treatments for infertility and to develop new and safer contraceptives that target endometrial receptivity.

IMPLANTATION The endometrium is unique as one of the few tissues into which an embryo will not attach and grow, except for a narrow period of uterine receptivity.4,5 The endometrium of most mammals undergoes a series of developmental changes in response to ovarian steroids that result in a defined period of receptivity towards embryo implantation.6,7 The putative “window” of implantation, as first suggested by Finn,8 has been demonstrated in both animal models9–12 and in humans.13,14 In women, the stages of implantation were documented in hysterectomy samples taken during the late secretory phase of women suspected of being pregnant.15 Of the 34 embryos identified in these specimens, all of the

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Fig 29.1 Photomicrographs of endometrium from the proliferative (A) and secretory phase (B). The proliferative phase is marked by mitotic activity in the glandular epithelium and straight, round, glandular structures. The secretory phase is noted for secretions from the glandular epithelium and eventually pseudodecidual changes in the stromal compartment. Also shown is an early (C) implantation site during stage 5a of implantation. At this stage the maternal vasculature remains intact but becomes surrounded by the expanding syncytium. This examples of a human implantation site was generously provided and photographed from the Carnegie Collection by Dr Allen Enders (University of California, Davis, CA). embryos that had undergone attachment in women who were from secretory phase day 21 or beyond; uterine material obtained prior to day 20 harbored embryos that were all still free floating within the uterine cavity or still within the fallopian tube. A more recent confirmation of a “window” of implantation in humans came from studies by Navot and coworkers. These investigators described the early rise in human chorionic gonadotrophin (hCG) as an indicator of early attachment.16 Further studies from this group used donor embryos replaced into hormonally prepared recipients from different stages of the secretory phase. Again, these results support the concept of a defined period of uterine receptivity that corresponds to cycle days 20 to 24.13,17 All of these studies define a time of implantation that corresponds surprisingly well to other markers of uterine receptivity described later in this chapter. The development of uterine receptivity is driven by ovarian steroids, estrogen, and progesterone.18 The endometrium undergoes marked developmental changes during the proliferative (Fig 29.1A) to secretory (Fig 29.1B) phases of the cycle. Estradiol is a mitogenic hormone that leads to growth of the endometrial lining, and induction of receptors for estrogen and progesterone.19,20 After ovulation, progesterone transforms the endometrium into the secretory structure that nurtures the early blastocyst and prepares for its attachment and ingrowth (Fig 29.1C). This transformation is accompanied by sequential and well-orchestrated expression of specific genes that both facilitate and sometimes limit the ability of the blastocyst and trophoblast to invade into the uterine lining. The first measure of the endometrial capacity for implantation that was used included the developmental histology changes, collectively known as

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“endometrial dating”.21 This collection of histologic criteria are shown in Figure 29.2 and have been used continuously as the “gold standard” for endometrial assessment for the past 50 years. The use endometrial dating lead to the description of the first recognized defect in

Fig 29.2 Histologic “dating” criteria of Noyes et al.21 The set of histologic criteria shown here have been used for the past 50 years as the method of choice to assess the endometrium and its response to the

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hormonal milieu. Reproduced with permission from the American Society for Reproductive Medicine. the endometrium, namely luteal phase deficiency (LPD).22 The diagnosis of LPD is a heterogeneous condition that is related to subfertility and is thought to be the result of inadequate hormone stimulation or endometrial response.23 This common yet controversial entity is often found in couples with infertility and may be the most common cause of recurrent pregnancy loss.24 The usefulness of histologic dating as a meaningful biomarker remains problematic because of high inter- and intraobserver variation, and dating alone may miss other defects in endometrium that are not reflected in histology or changes in morphology. Such occult defects may exist independent of histologic delay and may account for many cases of unexplained infertility.25,26 ARE BIOMARKERS OF ENDOMETRIAL RECEPTIVITY NECESSARY? Now 50 years after Noyes,21 we still rely on histologic evaluation of the endometrium to assess its function. The use of “endometrial dating” is, however, not a perfect assay of the endometrium.27 As the criteria were based entirely on biopsies from infertile women rather than from fertile subjects, there are concerns about the reliability and context of these indices. These studies were performed at a time prior to the modern practice of timing the biopsy to the mid-luteal urinary surge in luteinizing hormone (LH). This has lead to a shift in the timing of biopsy from late luteal phase to a time more relevant to implantation, the mid-secretory phase. As recently reported, a surprising consequence of this has been the appreciation that mid-luteal histology is actually a hypervariable period within the cycle, especially with regards to the glandular elements.28 What has been considered abnormal in the past (gland-stromal dyssynchrony) now seems to be part of normal differences between cycles from woman to woman. This variability has been described for another morphological feature, namely that of pinopods. As discussed below, these microprojections or blebs that extend from the luminal surface have a one to two day lifespan, but their timing of expression is variable between cycles.29 The establishment of appropriate biomarkers for the assessment of endometrial function may address other concerns associated with endometrial dating. Histology independent measures of the functional state of the endometrium that accurately predicts the potential for successful pregnancy will reduce our reliance on empiric therapies and lessen the frustration for patients with “unexplained” infertility or recurrent pregnancy loss. Biomarkers are already providing clues into the regulatory mechanisms of implantation.30 The appropriate biomarker or

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panel of biomarkers will provide early diagnosis and treatment, including the appropriate use of assisted reproductive technologies (ART).

Table 29.1. List of selected markers of uterine receptivity. Biomarker Predominant cell Function type Hormones/Receptors Calcitonin Glandular and Enhance embryo luminal epithelium quality/phenotype IGF-II Glandular and mitogenic luminal epithelium Progesterone Glandular Suppress uterine receptors (PR) epithelium receptivity Cytokines/growth factors/receptors CSF-1 Luminal epithelium Leukemia inhibitory Glandular and Enhance embryo factor (LIF) luminal epithelium quality/phenotype Heparin binding Glandular and Enhance embryo EGF luminal epithelium quality/phenotype IL-1 system Glandular and Enhance embryo luminal epithelium quality/phenotype Cell adhesion molecules Unknown/?Role In cell αvβ3/α4β1 integrins Glandular or luminal epithelium attachment Trophinin/tastin Glandular and Cell attachment luminal epithelium MAG Glandular and ? luminal epithelium MUC-1 Glandular and Anti-adhesion? luminal epithelium Decidual proteins IGF-BP1 Decidua Controls invasion of trophoblast Cadherin-11 Decidua Attachment/ adhesion Transcription factors HOXA10 Epithelium and Regulates gene expression stroma HOXA11 Stromal Regulates gene expression Structural features Pinopods Luminal epithelium Reduce intraUterine fluid/

References

147 72 20,140

148 68,149 30,110 150

41,43 53 60 151

72 152 120, 145 121 153

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Attachment Platform? Ultrasound indices

Serum markers Glycodelin (PP14)

Endometrial thickness or echogenicity

(From glandular epithelium) Serum progesterone Corpus luteum

154, 155

Immunologic suppression, 156 blocks fertilization? Prepares endometrium 157

AVAILABLE BIOMARKERS OF UTERINE RECEPTIVITY A list of available candidate biomarkers that can be used to assess uterine receptivity is growing rapidly and include immunohistochemical markers, ultrastructural components, and serum markers. A selection of the best characterized candidate biomarkers is provided in Table 29.1. Each of these factors maintain a temporal pattern of expression that can be viewed in the context of the putative window of implantation on cycle day on 20 to 24 (Fig 29.3). While certain biomarkers such as calcitonin or leukemia inhibitory factor (LIF) are aligned closely within this window, others have a pattern of expression that is inversely related to the time of maximal uterine receptivity (epithelial progesterone receptors (PR)). Selected biomarkers are described in greater detail below, grouped by category. STEROID AND PEPTIDE HORMONES Progesterone (P4) is the major steroid hormone of pregnancy and is a product of the corpus luteum that forms following ovulation. The production of P4 begins after the LH surge, and rising serum concentrations of P4 are primary responsible for a myriad of progestin induced proteins and define the conversion from a proliferative to a secretory endometrium. As a marker, P4 has been extensively studied, primarily to diagnose LPD,

Fig 29.3 The pattern of selected markers of uterine receptivity relative to the putative “window of implantation” (shaded area) is shown. Note that some markers align closely with this window (co-expression of certain

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integrins, calcitonin, LIF), while others are expressed before the window of implantation (HB-EGF). Other markers are expressed after the window has effectively closed (glycodelin). These patterns are indicative of the regulation and mechanism for the establishment of uterine receptivity. Each biomarker is ultimately controlled by serum ovarian steroids (estradiol and progesterone) and therefore the establishment of uterine receptivity and successful pregnancy is subject to variation and may be altered in the presence of an aberrant hormonal or paracrine milieu. Reprinted with permission from the American Society for Reproductive Medicine.21 a leading cause of recurrent pregnancy loss and infertility. Unfortunately, a single measurement of P4 may not accurately reflect cumulative P4 concentrations23 and does not correlate well with endometrial histologic dating.31 Although easy to measure, it is likely that only those concentrations of P4 that are grossly abnormal are reliable indicators of defective luteal function. Since normal serum P4 may also be accompanied by an inadequate endometrial response, the test may be falsely reassuring. Certainly, from the standpoint of the embryo, it is the endometrial response to P4 that is most critical and not the actual serum level of P4 that matters most. Calcitonin is a peptide hormone that is well known for its endocrine role in calcium homeostasis. Since the tissue of origin is traditionally the thyroid gland, the discovery that endometrium is a source of calcitonin was unexpected. Further, its expression by glandular and luminal epithelium coincides precisely with the time of implantation suggesting that its expression is hormonal dependent.32,33 It now appears that this peptide hormone functions as a paracrine factor arising from the endometrium, acting directly on the cells of the blastocyst, perhaps further embryonic differentiation.34 MEMBRANE-BOUND PROTEINS AND MUCIN5 Integrins are perhaps the best characterized of the immunohistochemical biomarkers of uterine receptivity.35 There are now over 22 separate integrins described. This class of cell adhesion molecules consists of heterodimeric pairs of peptides, always incorporating a and a β subunits. Together, the intact pair forms a membrane bound receptor that recognizes various extracellular matrix molecules as well as a variety of

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other CAMs and serves key roles in cell-cell and cell-substratum adhesion. There is now increasing interest in the use of integrins to assess uterine receptivity36–38 and significant data to suggest their involvement in both fertilization and implantation.39,40 The endometrium is an active site of integrin expression with both constitutive and cycle-dependent expression.41–45 At least three integrins have been reported to frame the window of implantation, co-expressed on glandular epithelium only during cycle days 20 to 24, corresponding to the putative window of implantation43 (Fig 29.4). The three integrins shown in this figure, the αvβ3 vitronectin receptor, the α1β1 collagen receptor and the α4β1 fibronectin receptor are all co-expressed during the time of uterine receptivity. The apical pole of the luminal epithelium expresses both αvβ3 and αvβ5 at the time of implantation.46 The former appears around mid-secretory phase of the cycle while αvβ5 increases during the early secretory phase. The localization to the apical pole of the

Fig 29.4 Relative Intensity of staining for the epithelial α4, β3 and al integrin subunits throughout the menstrual cycle and in early pregnancy. Immunohistochemical staining was assessed by a blinded observer using the semiquantitative HSCORE (ranging from 0 to 4) and correlated to the estimate of histological dating based on pathologic

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criteria or by LMP in patients undergoing therapeutic pregnancy termination. The negative staining (open bars) was shown for immunostaining of an HSCORE≤0.7, for each of the three integrin subunits. Positive staining for all three integrin subunits was seen only during a four day interval corresponding to cycle day 20–24, based on histologic dating criteria of Noyes et al.21 This interval of integrin coexpression corresponds to the putative window of implantation. Of the three, only the αvβ3 integrin was seen in the epithelium of pregnant endometrium. Reproduced with permission from the American Society of Reproductive Medicine.43 luminal epithelium suggests a role for these integrins in initial embryoendometrial interaction. These integrins recognize ligands containing the three amino acid sequence arg-gly-asp (RDG), implicated in trophoblast attachment and outgrowth.47,48 We have shown that neutralization of the mouse αvβ3 integrin reduces the number of embryos that will implant.49 Possible roles include signaling the embryo and stimulating the invasive phenotype of the embryo or placental cytotrophoblast.50 Other cell adhesion molecules have been examined in the endometrium at the time of implantation in either epithelial or stromal cells as possible participants in embryo/trophoblast interactions. These include the hyaluronic acid receptor, CD 44,51,52 trophinin,53 and cadherin-11.54,55 CD44 is present in various isotypes and is more strongly expressed in the secretory endometrium and decidua. Trophinin is a novel cell adhesion molecule that has been well described in the mouse53 and has been shown to be present in human endometrium as well. It is expressed on the luminal surface in both rodents and humans and may mediate cell-cell interaction between epithelial cells from maternal and epithelial surfaces. Cadherin-11 is another unique member of the cadherin family of CAMs. It is interesting since it is both an epithelial marker as well as being expressed in a cycle dependent manner in the decidua.55 This cadherin is also expressed on the epithelial component of the trophoblast suggesting a role in endometrial-embryonic interaction.54 Mucins are large glycoproteins that coat luminal and glandular surfaces. These complex glycopeptides have been suggested to have utility as detectable markers for both LPD and unexplained infertility.56,57 MUC-1 is a mucin that has been suggested to present a barrier to implantation when the endometrium is non-receptive and must be removed at the time of implantation in both rodents58 and primates.59 In

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humans, the situation for MUC-1 is less clear, since it is present throughout the secretory phase in normal cycles and does not disappear at the time of implantation. An antibody, D9B1, recognizing an oligosaccharide epitope, detected reduced expression of this marker in women with unexplained infertility compared with fertile controls.57 MAG is another mucin recognized by mouse ascites fluid, that has been studied in women with infertility and also appears to detect those with poor reproductive outcome.60 SECRETED PROTEINS, GROWTH FACTORS AND CYTOKINES One of the first and most abundant endometrial products is PP14, now referred to as glycodelin.61 This product of the endometrial epithelium protein has been shown to reduce sperm binding to the zona pellucida62 and was thought to have immune regulatory roles.63 Glycodelin may play a greater part after pregnancy has been established and thus may not be as useful in scrutinizing the initial phases of uterine receptivity during the putative window of implantation.64 Although it can readily be measured by immunohistochemistry, its usefulness has been primarily confined to serum measurements. Glycodelin appears to be reduced in women with LPD65 and may be reduced in those with recurrent pregnancy loss.66 Several cytokines and growth factors including LIF,67–69 heparin binding epidermal growth factor (HB-EGF),70,71 and insulin-like growth factor II (IGF-II)72 appear in the endometrium at or slightly before the receptive period in women and may be useful as markers of a receptive endometrium. LIF was one of the first cytokines found to be critical for implantation. It was originally shown to induce differentiation of a myeloid leukemia cell line, M1.73 For those who study implantation, interest in this cytokine was significantly increased by the observation that LIF appears in the mouse uterus on day 4 of pregnancy, corresponding to the day that embryos implant in this species.67 It was observed that female LIF −/− homozygous mice were infertile, while the fertility of the male mice was unaffected. Normal free-floating blastocysts found within the uteri of the LIF −/− females, failed to implant unless transferred to the uteri of normal female mice.68. Exogenous LIF partially reverse the defect in implantation in the LIF −/− mice. It now appears that LIF is essential for decidualization as attempts to induce decidualization in LIF −/− mice was unsuccessful.74 In humans, LIF is expressed during the window of implantation69,75 and may be reduced in some women with infertility.69 IL-1 FAMILY Interleukin-1 (IL-1) represents a family of peptides composed of IL-1α (159 amino acids), IL-1β (153 amino acids) and an inhibitor called IL-1 receptor antagonist (IL-1ra; 152 amino acids).76 IL-1β is produced by the

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endometrial stroma77 and by macrophages,78 in oocytes and embryos.79,80 The IL-1 system has also been examined in implantation sites.81 IL-1β, IL1R tl, IL-1 receptor antagonist (IL1-ra) were localized to macrophages in the villous trophoblast. The maternal-trophoblast interface and the maternal decidual during early pregnancy. In the endometrium, IL-1 modulates epithelial cell function supporting the theory that the embryo directly facilitates its own implantation82 and facilitates decidualization.77 The receptors for IL-1β include two subtypes termed type I (IL-1R tl)83 and type II (IL-1R tll).84 These receptors for IL-1 have been identified in the epithelial component of the human endometrium.85 Simón86 measured IL-1R tl throughout the menstrual cycle in human endometrium with significantly elevated expression during the mid and late secretory phase. They have shown that the IL-1R tl is increased by exposure to IL-1β.87 Simon et al found IL-1ra present throughout the menstrual cycle, localized primarily to the endometrial epithelium but present at significantly higher concentrations during the follicular phase compared with the earlier and mid secretory phases.88 In mice IL-1ra has been used to successfully block implantation suggesting a critical role for IL-1 in this process.89 Implantation rates in female mice injected with IL-1 receptor antagonist were much lower, independent of toxic effects of this compound to the embryo, suggesting an important role for IL-1 during implantation. Other postulated functions of IL-1 during implantation include its stimulatory effect of prostaglandin E290 and the stimulatory effect of IL1 on hCG release by human trophoblast.91,92 IL-1β inhibits stromal cell growth93 and inhibits attachment of blastocytes to fibronectin while enhancing blastocyst outgrowth.94 IL-1β95 and IL-1α96 have also been reported to be toxic to early embryos. Simón et al have shown that IL-1 stimulates integrin expression on endometrial epithelium, in vitro,82 suggesting that the embryo may play a part in endometrial protein expression at the time of implantation. The precise role and essential nature of this cytokine ultimately awaits further investigation. THE EGF FAMILY The EGF family of growth factors now encompasses both EGF and TGFα as well as many EGF-like molecules including amphiregulin, heparin binding EGF (HB-EGF), and betacellulen.97–102 EGF acts through a specific receptor, ErbB. There are now several epidermal growth factorrelated peptide members of the ErbB family of receptor kinases that have been described. Four members include ErbB receptor (ErbB-1), ErbB-2, ErbB-3, and ErbB-4, each with individual specificity’s for the various ligands thus far described.103 EGF has been immuno-localized during the menstrual cycle in human endometrium, decidua and placenta.104,105 In the primate model, Fazleabas et al have shown that IGF-1 and EGF were present in the glandular epithelium of the endometrium and underwent changes in the implantation

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site of the species, suggesting a role in trophoblast invasion and/or decidualization.106 HB-EGF and the other EGF-like molecules are expressed concomitant with implantation in the mouse,70,107,108 along with the EGF receptors.109 Mouse embryos adhere to the transmembrane of endometrium HB-EGF,110 and such interaction has been suggested to be independent of EGF receptor binding.111 In the human HB-EGF is temporally expressed during the time of uterine receptivity.70,112,113 On the basis of recent studies, it is tempting to postulate that HB-EGF maintains a role in both adhesion and development in the embryo.110,114 HB-EGF has been shown to improve the development or quality of human embryos in vitro115,116 and may be a paracrine factor in human endometrium regulating other key epithelial gene products. OTHER IMMUNOHISTOCHEMICAL BIOMARKERS OF UTERINE RECEPTIVITY Other marker proteins that appear critical to implantation in rodents include the homeobox genes HOXA-10117,118 and HOXA-11.119 These transcription factors now seem to have relevance for human implantation as well.120,121 The HOXA genes are important in segmental development and are both expressed by the endometrium, specifically during the midsecretory phase of the menstrual cycles. They are now thought to be master regulatory genes that control other factors important for implantation. Another factor critical for implementation is the enzyme cyclooygenase-2, a rate limiting enzyme in prostaglandin synthesis.122 This factor also seems to be a promising as potential biomarkers in the endometrium of primates as well.123,124 Finally, the receptor for the cytokine IL-11 seems to be critical for implantation and decidualization.125 The similarity in phenotype between these gene knockout mice and the COX-2 and LIF knockouts emphasize the importance of decidualization but also suggest common pathways are being targeted in these gene mutation studies. STRUCTURAL FEATURES Pinopods represent an ultrastructural feature of mid-cycle endometrium that has been proposed as a reliable marker of receptivity.126 These bleblike projections from the apical pole of the luminal surface of the endometrium was first described by Psychoyos127 and are best viewed by scanning electron microscopy. The pinopods’ principal value resides in the correlation between the appearance of pinopods and the temporal correlation to the window of implantation, their spatial localization to the luminal surface,128 and the in vitro data showing interactions between embryo and pinopod.129 While these endometrial microprojections have been suggested to function in the absorption of luminal fluid from the uterine cavity,130 another purpose of pinopods may be to elevate the

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implantation surface toward the embryo above any anti-adhesion molecules (such as MUC-1) that have been associated with a blockade of implantation.58 Modification of the luminal surface could facilitate the interaction between embryonic and endometrial adhesion molecules.131–134 CLINICAL USE OF BIOMARKERS OF ENDOMETRIAL RECEPTIVITY Endometrial biomarkers, such as those described in this chapter, have been felt to hold promise as diagnostic tools for the evaluation of causes of infertility and to better understand unexplained recurrent pregnancy loss. Pinopods, for example, have been extensively examined in ovarian stimulation protocols including superovulation135 or estrogen replacement protocols,29 to explore how cycles using ART compare to normal menstrual cycles. LPD is a major cause of infertility owing to inadequate progesterone concentrations or a diminished response to ovarian steroids.23 It is known that LPD is frequently associated with recurrent pregnancy loss136 and commonly diagnosed in women with unexplained infertility.26,137 Without synchronous development of the endometrial and embryo, successful implantation may not occur.138 Some of the biomarkers used to examine this entity include mucin epitopes,56 prolactin,139 and glycodelin,65 each advocated as potentially useful adjuncts for the diagnosis of LPD. PR, which are normally downregulated on endometrial epithelium by the time of implantation,20 are also potentially useful for the diagnosis of LPD. We found that women with histologic delay and LPD frequently had a greater degree of epithelial PR expression during the window of implantation. This increase in PR was associated with a decrease in other markers such as the αvβ3 integrin.140 The endometrial integrins have been extensively studied as potential markers of uterine receptivity.41,141 Given their precise expression during the menstrual cycle, three integrins, α1β1, α4β1 and αvβ3 were examined as an alternative for endometrial dating.28 Although they appear to have utility along with the criteria of Noyes et al,21 their use alone does not appear to be sufficient to “date” endometrial progression. The expression of the αvβ3 integrin may have utility for the assessment of uterine receptivity.41 The appearance of the αvβ3 integrin at the time of implantation offers a useful internal marker of endometrial progression that is consistently missing in the presence of LPD and associated histologic delay. In addition, the successful treatment of LPD with exogenous hormonal support results in the return of endometrial αvβ3 expression.26,140 Beyond this delay in histologic development, evidence also suggests that this integrin can be missing without any associated histologic delay. The existence of these otherwise occult defects have been suggested by studies of patients with endometriosis,44 hydrosalpinx,142 and polycystic ovary syndrome.28,143

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The existence of uterine receptivity defects has also been advanced by the study of other endometrial biomarkers, including the mucins,56,57,60 endometrial bleeding associated factor (ebaf),144 PP14,64 LIF,69 and HOXA10.145,146 There is now considerable interest in establishing the nature of such defects. A better clarification of their existence, prevalence, and effective means to treat such defects will lead to better diagnosis and treatment of unexplained infertility and recurrent pregnancy loss, and improve the selection criteria for in vitro fertilization.

FUTURE DIRECTIONS Now, having entered the 21st century, clinicians and scientists continue to debate the relative importance of embryonic versus endometrial quality. It is likely that implantation failure accounts for a subset of the growing number of couples with infertility. Certainly, we will find that implantation failure is multifactorial in its origin and a problem that had often gone unrecognized. With microarray DNA chip technology and continued discovery of critical factors and cofactors involved in implantation, we envision that a test will be soon be discovered that can reliably measure key factors endometrial tissue or uterine flushings. Any future progress will require extensive clinical investigation and a consensus by the scientific community regarding the definition of true defects in uterine receptivity. What criteria will be accepted to prove the existence of such defects and what treatments are best used to correct these defects represent other unresolved yet important issues for the use of uterine receptivity biomarkers.

ACKNOWLEDGEMENTS This research was supported by NICHD/NIH through cooperative agreement U54 HD30476 (BL) as part of the Specialized Cooperative Centers Program in Reproduction Research, the National Cooperative Program on Markers of Uterine Receptivity for Blastocyst Implantation (HD 34824; BL).

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90 Cole OF, Sullivan MHF, Elder MG. The “interleukin 1 receptor antagonist” is a partial agonist of prostaglandin synthesis by human decidual cells. Prostaglandins (1993); 46:493–8. 91 Steele GL, Currie WD, Leung EH, Ho Yuen B, Leung PCK. Rapid stimulation of human chorionic gonadotropin secretion by interleukin1β from perifused first trimester trophoblast. J Clin Endocrinol Metab (1992); 75:783–8. 92 Yagel S, Lala PK, Powell WA, Casper RF. Interleukin-1 stimulated human chorionic gonadotropin secretion by first trimester human trophoblast. J Clin Endocrinol Metab (1989); 68:922–95. 93 Van Le L, Oh ST, Anners JA, Rinehart CA, Halme J. Interleukin-1 inhibits growth of normal human endometrial stromal cells. Obstet Gynecol (1992); 80:405–9. 94 Haimovici F, Hill JA, Anderson DJ. The effects of soluble products of activated lymphocytes and macrophages on blastocyst implantation events in vitro. Biol Reprod (1991); 44:69–75. 95 Hill JA, Haimovici F, Anderson DJ. Products of activated lymphocytes and macrophages inhibit mouse embryo development in vitro. J Immunol (1987); 139:2250–4. 96 Fakih H, Baggett B, Holtz G, Tsang KY, Lee JC, Williamson HO. Interleukin-1: a possible role in the infertility associated with endometriosis. Fertil Steril (1987); 47:213–7. 97 Fisher DA, Lakshmanan J. Metabolism and effects of epidermal growth factor and related growth factors in mammals. Endocr Rev (1990); 11:418–42. 98 Savage CR, Jr, Inagami T, Cohen S. The primary structure of epidermal growth factor. J Biol Chem (1972); 247:7612–21. 99 Shoyab M, Plowman GD, McDonald VL, Bradley JG, Todaro GJ. Structure and function of human amphiregulin: a member of the epidermal growth factor family. Science (1989); 243:1074–6. 100 Watanabe T, Shintani A, Nakata M, et al. Recombinant human betacellulin. Molecular structure, biological activities, and receptor interaction, J Biol Chem (1994); 269:9966–73. 101 Marquardt H, Hunkapiller MW, Todaro GJ. Rat transforming growth factor type 1: Structure and relation to epidermal growth factor. Science (1984); 223:1079–82. 102 Higashiyama S, Abraham JA, Miller J, Fiddes JC, Klagsbrun M. A heparin-binding growth factor secreted by macrophage-like cells that is related to epidermal growth factor. Science (1991); 251:936–9. 103 Beerli RR, Hynes NE. Epidermal growth factor-related peptides activate distinct subsets of ErbB receptors and differ in their biological activities. J Biol Chem (1996); 271:6071–6. 104 Hofmann GE, Scott RTJ, Bergh PA, Deligdisch L. Immunohistochemical localization of epidermal growth factor in human endometrium, decidua, and placenta. J Clin Endocrinol Metab (1991); 73:882–7.

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105 Hofmann GE, Drews MR, Scott RTJ, Navot D, Heller D, Deligdisch L. Epidermal growth factor and its receptor in human implantation trophoblast: immunohistochemical evidence for autocrine/paracrine function. J Clin Endocrinol Metab (1992); 74:981–8. 106 Fazleabas AT, Hild-Petito S, Verhage HG. Secretory proteins and growth factors of the baboon (Papio anubis) uterus: Potential roles in pregnancy. Cell Biol Int (1994); 18:1145–54. 107 Das SK, Chakraborty I, Paria BC, Wang X-N, Plowman G, Dey SK. Amphiregulin is an implantation-specific and progesterone-regulated gene in the mouse uterus. Mol Endocrinol (1995); 9:691–705. 108 Das SK, Das N, Wang J, Lim H, Schryver B, Plowman GD, Dey SK. Expression of betacellulin and epiregulin genes in the mouse uterus temporally by the blastocyst solely at the site of its apposition is coincident with the “window” of implantation. Dev Biol (1997); 190:178–90. 109 Lim H, Das SK, Dey SK. erbB genes in the mouse uterus: Cellspecific signaling by epidermal growth factor (EGF) family of growth factors during implantation. Dev Biol (1998); 204:97–110. 110 Raab G, Klagsbrun M. Heparin-binding EGF-like growth factor. Biochim Biophys Acta Rev Cancer (1997); 1333:F179–F199. 111 Paria BC, Elenius K, Klagsbrun M, Dey SK. Heparin-binding EGFlike growth factor interacts with mouse blastocysts independently of ErbB1: a possible role for heparan sulfate proteoglycans and ErbB4 in blastocyst implantation. Development (1999); 126:1997–2005. 112 Birdsall MA, Hopkisson JF, Grant KE, Barlow DH, Mardon HJ. Expression of heparin-binding epidermal growth factor messenger RNA in the human endometrium. Mol Hum Reprod (1996); 2:31–4. 113 Leach RE, Khalifa R, Ramirez ND, et al. Multiple roles for heparinbinding epidermal growth factor-like growth factor are suggested by its cell-specific expression during the human endometrial cycle and early placentation. J Clin Endocrinol Metab (1999); 84:3355–63. 114 Tamada H, Higashiyama C, Takano H, Kawate N, Inaba T, Sawada T. The effects of heparin-binding epidermal growth factor-like growth factor on preimplantation-embryo development and implantation in the rat. Life Sci (1999); 64:1967–73. 115 Martin KL, Barlow DH, Sargent IL. Heparin-binding epidermal growth factor significantly improves human blastocyst development and hatching in serum-free medium. Hum Reprod (1998); 13:1645–52. 116 Sargent IL, Martin KL, Barlow DH. The use of recombinant growth factors to promote human embryo development in serum-free medium. Hum Reprod (1998); 13 (Suppl. 4) 239–48. 117 Benson GV, Lim HJ, Paria BC, Satokata I, Dey SK, Maas RL. Mechanisms of reduced fertility in Hoxa-10 mutant mice: Uterine homeosis and loss of maternal Hoxa-10 expression. Development (1996); 122:2687–96.

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118 Satokata I, Benson G, Maas R. Sexually dimorphic sterility phenotypes in Hoxa-10 deficient mice. Nature (1995); 374:460–3. 119 Taylor HS, Vanden Heuvel GB, Igarashi P. A conserved Hox Axis in the mouse and human female reproductive system: late establishment and persistent adult expression of the Hoxa cluster genes. Biol Reprod (1997); 57:1338–45. 120 Taylor HS, Arici A, Olive D, Igarashi P. HOXA 10 is expressed in response to sex steroids at the time of implantation in the human endometrium. J Clin Invest (1998); 101:1379–84. 121 Taylor HS, Igarashi P, Olive DL, Arici A. Sex steroids mediate HOXA11 expression in the human peri-implantation endometrium. J Clin Endocrinol Metab (1999); 84:1129–35. 122 Chakraborty I, Das SK, Wang J, Dey SK. Developmental expression of the cyclo-oxygenase-1 and cyclooxygenase-2 genesin the periimplantation mouse uterus and their differential regulation by the blastocyst and ovarian steroids. J Mol Endocrinol (1996); 16:107–22. 123 Jones RL, Kelly RW, Critchley HOD. Chemokine and cyclooxygenase-2 expression in human endometrium coincides with leukocyte accumulation. Hum Reprod (1997); 12:1300–6. 124 Kim JJ, Wang J, Bambra C, Das SK, Dey SK, Fazleabas AT. Expression of cyclooxygenase-1 and -2 in the Baboon endometrium during the menstrual cycle and pregnancy. Endocrinol (1999); 140:2672–8. 125 Robb L, Li RL, Hartley L, Nandurkar HH, Koentgen F, Begley CG. Infertility in female mice lacking the receptor for interleukin 11 is due to a defective uterine response to implantation. Nature Med (1998); 4:303–8. 126 Psychoyos A, Nikas G. Uterine pinopodes as markers of uterine receptivity. Assisted Reprod Rev (1994); 4:26–32. 127 Psychoyos A, Mandon P. Etude de la surface de l’epithelium uterin au microscope electronique a balayage. C R Hebd Seances Acad Sci Paris (1971); 272:2723–9. 128 Martel D, Frydman R, Sarantis L, Roche D, Psychoyos A. Scanning electron microscopy of the uterine luminal epithelium as a marker of the implantation window. In: Yoshinaga K. eds Blastocyst Implantation. Boston: Adams Publishing Group, (1993):225–30. 129 Bentin-Ley U, Sjögren A, Nilsson L, Hamberger L, Larsen JF, Horn T. Presence of uterine pinopodes at the embryo-endometrial interface during human implantation in vitro. Hum Reprod (1999); 14:515–20. 130 Anderson TL. Biomolecular markers for the window of uterine receptivity. In: Boston: Adams Publishing Group (1993):219–24. 131 Campbell S, Swann HR, Seif MW, Kimber SJ, Aplin JD. Cell adhesion molecules on the oocyte and preimplantation human embryo. Hum Reprod (1995); 10:1571–8.

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132 Carson DD, Tang J-P, Julian J. Heparan sulfate proteoglycan (perlecan) expression by mouse embryos during acquisition of attachment competence. Dev Biol (1993); 155:97–106. 133 Sutherland AE, Calarco PG, Damsky CH. Expression and function of cell surface extracellular matrix receptors in mouse blastocyst attachment and outgrowth. J Cell Biol (1988); 106:1331–48. 134 Sutherland AE, Calarco PG, Damsky CH. Developmental regulation of integrin expression at the time of implantation in the mouse embryo. Development (1993); 119:1175–86. 135 Kolb BA, Paulson RJ. The luteal phase of cycles utilizing controlled ovarian hyperstimulation and the possible impact of this hyperstimulation on embryo implantation. Am J Obstet Gynecol (1997); 176:1262–7. 136 Serle E, Aplin JD, Li T-C, et al. Endometrial differentiation in the peri-implantation phase of women with recurrent miscarriage: A morphological and immunohistochemical study. Fertil Steril (1994); 62:989–96. 137 Blacker CM, Ginsburg KA, Leach RE, Randolph J, Moghissi KS. Unexplained infertility: evaluation of the luteal phase; results of the National Center for Infertility Research at Michigan. Fertil Steril (1997); 67:437–42. 138 Wilcox AJ, Baird DD, Wenberg CR. Time of implantation of the conceptus and loss of pregnancy. N Engl J Med (1999); 340:1796–9. 139 Kauma S, Shapiro SS. Immunoperoxidase localization of prolactin in endometrium during normal menstrual, luteal phase defect, and corrected luteal phase defect cycles. Fertil Steril (1986); 46:37–42. 140 Lessey BA, Yeh IT, Castelbaum AJ, et al. Endometrial progesterone receptors and markers of uterine receptivity in the window of implantation. Fertil Steril (1996); 65:477–83. 141 Klentzeris LD, Bulmer JN, Trejdosiewicz LK, Morrison L, Cooke ID. Beta-1 integrin cell adhesion molecules in the endometrium of fertile and infertile women. Hum Reprod (1993); 8:1223–30. 142 Meyer WR, Castelbaum AJ, Somkuti S, et al. Hydrosalpinges adversely affect markers of endometrial receptivity. Hum Reprod (1997); 12:1393–8. 143 Lessey BA, Appa Rao KBC, Lovely LP, Gui Y. Elevated androgen receptor expression in women with poly cystic ovarian syndrome (PCOS). Biol Reprod, submitted. 144 Tabibzadeh S, Shea W, Lessey BA, Satyaswaroop PG. Aberrant expression of ebaf in endometria of patients with infertility. Mol Hum Reprod (1998); 4:595–602. 145 Gui Y-T, Zhang J, Yuan L, Lessey BA. Regulation of Hoxa-10 and its expression in normal and abnormal endometrium. Mol Hum Reprod (1999); 5:866–73.

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146 Taylor HS, Bagot C, Kardana A, Olive D, Arici A. HOX gene expression is altered in the endometrium of women with endometriosis. Hum Reprod (1999); 14:1328–31. 147 Kumar S, Zhu LJ, Polihronis M, et al. Progesterone induces calcitonin gene expression in human endometrium within the putative window of implantation. J Clin Endocrinol Metab (1998); 83:4443–50. 148 Pollard JW, Hunt JS, Wkitor-Jedrzejczak W, Stanley ER. A pregnancy defect in the osteopetrotic (op/op) mouse demonstrates the requirement for CSF-1 in female fertility. Dev Biol (1991); 148:273– 83. 149 Dunglison GF, Barlow DH, Sargent IL. Leukaemia inhibitory factor significantly enhances the blastocyst formation rates of human embryos cultured in serum-free medium. Hum Reprod (1996); 11:191– 6. 150 Simón C, Piquette GN, Frances A, Polan ML. Localization of interleukin-1 type I receptor and interleukin-1 beta in human endometrium throughout the menstrual cycle. J Clin Endocrinol Metab (1993); 77:549–55. 151 Carson DD, DeSouza MM, Regisford EGC. Mucin and proteoglycan functions in embryo implantation. Bioessays (1998); 20:577–83. 152 Chen GTC, Getsios S, MacCalman CD. Cadherin-11 is a hormonally regulated cellular marker of decidualization in human endometrial stromal cells. Mol Reprod Dev (1999); 52:158–65. 153 Nikas G, Psychoyos A. Uterine pinopodes in peri-implantation human endometrium—Clinical relevance. Ann NY Acad Sci (1997); 816:129– 42. 154 Noyes N, Liu H-C, Sultan K, Schattman G, Rosenwaks Z. Endometrial thickness appears to be a significant factor in embryo implantation in in-vitro fertilization. Hum Reprod (1995); 10:919–22. 155 Oliveira JBA, Baruffi RLR, Mauri AL, Petersen CG, Borges MC, Franco JG, Jr. Endometrial ultrasonography as a predictor of pregnancy in an in-vitro fertilization programme after ovarian stimulation and gonadotrophinreleasing hormone and gonadotrophins. Hum Reprod (1997); 12:2515–8. 156 Julkunen M, Koistinen R, Sjoberg J, Rutanen EM, Wahlstrom T, Seppala M. Secretory endometrium synthesizes placental protein 14. Endocrinol (1986); 118:1782–6. 157 Jones GS, Aksel S, Wentz AC. Serum progesterone values in the luteal phase defects. Obstet Gynecol (1974); 44:26–34.

30 Data management and interpretation—computerized database for an ART clinic: hardware and software requirements and solutions Giles Tomkin, Jacques Cohen

INTRODUCTION Assisted reproduction is a field that is ideally suited for advanced data assessment although statistical evaluation is hampered by considerable complexity. The amount of data generated per single attempt can be large when including records for single cell genetics, immunology, cryopreservation, etiology, and follow up. The further separation of data from a single patient procedure into records for each embryo markedly complicates data analysis, making preimplantation embryology ideally suited for computerization. Unfortunately, the simple purchase of one or more computers alone does not provide the solution. A database system includes a good deal more than that; provision must be made to finance professional advice for networking setup and hardware, programming, system maintenance, upgrading and scaleability, programming changes, data backup and security. Only when the system is in place and a certain amount of data have been entered, can one expect to return useful results for regular patient procedure reporting, summary reports and associated functions. Data collection, storage, management, verification, and analysis can be complex and time consuming. Also, many of the computer based tasks are new to the personnel assigned to fulfill them. In vitro fertilization (IVF) programs that are prepared to make allowance for these problems in their budget can see excellent returns on their investment. An unexpected, yet major, hurdle to overcome is the level of cooperation between the embryo laboratory and the clinical and medical side of an IVF program. Because a fully functional networked computerized database system is pretty expensive, it needs planning and preparation like any other major clinical acquisition. Purchases of this nature in sizeable clinics are often politically problematic, as some will believe this to be a waste of resources and others will want to include extensive sections of data entry for research purposes or quality assurance, to name but two of

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the potential areas of disagreement. Such difficulties often cause unreasonable delays in obtaining computerized database systems. A further problem is that few reproductive specialists have sufficient expertise to distinguish an excellent software solution and management plan from one that is barely adequate. It is therefore often more realistic for any group to get itself started separately, at whatever level, by buying their own hardware and creating their own database system. Later on, moves can be made to expand and integrate the systems. This is actually easier than it sounds, because a start can be made with inexpensive hardware, and just one dedicated staff member who is prepared to “read the manual”. The above situation occurs frequently enough that no commercial software system is as yet available that readily satisfies the majority of IVF programs. While there are some commercially produced databases available, problems related to clinic specific differences have prevented widespread application. Moves are being made to address this problem in Britain and Denmark, but not in the United States, as far as is known.1 However, the moves proposed will simply correlate data rather than provide adequate software. Each side within each IVF group generally has to create its own database solution; and, given the scarcity of knowledge about programming and relational database concepts, these in house systems can fail notably in two major areas. They do not permit adequate analysis for a wide range of factors; and they cannot be easily transferred to a new environment, namely other clinical programs. Nevertheless, even poorly planned program solutions will usually give a reasonable level of satisfaction in data storage and reporting; anything computerized is better than pulling individual charts for every new billing process or study. But it is very important that people starting up new systems should gain some instruction, however minimal, in basic database and program design before they start. When possible they should discuss the scope of their overall plan with the best database expert they can find, so that a framework can be made to which elements can be easily added. Here, we will describe our own laboratory system as it has been used between August 1995 and August 1999 at the Institute for Reproductive Medicine and Science at Saint Barnabas Medical Center, Livingston, New Jersey, USA. We will attempt to provide some guidance to avoid the major pitfalls in database design for IVF. We do not attempt to cover appointment calendars, clinical personnel and space management, salaries, invoicing, billing, and insurance claim management. Several commercial packages are suitable for these purposes, which can be readily tailored to a clinic’s needs; they are mainly distinguished by price, although there will be differences between individual geographic localities.

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DATABASE OBJECTIVES Our IVF laboratory database system is designed with two main objectives for the improvement of a patient’s treatment. The principal reason is to enter and review patient data in real time—to minimize paper form filling and later historical data entry—by using networked computer workstations in the laboratory and elsewhere. Users in each group (doctors, nurses, embryologists, and scientists) can review patient charts and enter data in their own area of work as needed. The printed reports form the basis of the patient chart for their IVF procedure and require a minimum of handwritten annotations. Secondarily, we can perform scientific research analysis on embryo development according to an exceptionally wide range of criteria, as we observe and record individual oocytes or embryos. A database can serve as a quality control tool because data can be analyzed concurrently, showing comparisons for periodic fluctuations in development speed, incidence of multinucleation, etc., for gametes and embryos, as well as standards for tracking medium lot numbers for culture and equipment such as incubators. Analysis can also provide for personnel assessment according to types of procedure performed in narrow and comparable populations according to predetermined parameters such as previous attempts, etiology and age, so that individuals can concentrate on their best skills and either improve or desist in other areas. Also, analysis can provide research for determining results for new or changed procedures and adjusting or streamlining existing systems to improve embryo development. Additionally, our program improves gamete handling in the laboratory, as it never allows embryos to be overlooked, which may happen both in the culture vesicle and on paper forms when large numbers of oocytes are obtained for a single procedure.

DATABASE SYSTEM As the so called IBM compatible personal computers are ubiquitous today, it would be unreasonable to look elsewhere for hardware. Our network has been built up piecemeal over the years on personal computers, and is now MS Windows NT based, with MS Windows NT or MS Windows 95/8 on most workstations, and we use MS Access with the program suite MS Office (Microsoft Corporation, Seattle, WA, USA). With in house

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Fig 30.1 Network file layout. This diagram shows the layout of our database on a computer network. The plan would be very similar for any networked database. The names are: “Forms” File=The file with the programming and forms for data input; “Data Tables”=The data tables file, holding all information; “Summary Reports” file=Programming and result forms to present data in summary reports.

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programming we have created a suite of tables, forms, and reports in MS Access tailored to the requirements of clinical embryology (EggCyte™, ART Institute of NY and NJ, Livingston, NJ, USA). This is by no means to recommend the above systems as being the “best”; it is safe to say that any “best” system could be judged as such only for a single season. Thereafter, a newer system is sure to appear that is faster, cheaper, more easily managed, or have some other currently unknown advantage. It was selected on the assumption that the most prevalent system would make it easy to find people who could manage it; and because there are many such people, reasonable expertise would be available at a minimum cost. Also, it is essential for safety that a system be developed or obtained that can be serviced readily by other database experts not familiar with reproductive medicine. In other words, the basic underlying hardware and programs for any system should be in widespread use. Whether a database is “standalone” on a single machine, or networked to computers in offices all over the United States, the system is the same—a “data tables” file receives the information into tables which is entered through “forms” files, and a “summary reports” file performs calculations for summary reports (see Fig 30.1). The “forms” and “summary reports” files link to the “data tables” file on the server by a set of pointers. These links between “forms” and “data tables” are hidden from the user in a single, standalone database file; but when two or more computers are involved, the data tables and forms are in separate files, which must be linked. In this way, many copies of the forms file can be used at once from different work stations, all of which link or connect to the single data tables file on the server.

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Fig 30.2 EggCyte™ forms scheme. The layout of data entry forms in the database. This simplified diagram indicates which data tables underlie the forms, showing the first two or three letters of each one’s name. Thaw procedures “move” down the left side. All other procedures developmental days “move” down the right hand side.

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As this is crucial to the understanding of any such system, let us explain the above another way. The purpose of the data tables file is to receive all data entered via the forms file. The purpose of the forms file is to facilitate data entry. Forms are simply a list of questions through which the data are entered, and programming ensures that each item goes to the right slot or field in the data tables file (Fig 30.2). The forms file links to the data tables file. Once data are entered, reports for individual procedures can be printed in a layout suitable for the patient’s chart. The forms file also has a set of procedure reports, which print out a detailed description of all the data entered for any given procedure. These form the basis of the patient’s file, and are not to be confused with the summary reports in the summary reports file, next. The summary reports file provides evaluation of procedure data for many different purposes. Only certain workstations have the summary reports file installed because only certain users need to see these results (Fig 30.1). The summary reports file generates compiled and often complex data reports using summarized information from many procedures into a table of figures. Users select different parameters depending on which type of report is wanted, and the result considers only procedures that fit the selection criteria. Finally, we have a fourth little analysis file whose purpose it is to permit individuals to create their own queries and reports however they choose. The file contains minimal programming, sufficient only to link all the required data tables. This means that all the data tables show up in this file and can be used in queries. To prevent users making errors in the main tables, this file links only to a copy of the “data tables” file that has been renamed as a “safe local copy” to distinguish it from the server file. The analysis file is very small and multiple copies can be made, so that users can apply one copy for each study project.

SYSTEM COSTS We mentioned earlier that reasonable expertise would be available at a minimum cost. This refers to the maintenance of an existing system. But during the design and set up stages of any database project and irrespective of the system, high level expertise should be called on. Although this is harder to find and can be costly, it is an essential requirement; but one that is only required at the beginning. Thereafter, mortals of lesser knowledge and cost can help the brave staff database person with programming problems. Oddly enough, speed of the system and actual hardware costs actually mean very little overall. While your system may be cheap and slow when you get started, the next few years will bring much faster machines for the same small money. These will probably be added to your earlier machine(s), which can usually be upgraded to some extent, and remain

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useful member(s) of your growing network. Your budget will undergo amazing expansion also, as administration realizes little by little how many man hours are saved on the one hand, and, on the other, how useful and far reaching your analysis of historical data has become. But the actual hardware costs will always be overshadowed by the salary cost of personnel who are managing the system. Even if it is only one person, they will have to dedicate a substantial proportion of their time, and therefore their salary, to the computer(s) and data, the total amount will quickly overtake the hardware cost as time flies by. There is no escaping it: clean, accurate data entered in a timely fashion cost a good deal of personnel hours, and any skimping results in untrustworthy data can undo the whole effort.

THE CENTRAL DESIGN AND PLAN It is essential to start out with a good plan. This is because the concepts behind relational databases exist only in computers. They cannot exist in the real world because printed file cards cannot contain data that slides around. Relational databases however, can present data in any form whatsoever, as long as the basic setup follows essential, simple rules. Relational databases work by recording pages of information in separate tables, and linking relevant records by key fields common to each table. This means that the data is repeated in each table for the key field(s) only, such as a procedure number, which is the same in each table. But one table can hold demographic information, while another holds procedure information. The procedure number, common to each, permits the use of a link from one table to another—this is the relation between them. In the EggCyte™ database, the links between tables are maintained successively and respectively by a patient identification number (usually the social security number when available or an invented code when not), then procedure number (proc.), and oocyte/embryo number (proc./emb.), and finally by proc., emb. and check number (proc./emb./chk.). At our laboratories, oocytes and embryos are numbered individually and tracked by marks on the base of the dishes throughout their laboratory time. The individual numbering starts at insemination for intracytoplasmic sperm injection (ICSI) procedures or at the day 1 checks for IVF procedures. Oocytes and embryos are placed in their individual spots in the culture dishes and thereafter separately tracked with data tables for each day of development, just as in a daily journal. If additional checks are performed on a given embryo in a single day, other entries for that embryo are entered for that day’s table of records; hence the last index item, “chk.— proc./emb./chk.” (Fig 30.3). Preimplantation genetic tests involving cells obtained from the eggs or embryos use the same numbering system but with “cell” instead of “check—proc./emb./cell.”

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Fig 30.3 Linked key fields. Each panel in the diagram represents a table and their field names in the database. The lines connecting the panels indicate successive table linking according to the key fields in the tables. Table “2A_RET” (Retrieval) has only a single procedure number as key field (link “A”) while table “4A_INS” (Insemination) has two key fields whose combination in any one record must be unique (link “B”). Link “C” shows three key fields whose combination must be unique in tables “6A_D1” (Day-1) and “7A_D2” (Day-2). Note that the field names always identify the table to which each belongs—this system is of paramount importance for data analysis. Once the key fields are assigned during table creation, automatic monitoring by the database program prevents record duplication in these key fields, and even millions of records across many tables can be handled without a hitch. Developmental information is added, record by record, to a new table for each day in culture. If extra events such as cryopreservation or biopsy are applied to embryos, then separate tables for each of these events record this data, linked by procedure and embryo number. If new items are to be recorded for any developmental day, new fields can be added to the existing tables; if culture is to be extended for further days, new tables can be added. This is a clean design method that

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can be readily understood by the ordinary humans who will be managing the data. Note that the field names always identify the table to which each belongs—this system is of paramount importance for data analysis.

SPACE REQUIREMENTS Considering the space requirements on the computer’s hard disk, field sizes should be limited where possible. There are many fields that will not have data entered for any given procedure, and will therefore be empty space, creating extra bulk in the database. For an embryo replaced on day 3, there are approximately 275 fields to record information in our database, including about 53 key linking fields. Thus about 220 fields are for developmental data on each egg or embryo. This excludes semen analysis data, cryopreservation data, but includes possible polar body biopsy, coculture, and blastomere biopsy information. Typically, no more than 140 of these are filled in; so our database carries nearly 80 empty fields for each embryo record. We limit the size of fields as far as possible by programmatically returning only two letters for each record for each item entered, using adjustable “drop lists”, which are all stored in single table. Staff names are four letters only, being the first three of the last name and the initial, in a separate table. There is another way of designing a database that is much more compressed, as it has almost no empty space at all, but requires much more programming. This is designed with very few tables, and the field name for each data item is entered as a part of the data entry. This system means that only those “fields” that are filled in actually occupy space in the database; and, under certain circumstances, this does allow fields to be added “on the fly” by users, which is a very attractive feature. Handling such a system is impenetrably clever for any but programmers because none of the data relation are obvious. Furthermore, any changes in the features require intensive programming. This makes it expensive to maintain and difficult to change, as well as effectively preventing data research studies by modest users applying queries because the relationships between the data tables and fields are hard to understand. Overall database size is becoming less of a problem as hard disk space geometrically increases, but all possible measures should always be taken to keep the size down, as small overall file size vastly simplifies daily handling. Our data tables file has recently reached 60 megabytes in size, and contains data for about 6000 procedures and 90000 embryos. Although hard disk sizes in early 2000 are commonly 10 gigabytes, we may nevertheless have to begin archiving measures in another few years, when it reaches about 100 megabytes in size. This is because the programming has to look through all records when finding data that relates to a procedure; as quick as computers are, the time taken becomes unacceptable as the number of procedures rises into the many thousands.

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However, it is always good practice to limit size wherever possible for ease of daily management; because present practice requires copies for backup and file compression and repair; on smaller files this takes minutes; on large files this can take hours, causing serious delays in potentially critical situations.

ANALYSIS WITH QUERIES Analysis is normally performed by creating selection lists called “queries” from each table. When multiple tables are included in a single query, each table must be linked to the others by the correct key fields as described previously. These queries can be individually named and saved for later reference, and thereby form the basis for all database programming, reporting, and analysis. When considering this type of analysis, the importance of including the table name in each field can be easily understood. Research using queries requires understanding of individual tables and fields in the database, so it is always important to document the structure and relationship between data tables, as well as their field names and purpose.

TABLE AND FIELD NAMES Database users need all the help they can get, and a coherent naming scheme is very important. Field names should always be short and without spaces so that they can fit in the top of a narrow column because their contents are usually a number or a few letters. Data table names should have an even shorter reference code in their name, which is repeated in the name of each field of that table; this makes each name unique (Figs 30.3, 30.4). This seeming trifle becomes very important as queries and programs become more complex over a large number of tables. Most database programs expect short code names, and therefore also have a larger space for a description of the function of each field; all of these descriptions should be carefully entered. Even so, a separate file should be made listing each table and field and their purpose, so that users can refer to this outside documentation during query creation or program maintenance. In computer programming, it is customary to include a code in the field name which indicates the type of field, being numeric integer, numeric decimal, alphanumeric, date, time, etc. This is possible, but in our opinion, makes the field names too long and therefore cumbersome. Knowing to which table the field belongs is critically important, so that users can readily understand the relation between the items they are using.

Fig 30.4 Simplified table layout. This shows actual tables that can be used in a simplified database which tracks individual eggs and embryos. The naming scheme follows the suggestions outlined in the text. Excepting tables A1 through A3, records are either one per procedure or one per oocyte.

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Further tables can easily be added and linked appropriately. I wish I had used this naming scheme for EggCyte, but it is too late now.

PROCEDURE TYPES AND NUMBERING Since the start of our program in 1995, each year is divided into four cycles of procedure handling with a two or three week break between each. Our procedure numbering follows these cycle numbers, so that the first REGULR (sic) procedure of the first three month cycle would be 1– 001, the first THAW procedure 1–701, and the first DONOR being 1– 801. The last three digits indicate the procedure type (Table 30.1). At the time of writing, we are in cycle 20, so our procedure numbers are now 20nnn, while the actual number of procedures performed is about 6000. However, it is the procedure type only, and not the numbering system, that is used throughout the database by queries and in the programming to separate the procedures. This is because the time may come when unexpected numbers of a single procedure type use up the available numbers in a particular cycle, and force a temporary change in numbering. They are formatted here with a “dash” for clarity, but the database stores these as integer numbers.

TYPICAL DATA ENTRY The manual for the EggCyte™ database program is over 250 pages of text and pictures, so here we can only show a few of the screen forms to give an idea of some data entry solutions. Data is typically entered in spreadsheet style for the development days, both after a retrieval or thaw procedure. Once the number of oocytes or embryos has been entered and confirmed for either a retrieval or thaw procedure, new records are automatically created in the appropriate tables. The OutCome tables, either “8C_OUT” or “9E_OUT,” are for the retrieval or thaw procedures respectively (Fig 30.5). As development continues, Outcomes are returned here, one for each entry, and it is these outcomes that give the control within the program; if no outcome is present for a given embryo, then it is still in culture. On

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Table 30.1. Procedure numbering system. The “n” for each number range indicates the “cycle” since program inception. While formatted with a “dash” for clarity, the database stores these as integer numbers. The numbers are for easy identification by personnel only; they do not form part of programming requirements in the database. Procedure numbering system Procedure type Database code Range of procedure numbers Regular IVF or ICSI REGULR n-001 to n-499 Oocyte recipient RECIP n-600 to n-699 Part donor, with own regular proc. PRTDNR Has 2 IDs (a) Oocyte donor DONOR n-800 to n-899 Thaw cycle THAW n-700 to n-799 Cytoplasmic recipient CYTREC Has 2 IDs (b) Addit. recipient proc. for Cyto.recip. RECIPX n-600 to n-699 Gamete intrafallopian transfer GIFT n-001 to n-499 Gestational provider GPROV n-001 to n-499 Gestational carrier GCARR n-001 to n-499 All embryos to cryo (pre-planned) CRYO n-001 to n-499 (a) 1st proc is DONOR, 2nd is REGULR (b) CYTREC ID range as for REGULR

Fig 30.5 EggCyte™ table scheme. This shows the scheme for tables as used in our laboratory. Forty-one tables track embryo development and supply data for analysis and reporting. The tables “8C_Out” and “9E_Out” record outcomes for each gamete, according to time in culture. If I could do it all again I would use the naming scheme in the simplified table

layout, where the characters precede the numerals.

Fig 30.6 Typical data entry form. This shows the data entry form for day 2 and day 3. The current procedure is selected from either of the fields at top left. Data previously entered for that procedure are reviewed in the top part, and new data are entered in the lower part. The form shows a day 3 check being performed prior to embryo replacement. The principal tables that underlie this form are “7A_D2”, “8A_D3”, “6B_COC”, and “8B_BIOP” from the EggCyte scheme. The top windows show the status of the procedure and “buttons” to view other days, printed patient reports or to exit the form. Buttons on the lower part can reorder the embryos in the form, create records for extra checks, create blank blastomere biopsy or embryo coculture records. Embryo outcomes are chosen, including selection for replacement. The cursor “focus” is located where the two gray arrows indicate the cytoplasm column. The little “down arrow”

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in that box indicates that a drop list is available for selection here. The single arrow to the left shows where the column description appears. selecting the patient procedure in the on screen form, records are automatically created for those embryos that continue in culture. The user sees yesterday’s information in the top sheet and enters new items in the lower sheet as forms combine data from multiple tables. A similar format is used for all developmental days and for days after thaw. Many more columns for data entry are available by moving the on screen slide-bars, and a “copy down” function copies data across all embryos records when this will facilitate data entry (Fig 30.6).

CRYOPRESERVATION DATA ENTRY Outcomes indicating cryopreservation must be written back to the outcome table “8C_OUT” before the cryopreservation data can be entered. Then the screen form for cryopreservation is called, and the patient procedure number is selected. The programming seeks any embryos with outcomes marked for cryopreservation for that procedure and automatically creates records. Data is entered in a spreadsheet format, using the drop down lists. A tick box column indicates if any embryos have been thawed later on, so that those remaining in cryopreservation can be automatically counted at any time. Special conditions such as removal to another location, donation or other removal requirements are handled in another form called over this one (Fig 30.7).

THAW PROCEDURE DATA ENTRY The embryo thaw and subsequent development until replacement is handled by the series of tables seen on the left side of the EggCyte™ table scheme (Fig 30.5). The actual thaw form must account for many different conditions, including the tracking of the original procedure number from which the embryo was derived; the possibility of embryos donated from another patient; and the introduction of embryos into the database from other clinics that patients may bring with them. At thaw, embryos can be at any developmental stage, so the tables for days of thaw do not represent developmental days, simply the time after thaw. Also, embryos may have to be returned to cryopreservation if too many unexpectedly survive in a given procedure (Fig 30.8). The key fields that make up the index of the thaw development tables need one extra field for the original procedure number from which the embryo came; the index now has four parts— “Thaw ID”, “Orig Proc ID”, “Emb Num”, “Check”.

Fig 30.7 Cryopreservation data entry form. This is a partial view and does not show the slider bars nor all the data columns. The main tables that underlie this form are “7B_CRY” and “7C_SCON” from the EggCyte scheme. The left pointing arrow indicates the column that shows whether an embryo has been thawed and is no longer present. The right pointing arrow indicates the button to call the “special conditions” form, which records items such as removal from the institute to another place, availability for research or donation, or destruction, according to patients’ wishes. The small table at top right shows who originally entered the data, who checked it the first time, and that it is pending a third check.

Fig 30.8 Thaw data entry form. This form has five main underlying tables: “9A_THW”, “7B_CRY”, “9B_THW”, “9F_COC”, and “9G_BIOP”. When the patient procedure is chosen in the left side selection fields, both lower forms appear blank until the source of the embryos for thaw is chosen in right hand side procedure selection fields, and could be from “Outside Cryo” or OutCry, the button indicated with the arrow. When the source is chosen, the left side selection fields gray out, preventing change, and the top records appear, from which the embryos to be thawed are ticked (left pointing arrow). The right hand side procedure selection fields permit embryos to be chosen from multiple procedures. The button “(4) do thaw” is hit to create records in the lower form. Thawed embryo records always retain their original procedure numbers. Other buttons switch the lower form to either blastomere biopsy or coculture forms. Note the bottom form leftmost column showing the original procedure number.

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If embryos are brought from outside the clinic, a button named “OutCry” (for Outside Cryo) is used, and the number to be thawed is entered. The ensuing records are automatically supplied with negative original procedure numbers and can therefore easily be isolated for independent analysis.

REPLACEMENT AND RESULT DATA ENTRY The replacement procedure form provides for selection of embryos from day 2 onwards and any thaw day. It also permits embryos to be chosen from any current procedure for the replacement in the rare but not impossible case of last minute donation. In EggCyte™, embryos for replacement are selected by choosing the appropriate OutCome from the developmental day, either a first choice “1-Replacable” or default second choice “2Replacement possible” When these selections are made, the “do Replacement” button is pressed, and records are created for the replaced embryos, in which their individual status can be noted. As there can always be slips ‘twixt cup and lip’, other fields record the number

Fig 30.9 Replacement entry form. The main underlying tables are “11A_REP” and “11B_REMB”. The patient procedure is chosen in the left side selection fields and the source of the embryos for replacement defaults to the same person and procedure

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number in the right hand side procedure selection fields. The right hand side selection fields are for the rare, but not impossible last minute donation of embryos. Any developmental day table can be chosen using the “other days” button (top right arrow). Selection for replacement is made by changing the “Outcome” (top left arrow). When the “do replacement” button is hit, records are created for the chosen embryos in the lower table. Multiple selections from other procedures or days can be made if unusual circumstances require it. The cursor focus is in the “cytoplasm” column of the lower table, as described at lower left. of embryos loaded in the catheter, returned (intact or empty zona) and the final number replaced (Fig 30.7). The pregnancy result for each procedure (except DONOR) is always entered in the results table “12B_RES” (Fig 30.5), even if that procedure failed to have a replacement such as in the case of a deliberate cryopreservation procedure, when all embryos are cryopreserved. A field in the results table indicates if a replacement took place, and a comment field explains why, if there was no replacement. This logic is not altogether obvious, but is done to keep numbers of results (except for DONORS) the same as the number of procedures as first undertaken in our laboratory. Procedures with two procedure numbers, such as for cytoplasmic replacement and partial donor patients require individual attention, but are easily separated using their procedure type when data analysis is performed.

SUMMARY REPORTS Reports fall into two main categories, the first being straightforward patient procedure reports (diagnostic as well as therapeutic), where all the data recorded for a procedure are displayed in a few tightly formatted pages. The second type is for procedure summaries, including such things as total count of procedure types, numbers of eggs fertilized, pregnancy results, and so forth. The second type is by far the most interesting because doctors and scientists can use these items to judge the behavior of individual equipment and material lot numbers; the effectiveness of stimulation protocols, laboratory procedures, and skill of both doctors and embryologists.

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The summary reports file is connected to a copy of the data tables file as distinct from the real data tables file on the server. For up to the minute data, the data tables file can be updated (copied down from the server) just prior to running the reports. This provides the fastest report generation because computers working with local files are much faster than through the network, and complex reports can take time to generate. These reports are so powerful in terms of clinical handling, it is worth describing some individually. Overall Pregnancy Results: This gives implantation by embryonic sac or by fetal heartbeat between selected procedure numbers, maternal age, and procedure type(s). Doctors performing retrievals and replacements can be included or excluded. Individual Doctors: This report tracks the activity, performance and effectiveness with pregnancy results for the selected age groups and procedure numbers for each practicing doctor. This report is comparative, and is worked in sets, by using a spreadsheet for overall comparisons by taking the same parameter set for all doctors. Individual Embryologists: This tracks the activity, performance, and effectiveness with pregnancy results for each embryologist within the selected parameters, including retrieval and replacement staff doctors (or all). It has separate categories for each of the embryologists’ activities: some for all the embryos of a procedure, such as in inseminations, or retrievals; others for individually handled embryos, such as during ICSI, cryopreservation, or fragment removal; and a special side table for ICSI results. Resulting numbers of embryonic sacs and fetal heart beats are compared with the overall average for each category, with the differences expressed in percentages. This report is comparative, and is worked in sets by using a spreadsheet for overall comparisons by taking the same parameter set for all embryologists. Grouped pronuclei (PN): Parameters for procedure numbers and type, age group, incubators, and embryo coculture return a bar chart dividing the procedures into 10 groups, giving fertilization results according to numbers of PN as percentages—1 and under, 2, and 3 and over. Ideally, each bar in the chart should be the same length as each category in the other nine groups. The differences in bar length indicate overall variation, from which the user can determine which particular set of procedures shows any unreasonable variation, positive or negative. Alerted thereby, the user can attempt to track down the laboratory differences in those procedures that may be the cause. Grouped Embryo Development: Similar to the above report, this gives bar-chart development results according to numbers of day 2 multi-nucleation, day 3 cell numbers, and fragmentation in a bar chart. Grouped Implantation Rates: Similar to the above report, this gives bar-chart implantation results according to numbers of embryonic sacs and fetal heart beats. Multiple Pregnancies: Parameter selection includes procedure numbers, maternal age, numbers of embryos replaced and type of election, developmental day of replacement, and, finally, the number of IVF attempts the patients may have had; the report returns percentage rates and numbers accordingly. This report is helpful in real time, because specific patient data prior to embryo transfer can be filled in, to build a set of patients and their results which resemble the patient in question. Once

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the common parameters are entered, the team members may decide to reduce or increase embryo numbers for replacement in order to optimize outcome while limiting multiple pregnancy. No separate report for government or other officials has been included in our database because their requirements change every year, as their knowledge grows and treatments develop into new areas. It is therefore much simpler to merely generate individual report numbers during an annual query session. The laboratory manager should nevertheless keep an eye on the official reporting requirements to be sure that their program’s database includes all their needs. If the above design guidelines are followed, there should be no problems adding fields or even tables to adapt to new requirements. Many other studies and types of research are performed, including one attempt to determine how the frequency of incubator door opening affects embryo development and pregnancy outcome according to the crowding in a given incubator. In the absence of mechanical door opening counters, we derived the frequency of door openings, or DOPS, programmatically, according to the procedure dates and incubator numbers from the database. This will never be included as a regular ongoing report because the resulting program functions take over 10 hours to run on a Pentium-2, 350mHz processor with 256mB of RAM—impractical for normal users, to say the least. However, unexpectedly, the study appears to indicate that pH and temperature settings may be suboptimal in general, requiring further in depth studies of biophysical conditions for culturing human gametes and embryos in vitro. Our system tracks eggs and embryos individually in multiple tables, and is probably the most complex and detailed laboratory system of any clinic to date (Fig 30.5). However, individual tracking can be performed in a much simpler way by using one large record for each egg. All the other normal database rules apply, but instead of having over 40 database tables, the number is reduced to about 15 (Fig 30.4). As an aside, we must admit that the naming scheme shown in the simplified table scheme is better than the one chosen for the EggCyte™ table scheme; hindsight provides 20–20 vision; however, so much programming has been built on the original names that change is now impractical. The table “E1_Ooc,” which contains all the fields for individual embryo development, is limited to a maximum of 250 fields, because this is the limit the Microsoft Access database program will permit. It would nevertheless be advisable to create more tables rather than use this full amount. This certainly applies to activities such as coculture or embryo biopsy, because these items are not applied to all embryos. Many clinics do not number eggs individually; but while this makes the data entry and handling even simpler, it seriously restricts the possibilities of analysis and reporting where embryonic development is concerned, including preventing embryo selection based on single performance events.

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In conclusion, it remains to strongly emphasize the enormous advantages of even limited computerized databases. Getting started is the hardest part, mainly because everyone wants to know exactly what the end results will be in advance; and this is simply not possible. Usually, the end results are much greater than expected: our system is still providing surprises about what can be done after nearly five years, and even appears underused as far as quality assurance tracking is concerned.

REFERENCES 1 Jenkins JM, Howden-Leach H. Information interchange between computer systems utilising the British Fertility Society infertility practice classification. Presented at the International Symposium on Health Information Management and Research, 5–7 April 1995, Sheffield (SHIMR 95). (http://www.bris.ac.uk/Depts/ObsGyn/it_med/bfs_inf.htm)

31 Evidence based medicine Salim Daya

The rapid proliferation in medical knowledge and the relative ease of access to this information have resulted in healthcare practitioners and consumers being overwhelmed with the volume of material that has to be evaluated from a utility perspective. Furthermore, the decision making process for practitioners is becoming increasingly reliant on using valid evidence to develop clinical care plans. Learning to access, interpret and apply this knowledge appropriately may be a daunting task but is fundamentally important to evidence based medicine, a concept that has a historical background dating to 1910 when it was reported that the new foundation of medicine was based on biomedical sciences, which, at that time, served as the best available evidence on which medical decisions were made.1 The present day focus on evidence based medicine has evolved significantly and relies on a systematic approach to gathering and appraising the evidence and applying the results of such a review to clinical care. It has been defined as the conscientious, explicit and judicious use of the best evidence that is currently available to make decisions about the care of individual patients.2 This definition emphasizes that judgment is required on the part of the clinician to incorporate into clinical care the evidence, that has been sifted and rendered valid after evaluating it using the necessary tools within an explicit and rigorous framework. By applying the rules of evidence to systematically gathered information, it is anticipated that the practice of medicine will lead to an improvement in patient outcomes and be more cost effective.

COMPONENTS OF EVIDENCE BASED MEDICINE The traditional approach to clinical decision making has accepted pathophysiology as the foundation for clinical practice, with individual clinical experience providing the basis for making diagnosis, offering treatment and counseling patients on prognosis of the disease. Reliance on medical training and common sense have been sufficient to enable a clinician to evaluate new tests and therapies. The present day approach assumes that, when possible, clinicians use information derived from well conducted studies that have been identified

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systematically and evaluated using the rules of evidence. In this context, an understanding of the pathophysiology of the underlying disorder is still necessary but is insufficient for the practice of medicine. The starting point requires clear and precise formulation of the clinical problem so that appropriate search strategies can be devised to identify the relevant information. Once the information has been identified, and retrieved it has to be evaluated for its validity before the data can be extracted and analysed and the results applied to the care of the patient. The clinical problem is formulated in the form of a question and can involve any aspect of medical care including therapy (for example, is salingectomy prior to in vitro fertilization (IVF) efficacious in achieving pregnancy in women with infertility resulting from hydrosalpinges?), diagnosis (for example, what is the accuracy of sonohysterography in the assessment of tubal patency in infertile women), prognosis (for example, what is likelihood of pregnancy in women with endometriosis undergoing IVF?), causation (for example, is there a causal relation between the use of fertility drugs in women with ovulatory disorders and the subsequent development of ovarian cancer?), and economics (for example, is the use of intravenous immunoglobulin cost effective in achieving successful pregnancy when used to treat women with unexplained recurrent miscarriage?).

EVIDENCE BASED PRACTICE The practice of evidence based health care is a multistep process requiring time and energy at each step.3 The process begins with defining, in clear, concise, and explicit terms, the question that needs to be answered. The question should identify the target population, the experimental intervention or exposure being considered and the control intervention or exposure (or lack of) against which it is being evaluated, and the outcome of interest. The next step required the identification and gathering of the evidence needed to answer the question. A librarian can play a key part in this process by not only providing the relevant literature that may contain the evidence being sought but by teaching practitioners on how to effectively and efficiently search for the evidence in the healthcare literature. The third step utilizes evaluation skills in conjunction with basic knowledge and previous clinical experience to critically appraise the identified studies to determine their validity. The fourth step is the judicious application by the practitioner of the evidence that best addresses the clinical problem so that decisions about a plan of care can be facilitated. The final step, and one that is often not considered, is the evaluation of the whole process so that improvements can be made the next time a clinical question is encountered.

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This process, if undertaken on a regular basis, will equip clinicians with the necessary tools to help them in accessing the current best evidence so that the care they provide can be maximized.

THE NEED FOR EVIDENCE BASED PRACTICE There are several reasons why evidence based decision making has become necessary for clinical care. KNOWLEDGE AND CLINICAL PERFORMANCE DETERIORATE WITH TIME An inverse correlation exists between a physician’s knowledge of up to date care and the length of time that has elapsed since his or her graduation from medical school. This disconcerting fact has been observed repeatedly and has been documented using a variety of clinical care scenarios.4,5 The decision to initiate treatment is more closely linked to the number of years since the physician’s graduation than to the severity of the target organ damage in the patient.6 The consequence of not keeping up with advances in medicine is that physicians become progressively out of date and unable to provide appropriate clinical care to their patients. FAILURE TO ACCESS NEW INFORMATION Although the necessity of keeping up to date with clinically important information is well accepted, failure to do so is a general phenomenon among physicians. This discrepancy between what is required and what is actually done to access information has been the focus of several studies. In one study, direct observation during a half day period of the behaviour of general practitioners as they evaluated patients identified up to 16 different instances when new and clinically important information was required; a rate of two questions for every three patients that were encountered.7 Thus, in a typical half day of practice, many medical decisions would have been altered if clinically useful information about them had been readily available. Despite this need, in only 30% of instances was information obtained in the clinics or offices where the physicians worked. In most of these cases the need was met by consulting colleagues and not by referring to books and journals as had been claimed by physicians who were asked about how the needs for clinically important information were usually met.8 The demands of a busy practice make it increasingly difficult to keep abreast of the field of medicine, especially when it has been estimated that at least 19 new articles become available daily to general physicians.9 The sheer volume of clinical literature overwhelms physicians trying to focus

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their attention to the material relevant to their practices. Even enthusiastic academic physicians spend only a few hours a week trying to keep up with new information, much of which is not valid or relevant to clinical practice. This fact is somewhat disturbing in light of recent observations that indicated that up to 75% of postgraduates in their first year had not read anything about the problems presented by their patients during the previous week and were being taught by senior consultants, up to 40% of whom had not read anything either.7 FAILURE TO IMPROVE CLINICAL PERFORMANCE THROUGH TRADITIONAL CONTINUING EDUCATION PROGRAMS In an effort to try and keep up to date, increasing emphasis is being placed on continuing education (CE) programmes. Several medical societies have introduced systems for accumulating and tracking study credits, which reflect the amount of time spent participating in CE activities. In Canada it has been suggested that malpractice insurance premiums should be reduced for physicians who regularly attend CE programs because of the belief that such individuals are more likely to provide appropriate care than those who do not participate in such activities. Unfortunately, the results of studies evaluating the efficacy of CE programs have been disappointing. Systematic reviews of randomized trials showed that traditional, instructional CE failed to modify clinical performance of physicians and was ineffective in improving the health outcomes of their patients.10

GENERATING EVIDENCE WITH APPROPRIATE STUDY DESIGNS There are many types of research study that can be undertaken to address specific healthcare issues raised by the question that is being asked. The study designs include simple descriptions of rare events (as with case reports and case series), cohort studies for the observation of the natural history of a disorder, case-control studies to identify causal associations, and experimental studies to establish a cause and effect relationship. Each study design has its strengths and weaknesses and their role in the area of infertility has previously been reviewed by the author.11 The experimental designs used to evaluate the efficacy of treatment will now be discussed in more detail. There are two main types of experimental study: randomized controlled trial and crossover study. Both involve random allocation of subjects to the experimental or control arms of the study but several differences exist between the two designs.

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RANDOMIZED CONTROLLED TRIAL The randomized controlled trial (RCT) is the standard against which all other designs are compared because it provides the strongest evidence for a cause and effect relationship and is subject to the least amount of bias. In such a study, the new or experimental treatment is compared with either a placebo or the current treatment as control (Fig 31.1), to determine whether an observed difference can be attributable to the experimental treatment. Evidence from such studies allows inferences to be made that are valid, reliable and convincing.

Fig 31.1 Design outline of randomized control trial. The subjects are selected from a target population on the basis of inclusion and exclusion criteria that are defined by the investigator so that homogeneity of the sample is assured. These criteria are important because they help determine how the outcome of the study can be applied to the population as a whole (generalizability). Two important issues in RCT design are the randomization process and blinding (or masking, as it is sometimes called). RANDOMIZATION Allocation to the experimental or control groups should be undertaken using a randomization scheme so that all subjects in the sample have an equal probability of being assigned to either group. Such a method ensures that, over the course of the study, any underlying factors that may affect the outcome are likely to be equally distributed in each group. There are many methods available to achieve randomization. The simplest one is to use a table of random numbers whereby, assignment to the two groups can be done on an even/odd number basis. Using such a table, subjects would be allocated to the experimental group if the next number

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in the sequence is an even number and to the control group if the next number is an odd number. This process is continued by following the sequence of numbers until the whole sample has been allocated. For three groups, one option for allocation would be to use numbers 1 to 3 for the first group, 4 to 6 for the second group, and 7 to 9 for the third group (the number zero would be ignored in this example). Unfortunately, group sizes may be unequal using the simple randomization method. To avoid the problem of unequal sizes, block randomization should be used whereby subjects are allocated in small blocks that usually consist of two to four times the number of groups, ie for two groups the block size may be four to eight. Block randomization ensures that there will be equal numbers of subjects in all groups. Thus, a block size of eight would contain four subjects randomly allocated to each of the two groups. It is important to ensure that the process of randomization is tamperproof so that the allocation process is not influenced by the investigator. The best solution would be to set up a separate randomization facility that is contacted when the subject is ready to be randomized. Alternatively, opaque, sealed, numbered envelopes containing the allocation group can be used with similar effect. These elaborate precautions for concealment of allocation are necessary to avoid bias that would cause an overestimation of the effect of treatment if the investigator was aware of the allocation sequence. BLINDING (OR MASKING) Whenever possible, the study should include blinding of subjects and investigators. In this way neither the study subjects (single blind), nor the investigator or anyone who has contact with the subjects has any knowledge of the group assignment (double blind). From the subject’s perspective, blinding eliminates any “placebo effect” from the active treatment so that any difference between the study groups can be attributed to the biologic effect of treatment. The mechanism of the placebo effect has not been identified but is believed to be exerted through the power of suggestion.12 Another problem with an unblinded study is that the investigator may give more attention to the subjects receiving the experimental treatment. This unintended intervention (called cointervention) may influence the outcome. Despite these problems, blinding may be difficult to achieve because the placebo, which has to be identical in appearance and administration to the active treatment, may be distinguishable from the active treatment. For example, blinding may not be possible in many surgical procedures or for treatment that has distinctive side effects (for example, prednisone). In these situations the placebo effect and risk of co-intervention should be borne in mind when analysing the outcome data. In situations where prior knowledge of the group assignment may influence assessment of the outcome data (ascertainment bias), blinding of

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the observers of the outcome also becomes necessary (the so called triple blind study).

Fig 31.2 Design outline of crossover study. STRENGTHS OF RCT The RCT provides the strongest evidence for a cause and effect relation and for evaluating efficacy of treatment. The study groups are comparable because confounding variables are likely to be equally distributed (balanced) between the two groups as a result of the randomization process. The assumption of random allocation underlying many statistical tests is maintained with an RCT. Consequently, the study is more likely to provide conclusive answers. WEAKNESSES OF RCT Controlled trials are often time consuming and expensive, especially when the outcome is an infrequent event (for example, pregnancy in patients with oligozoospermia). Sometimes ethical concerns arise because the control group given placebo does not receive a potentially effective treatment or the experimental group receives a potentially harmful treatment. CROSSOVER STUDY The crossover study is a special case of a randomized controlled trial that allows subjects to serve as their own controls. The RCT is a parallel groups study concerned with “between subject” comparisons in which each subject receives only one treatment. In contrast, the crossover study is a “within subject” study in which each subject receives more than one

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treatment (usually two treatments, or treatment and placebo, one after the other in random order). The simplest design utilizes a two period, crossover method in which each subject receives either the experimental (A) or control treatment (B) in the first period and the alternative treatment in the succeeding period (Fig 31.2). The order in which A and B are given to each subject is random so that approximately half of the subjects receive the treatments in the sequence AB, and the other half in the sequence BA. It is important that the effects of treatment in the first period do not carry over into the second period. Variability is reduced because the measured effect of treatment is determined by the difference in the subject’s response to the experimental and control treatments. Each subject is used twice thereby halving the sample size required for the study and allowing more precise evaluation of whether the subject does better on one or the other treatment. Thus, the between subject variation in estimating the effect of treatment is eliminated. In crossover studies, only short term responses, as measured during and at the end of each treatment period, are of interest. Any longer term effects may represent a carryover effect of the first treatment into the second treatment. If such carryover is likely, then a washout period during which no treatment or placebo are administered, should be included after the first treatment period. Also, the disease should be relatively stable so that the ability to respond remains unchanged from the first to the second treatment period. If the disease improves or deteriorates before commencement of the second period, interpretation of the overall treatment difference becomes difficult because the observed treatment difference is highly dependent on which treatment was given first. This period effect is a problem for crossover studies. Subject withdrawals from the study may seriously affect the analysis and interpretation of the results. A large number of dropouts after the first treatment period makes it possible only to compare the results at the end of the first period using a between subject analysis (as in the parallel RCT study). Consequently, the design becomes inefficient because a large enough sample size has not been used for this type of analysis. STRENGTHS OF CROSSOVER STUDIES The main advantage is that subjects serve as their own controls, thereby reducing any variability that may exist between subjects. Consequently, fewer subjects are required than for RCTs, and all subjects are assured of receiving treatment at least for some period of time. Because randomization is used, statistical tests can be used to analyse the results.

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WEAKNESSES OF CROSSOVER STUDIES Subjects who respond to the active treatment have to be taken off it to be given the placebo or the alternative treatment. Also, for some treatments, the washout period may be quite long, during which time subjects are often given placebo. Any period effect or carryover effect make interpretation of the data more difficult. Finally, the study design is not suitable for primary outcomes which are terminal events (for example, death and pregnancy) because if the event occurs in the first treatment period, the opportunity of receiving the alternative treatment is lost.13,14 Hence, no within-subject comparisons between the two treatments can be made.

THE ROLE OF META-ANALYSIS IN DECISION MAKING When faced with many studies addressing a particular clinical question one can combine the data into concise summaries that are easier to understand. This approach involves systematically gathering the information and then using statistical methods (metaanalysis) to pool the data so that an overall estimate of the effect of the intervention can be obtained. The resulting systematic review establishes whether the scientific findings are consistent and can be generalized across populations, settings and treatment variations, or whether the findings vary significantly by particular subgroups.15 The importance of statistical power, which enables a clinically important difference in outcome events between two interventions to be detected in a study, is not an intuitively obvious concept to many clinicians. Consequently, many studies that are undertaken often have inadequate sample sizes to allow inferences to be made with confidence. The following arithmetic calculation highlights the difficulty faced with conducting trials of sufficient power to test the efficacy of interventions in assisted reproductive technology. To detect a clinically important absolute difference in clinical pregnancy rate per cycle of 5% between the group with the new intervention and the control group (with expected pregnancy rates per cycle of 20% and 15% respectively), a sample of approximately 1450 subjects will be required (with α=0.05 and β=0.2 in a two-tailed analysis). Average clinics, with an annual volume of 200–300 cycles, will need to run the trial for at least five to seven years before accrual is complete and conclusive evidence can be obtained. Clearly, it may not be feasible to conduct a trial of this size in a single centre because its duration is likely to be prohibitive unless it is undertaken as a multicentered task. Instead, investigators run smaller studies (in the hope of detecting large differences in event rates) and when the magnitude of

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the effect detectable by such studies is not observed they erroneously conclude that no difference exists between the two interventions. By referring to a systematic review, one is likely to avoid making such premature (and often incorrect) conclusions about the benefit (or lack thereof) of a particular intervention. There are several steps that are important and necessary in conducting a good systematic review. These steps include: clearly specifying a research question; outlining a search strategy to identify and select relevant studies; assessing the validity of each study; extracting and pooling the data; and summarizing the results so that appropriate inferences can be made. The final common pathway for a systematic review is a quantitative summary of the data, or meta-analysis, which is a statistical procedure that integrates the results of several independent studies deemed on predefined criteria to be eligible for pooling. IDENTIFICATION OF STUDIES The search for studies should be comprehensive and involve several sources. The Medline database is an important first step, but even the most thorough Medline search is likely to miss many studies. Therefore, the search should include other data bases such as Embase, Science Citation Index, and the Cochrane controlled clinical trials register. Scanning the reference lists of selected publications and review articles often yields useful articles not identified in the initial search. Another important source of relevant studies is the “grey” literature, which includes theses, internal reports, non-peer reviewed journals, and pharmaceutical industry files. Abstracts of major scientific meetings should be scanned for trials that have recently been completed. Authors of primary studies may be contacted for more information and further clarification regarding the methods and results of their studies. Finally, peer consultation should be sought for any remaining articles. STUDY INCLUSION The method for selecting studies should be reliable and reproducible so that it can be replicated by others who wish to confirm the findings. The process begins with a clearly focused clinical question that outlines the population being studied, the active and control interventions being compared and the outcome event that is of interest to the investigators. Selection criteria are then established that are sensible, reflect the clinical question, and provide direction for an efficient and comprehensive search of the literature. The criteria for including studies in the review can be narrow or fairly broad. With narrow inclusion the risk for false positive or false negative results is increased because the amount of data in the review are limited. Such criteria also preclude the study of appropriate and clinically important subgroups. In contrast, broad inclusion criteria

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increase the likelihood of finding wide variation in the results of the different studies (heterogeneity), making analysis and interpretation of the results more challenging. Because such a search will usually identify more studies than are relevant to the clinical question, a process for sorting through the material is required so that the relevant studies can be retrieved. The specificity of the retrieval process will depend on the explicitness of the criteria established; any ambiguity will result in errors and reduce the accuracy of this process. The selection criteria should be specified in several categories. The type of patient to be studied should include a clear description of the disease or condition and its severity and the setting from which the population is drawn (for example, community or hospital). The main and control interventions should be specified with respect to the timing, dose, duration, and route of administration for medical treatments, and if the intervention is surgical then exact details of the procedure should be provided. The outcomes of interest should be unambiguous, clinically relevant, and defined clearly to avoid confusion and allow generalization of the findings. The type of design will depend on the clinical question being asked and can be in the experimental or descriptive domains. Although publication of studies in languages other than English may create problems with retrieval and translation, the decision must be made before beginning the search process whether non-English literature sources should be accessed. Meta-analysts can simplify the task of selecting articles from a large sample of studies by first reviewing all of the titles, then the abstracts and then the complete articles, excluding studies at each step that do not meet one or more selection criteria. Although this process is quite efficient, there is a risk of missing relevant articles, the content of which may not be clearly specified in the title or abstract. DATA SUMMARIZATION The data to be combined in a meta-analysis can be classified as either categorical or continuous in type. Categorical data are usually in binary format involving a yes/no categorization (for example, pregnancy or no pregnancy). Continuous data are expressed over a range of values (for example, serum concentrations of progesterone after administration of vaginal progesterone suppositories). Binary data can be summarized by using several measures of treatment effect including risk ratio and odds ratio, both of which provide estimates of the relative efficacy of an intervention, and risk difference, which describes the absolute benefit of the intervention. Continuous data can be summarized by the raw mean difference between the two groups if measured on the same scale (for example, serum progesterone), by the standardized mean difference when different

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scales are used (for example, different pain scales), or by the correlation coefficient between two continuous variables.16 GRAPHIC DISPLAY Results from each study are graphically displayed as point estimates and their confidence intervals. The odds ratio tree shown in Fig 31.3 displays the results summarized by the odds ratios and their 95% confidence intervals from 12 hypothetical studies that met prespecified inclusion criteria. The 95% confidence interval indicates that if the study was repeated 100 times, the interval would contain the true (but unknown) effect of the intervention on 95 occasions. The solid vertical line representing an OR of unity represents a null effect. If the 95% confidence interval includes this value (the horizontal line representing the confidence interval crosses the vertical line at OR=1.0), then the observed effect of the experimental manoeuvre is not statistically significant at the conventional level of P<0.05. The confidence interval in 11 out of the 12 studies crossed the vertical line indicating that the estimates of treatment effect were not statistically significant in these studies. A logarithmic scale is used for plotting the ORs, as shown in Fig 31.3, so that the confidence interval will extend symmetrically around the point estimate. ASSESSMENT OF STATISTICAL HETEROGENEITY IN THE EFFECT OF TREATMENT Before the data from the studies can be combined, it is necessary to determine whether the effect of treatment is homogeneous across all studies. This assessment of homogeneity involves calculating the magnitude of statistical diversity that exists in the effect of treatment among the different studies. Statistical heterogeneity may be attributable to two sources. First, study results can differ because of random sampling error. In other words, even if the true (but known) effect is the same in each study, the observed effect size will vary randomly around this true, fixed effect (within study variance). Second, each study sample may have been drawn from a different population, and even if each enrolled a large number of subjects, the effect size would vary as a consequence. These results differ because of between study variation and are called random effects. By examining the degree of homogeneity in the outcomes of the studies, using a statistical test based on the chi-square distribution, it can be determined whether the results of the study reflect a single underlying effect or a distribution of effects. If the test shows that significant heterogeneity is not present, then the differences among studies can be assumed to be a consequence of sampling variation and the data can be combined using a fixed effects model. On the other hand, in the presence of significant

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heterogeneity of treatment effect, a random effects model for combining the data is advocated. ESTIMATING THE COMBINED EFFECT OF TREATMENT The results of the different studies can be pooled by statistically combining them into an overall summary estimate. A simple arithmetic average of the results from all the studies

Fig 31.3 Odds ratio for successful outcome comparing experimental and control interventions in a hypothetical example. would give misleading results depending on the relative contributions of small and large studies. The results of the smaller studies are more likely to be influenced by chance and, therefore, should be given less weight in the combined estimate. The methods employed in a meta-analysis take this fact into consideration by using a weighted average of the results so that the larger studies have more influence than the smaller ones. In general, in the fixed effects model, which considers all variability to be due to random variation, each study is weighted by the inverse of its own variance, which is a function of the sample size and the number of events in the study. The random effects model includes the between study

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variance with the within study variance because it assumes that there is a different underlying effect for each study. Consequently, the confidence interval around the combined effect is wider than with the fixed effects model. Pooling the results of the hypothetical studies shown in Fig 31.3 using the fixed effects model produced a combined OR of 1.80 (95% Cl 1.15 to 2.34, P=0.004). Thus, the meta-analysis indicated that the experimental treatment was more efficacious than the control intervention. METHODOLOGIC QUALITY The methodologic quality of each study can be rated according to several predetermined criteria, such as method of randomization, concealment of treatment allocation, blinding of patients and investigators, completeness of follow-up of study subjects, and so on. It has been suggested that such quality scores should be incorporated into the meta-analysis so that the validity of the studies can be evaluated and ranked by methodologic rigor.17,18,19,20 Combined effect estimates then can be calculated for studies of similar quality thereby providing another type of sensitivity analysis. Theoretically, the better quality studies should provide more reliable effect size estimates. Although such an approach to meta-analysis seems logical, to date no scale or scoring system has been shown to consistently correlate with treatment efficacy.21 Nevertheless, there is some evidence to suggest that studies of poor quality may overestimate the effect of treatment.22,23 SUBGROUP ANALYSIS Subgroup analysis is a useful method to address supplementary questions but requires the data for the subgroups to be available for each study. This approach may also provide insight into the sources of clinical heterogeneity (variability resulting from clinical factors associated with the medical disorder being studied). However, it should be recognized that the power of subgroup analyses is reduced because the sample sizes are much smaller within the subgroups. Consequently, the results should be interpreted with caution but may be used as springboards to generate hypotheses worthy of further testing. PUBLICATION BIAS Publication bias occurs when the results of completed studies are unavailable for analysis. In some situations, studies may not be retrieved despite a thorough search of several data bases. Unfortunately, the amount of bias resulting from this problem has not been quantified. Another potential source of bias stems from the fact that studies with “negative” results (a null effect) are less likely to be published because either the

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investigators are not willing to submit them for publication, or the peer reviewers and editors are not impressed by the findings to warrant their publication.24,25 Although publication bias is difficult to eliminate, its presence may be suggested by two diagnostic techniques. The first technique, which is called the “Fail-Safe N” method, employs a statistical calculation that allows the estimation of the number of “negative,” unpublished studies that would have to exist to nullify the significance of the pooled estimate of the effect size. This procedure, which is based on a method of combining Z values, can estimate the number of such studies that have been tucked away in file drawers in investigators’ offices.26 From this information the meta-analyst has to make a judgment whether the “Fail-Safe N” is sufficiently large to render the possibility unlikely that this number of unpublished studies exists, or the number is so small that there is concern about the reliability of the combined results which should be interpreted cautiously. The second technique involves a visual exploration of the data using an inverted funnel plot.27 In this method, a scatterplot is used to display the relationship between the effect estimate of each study and its sample size. The funnel plot is based on the fact that precision in estimating the underlying treatment effect will increase as the sample size of the study increases. Thus, one could plot the precision of the study (calculated as the inverse of the standard error) against the effect measure (for example, odds ratio, risk ratio, etc.) as shown in Fig 31.4 for the hypothetical studies. The results from smaller studies would be expected to be widely scattered along the bottom of the graph, whereas the spread from larger studies would be less. Therefore, in the absence of bias, the plot will have the appearance of a symmetrically inverted funnel, whereas in the presence of bias, the funnel plot will be skewed and asymmetrical. Applying the funnel plot test to the data from the hypothetical studies, it can be seen quite readily from Fig 31.4 that the scatterplot follows a symmetrical, inverted funnel distribution. This graphical display provides reassuring evidence that the meta-analysis is likely to be free from publication bias. The funnel plot is a simple and useful visual test for the likelihood of bias in meta-analyses. However, the capacity to detect bias is limited when the assessment is based on only a few small studies. The results of such meta-analyses should be interpreted cautiously. CUMULATIVE META-ANALYSIS Cumulative meta-analysis is a good method for assessing the incremental effect of each study.28 It is defined as the repeated performance of metaanalysis to update the pooled effect by recalculating it each time a new study becomes eligible for inclusion with the previously collected series of studies. The accumulation of studies may proceed according to the year of completion or publication of the study, the event rate in the control

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group, the size of the study, the size of the difference between the treated and control groups in the study, some quality score that has been assigned to the study, or other covariates such as drug dosage or time to treatment.28 The sequential pooling may be undertaken in ascending or descending order.

Fig 31.4 Funnel plot example.

for

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Fig 31.5 Cumulative meta-analysis of hypothetical studies comparing experimental and control interventions. Cumulative meta-analysis is best interpreted in the Bayesian framework. The prior probability (the prior belief) is generated by the pooled results of all prior studies, and the posterior probability is derived by adding the results of the new study to those of the others. This posterior probability then becomes the new prior probability for more data to be added when they become available. Advantages of a cumulative meta-analysis include the determination of whether the pooled estimate has been robust over time and the point in time when statistical significance of the pooled result is reached. In this way, the benefit (or harm) of an intervention can be identified as early as possible by routinely updating the meta-analysis with each new study, thereby guiding clinical decisions in an efficient manner. The cumulative meta-analysis of the hypothetical data are shown in Fig 31.5. It can be seen that, a statistically significant difference was observed from the eighth study and, the pooled effect remained consistently significant thereafter. The magnitude of the pooled effect did not change much; the addition of more studies merely improved the precision of this estimate. Consequently, any further studies may be superfluous and unnecessarily costly, if not unethical, given that a significant treatment effect is evident from the meta-analysis.

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CONCLUSIONS The ever expanding volume of scientific literature requires efficient methods to identify valid studies that can influence clinical care. The integration of the best evidence into the decision-making process requires experience, judgment, and sufficient time to undertake a thorough critical appraisal of the information. Healthcare practitioners should learn the skills required to appraise the literature critically so that the best quality evidence can be selected and applied to clinical care within the context of the patient’s preferences. Evidence based clinical care is not merely an academic exercise but an approach geared towards improving clinical practice so that the patient can be best served while upholding the dictum of doing more good than harm.

REFERENCES 1 Flexner A. Medical education in the United States and Canada: A report of the Carnegie Foundation for the Advancement of Teaching. Institution: Carnegie Foundation, 1910. 2 Sackett DI, Rosenberg WMC, Gray JAM, Haynes RB, Richardson WS. Evidence-based medicine: what it is and what it isn’t. BMJ (1996); 313:71–2. 3 McKibbon A. PDQ evidence-based principles and practice. Hamilton: AB Decker Inc. 1999:2–3. 4 Ramsey PG, Carline JD, Inui TS. Changes over time in the knowledge base of practicing internists. JAMA (1991); 266:1103–7. 5 Evans CE, Haynes RB, Birkett NJ. Does a mailed continuing education program improve clinician performance? Results of a randomized trial in antihypertensive care. JAMA (1986); 255:501–4. 6 Sackett CE, Haynes RB, Taylor DW, Gibson ES, Roberts RS, Johnson AL. Clinical determinant of the decision to treat primary hypertension. Clin Res (1977); 24:648. 7 Sackett DL, Richardson WS, Rosenberg W, Haynes RB. Evidence based medicine—how to practice and teach EBM. New York: Churchill Livingstone, 1977. 8 Covell DG, Uman GC, Manning PR. Information needs in office practice: are they being met? Ann Intern Med (1985); 103:596–9. 9 Davidoff F, Haynes B, Sackett D, Smith R. Evidence based medicine. A new journal to help doctors identify new information they need. BMJ (1995); 310:1085–6. 10 Davis DA, Thompson MA, Oxman AD, Haynes RB. Changing physician performance. A systematic review of the effect of continuing medical education strategies. JAMA (1995), 274:700–5.

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11 Daya S. Infertility trials and study design. In: Templeton A, Cooke I, Shaughn O’Brien PM, eds. Evidence-based fertility treatment. London: RCOG Press (1998): 65–86. 12 Daya S. The placebo effect. Evidence-based Obstet Gynecol (2000); 2:1. 13 Daya S. Is there a place for the crossover design in infertility trials? Fertil Steril (1993); 59:6–7. 14 Khan KS, Daya S, Collins JA, Walter SD. Empirical evidence of bias in infertility research: overestimation of treatment effect in crossover trials using pregnancy as the outcome measure. Fertil Steril (1996); 65:939–45. 15 Mulrow CD. Rationale for systematic reviews. BMJ (1994); 309:597– 9. 16 Lau J, loannidis JPA, Schmid CH. Quantitative synthesis in systematic reviews. Ann Intern Med (1997); 127:820–6. 17 Chalmers TC, Smith H Jr, Blackburn B, et al. A method for assessing the quality of a randomized control trial. Control Clin Trials (1981); 2:31–49. 18 Mulrow CD, Linn WD, Gaul MK, Pugh JA. Assessing quality of a diagnostic test evaluation. J Gen Intern Med (1989); 4:288–95. 19 Detsky AS, Naylor CD, O’Rourke K, McGreer AJ, L’Abbe KA. Incorporating variations in the quality of individual randomized trials into meta-analysis. J Clin Epidemiol (1992); 45:255–65. 20 Moher D, Jadad AR, Nichol G, Penman M, Tugwell P, Walsh S. Assessing the quality of randomized controlled trials: an annotated bibliography of scales and checklists. Control Clin Trials (1995); 16:62–73. 21 Emerson JD, Burdick E, Hoaglin DC, Mosteller F, Chalmers TC. An empirical study of the possible relation of treatment differences to quality scores in controlled randomized clinical trials. Control Clin Trials (1990); 11:339–52. 22 Jadad AR, McQuay HJ. Meta-analyses to evaluate analgesic interventions: a systematic qualitative review of their methodology. J Clin Epidemiol (1996); 49:235–43. 23 Schultz KF, Chalmers I, Hayes RJ, Altman DG. Empirical evidence of bias. Dimension of methodologic quality associated with estimates of treatment effects in controlled trials. JAMA (1995); 273:408–12. 24 Dickersin K, Chan S, Chalmers JC, Sacks HS, Smith H Jr. Publication bias in clinical trials. Control Clin Trials (1987); 8:343–53. 25 Dickersin K. The existence of publication bias and risk factors for its recurrence. JAMA. (1990); 263:1385–9. 26 Rosenthal R. The “File Drawer Problem” and tolerance for null results. Psychol Bull (1979); 86:638–41. 27 Egger M, Smith GD, Schneider M, Minder C. Bias in meta-analysis detected by a simple, graphical test. BMJ (1997); 315:629–34.

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28 Lau J, Schmid CH, Chalmers TC. Cumulative meta-analysis of clinical trials builds evidence for exemplary medical care. J Clin Epidemiol (1995); 48:45–57.

32 Indications for IVF treatment: from diagnosis to prognosis Nicholas S Macklon, Math HEC Pieters, Bart CJM Fauser

INTRODUCTION Since the birth of Louise Brown, the first in vitro fertilization (IVF) baby, dramatic developments have occurred in 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 500000, resulting in 1 in 100 to 1 in 150 babies born in the Western world being conceived by IVF. 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 are still lacking. Despite the relative lack of data objectively supporting the use of IVF, it has attained a central role in the treatment of infertility throughout the world. Currently applied standard IVF protocols are expensive, complex, and not without risk. Those providing IVF therefore have a responsibility to offer it only when the indications are appropriate. In recent years, data have emerged which allow us to re-evaluate how we assess these indications. It has become clear that factors other than the cause of infertility itself are of importance in determining the chance of success from IVF. In this chapter we will review indications for IVF, consider those factors which may affect chances of success, and evaluate additional aspects which require consideration when deciding for whom IVF should be advised.

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CONVENTIONAL APPROACH: DIAGNOSIS AS THE INDICATION FOR IVF Since the development of IVF for clinical use, a consensus has developed as to what constitutes the primary medical indications. In the Netherlands, the Dutch Society of Obstetrics and Gynaecology has recently published guidelines for indications for IVF. Although some account is taken of the woman’s age, these indications are primarily diagnosis based (Table 32.1). The original indication for IVF, tubal disease, remains an important indication for IVF. In terms of numbers of patients treated, however, other indications have become more important. This is reflected in the similar frequency of indications revealed by independent databases (Fig. 32.1). Variations between databases may simply reflect differences in definition or population. Patients with low grade endometriosis may, for example, 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. Male factor infertility has become a major indication for IVF (Fig 32.1). In the context of IVF this encompasses both qualitative and quantitative sperm abnormalities. Both sperm motility and morphology are considered to be important for determining IVF outcome in this group.4,5 Our own experience has indicated the importance of the total motile count in this regard while showing that if fertilization occurs, then the outcome is the same as

Table 32.1. IVF indication list of the Dutch Society of Obstetrics and Gynaecology. 1. Tubal pathology • If tubal surgery is not a realistic option, IVF is the method of choice. • In case of impaired tubal function but no occlusion present, or after tubal surgery, IVF is the method of choice after an infertility duration of 2 years or longer. Depending on the woman’s 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

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4. Endometriosis • In case of mild or moderate endometriosis treat as unexplained infertility • In case of severe endometriosis 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.

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Fig 32.1 The percentage of IVF treatment cycles carried out for the five major indications are shown for independent databases. (Data for Cornell, New York, adapted from: Davis OK, Rosenwaks Z. In vitro fertilization. In: Adashi EY, Rock JA, Rosenwaks Z, eds. Reproductive Endocrinology, Surgery and Technology. Philadelphia: Lippincott Raven: (1996) 2322. the normozoospermic group (Fig. 32.2). The impact of motility on fertilization is corrected for by the use of ICSI (Fig. 32.3). Although ICSI has transformed the fertility prognosis for couples with severe male factor infertility, the appropriate indications for ICSI remain controversial.6 In the Netherlands ICSI tends to be restricted to treating severe oligoasthenospermy and total fertilization failure following IVF (Table 32.2). Other European centres 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.

Fig 32.2 IVF outcomes in the Rotterdam center as related to total motile sperm count (x106).

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Fig 32.3 IVF outcomes in the Rotterdam center as related to total motile sperm count (x106) when ICSI was performed.

Table 32.2. Indications for ICSI. • Total motile sperm count (TMC) <1 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 spermatazoa Table 32.3. Impact of cause of infertility on livebirth rate from IVF. Cause of Number of Livebirth rate (%) (95% Cl) infertility cycles Per treatment Per egg Per embryo cycle collection transfer Tubal disease 19096 13.6 (13.0 to 15.0 (14.5 to 16.5 (15.9 to 14.0) 15.6) 17.1) Endometriosis 4117 14.2 (13.2 to 15.9 (14.7 to 17.9 (16.6 to 15.3) 17.0) 19.3) Unexplained 12340 13.4 (12.9 to 15.2 (14.6 to 19.7 (18.8 to 14.1) 15.9) 20.5) Cervical 4232 14.2 (13.2 to 16.2 (15.1 to 18.8 (17.5 to 15.3) 17.4) 20.2)

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Adapted from reference 2. CAUSE OF INFERTILITY AND IVF OUTCOMES The extent to which the underlying pathology itself can affect the chance of success has attracted considerable attention in the literature. Initial reports indicated certain causes of infertility to be associated with a lower chance of success than others. The possible influence of endometriosis on IVF outcome has been a particular topic of controversy. Early reports from major IVF centres in Melbourne, Australia and Norfolk, USA indicated that IVF success rates in women were not adversely affected by endometriosis.7,8 These were followed by a number of studies which reported a significant decrease in the fertilization rate in vitro in women with endometriosis.9–11 However, more recent studies2 again cast doubt on the true impact of endometriosis on IVF outcome. The impact of tubal dysfunction on IVF outcome has been similarly controversial.12,13 The largest study yet published on the effect of the cause of female infertility showed no significant effect on outcome (Table 32.3).2 Further evidence for the lack of importance of the female cause of infertility in determining IVF success has since been provided by an analysis of the French National IVF registry (FIVNAT), in which only an isolated male factor significantly affected outcomes.14 The negative association between male factor infertility and cumulative conception and live birth rates has been observed in other studies.15 It seems reasonable to conclude that when considering which patient is likely to obtain successful treatment of their infertility with IVF, the cause of female infertility is of little relevance. This does not imply that infertility work up is obsolete, however.16 The cause of infertility has a role in determining the chance of spontaneous pregnancy, or a pregnancy following conventional, less complicated treatment. Moreover, the indication for IVF may affect the risk of complications associated with IVF treatment. For instance, patients with anovulation secondary to polycystic ovarian syndrome (PCOS) may be referred for IVF when mono-ovulation induction treatment has failed owing to hypo- or hyperresponse. Such patients are at particular risk of developing ovarian hyperstimulation syndrome after IVF treatment,17 and their ovarian stimulation and monitoring should be planned accordingly.

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 treatment (absolute infertility), and those with reduced fertility who still have a considerable

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chance to conceive spontaneously 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 most couples attending a physician for fertility problems, a clear diagnosis explaining their decreased or absent fertility cannot be found (also referred to as unexplained infertility).18 Proper conventional infertility management—especially ovulation induction—has been demonstrated to be more cost effective than IVF as first line therapy.16 Another randomized comparison in 250 couples between a single IVF cycle and 6 months of expectant management failed to establish any difference in pregnancy rates, when bilateral tubal occlusion was excluded.19 For the group of patients with more subtle abnormalities (such as endometriosis, minor tubal disease, oligospermia, or unexplained infertility), proper management should focus on prognosis rather than diagnosis. The prognosis of a given couple for spontaneous pregnancy should be weighted against pregnancy chances after more invasive treatment strategies such as intrauterine insemination (IUI) (with or without ovarian stimulation) or IVF. Factors shown to affect spontaneous pregnancy chances include duration of infertility, history of previous pregnancies, female age, and, to some extent, sperm quality.20,21 Combining these factors with the use of multivariate regression analysis, pregnancy rates within 1 year ranging from 10% to 70% can be predicted for a given couple. Similar models have been developed for chances of success of ovulation induction in anovulatory patients22 and IVF.2 Overall, the “medical indication” for treatment had no effect on IVF outcome.2,23 Instead, pregnancy chances were again determined by female age, duration of infertility, and previous pregnancy.2 Few randomized comparisons between IVF, IUI, and expectant management in unexplained infertile couples have been published. Spontaneous pregnancy chances in these untreated couples vary from 30% to 70% within 2 years.18 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 higher order multiple pregnancies) are unacceptably high.24 On the basis of a recent 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.25 Indeed, success rates per cycle of a given treatment should be weighed against costs, burden for the patient, and chances for

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complications in mother and child. Risks for finance driven overtreatment remain substantial. In conclusion, a true cause for the infertility cannot be found in the great majority of couples presenting with fertility problems. Therefore, causal treatment is only possible in a small proportion of patients. For the remaining couples a pragmatic prognosis oriented approach should be applied. Most importantly, 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 treatment. This becomes even more predominant over the years, since women in the Western world tend to postpone their wish for children. There is an urgent need for studies focusing on endocrine or ultrasound markers for ovarian ageing and their value as prognostic factors for pregnancy chances.

PROGNOSTIC FACTORS INVOLVED IN IVF Age: The social trend towards delayed childbirth is increasing the importance of ovarian ageing in relation to fecundity and fertility treatment.26 An age related decline in response to stimulation with gonadotropins and a reduction in the number of oocytes,27 oocyte quality,28 fertilization rates,29,30 and ultimately embryos31–33 has been well documented. The number of embryo’s available for transfer appears to be a crucial factor in determining the chance of success with IVF,34 and this is of equal importance in older women.32,35 In a study of the factors influencing the chance of success with IVF in women aged 40 years and above, Widra et al36 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 al37 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 40.38 Many studies point to 40 years of age as being a significant cut off for effectiveness of IVF.35,39,40,41,42 This age related effect on pregnancy rates is similar to that reported in donor sperm programs43 and chances for spontaneous pregnancy. A multiple regression analysis of factors influencing IVF outcomes, showed a predicted livebirth rate of 17% per cycle at age 30, falling to just 7% at 40 years, and 2% at 45 years of age.2 The effect of age was largely, but not completely, overcome by the use of donated oocytes. Establishing “cut off” ages above which admission to IVF treatment is denied has widespread support and is an approach used in the Netherlands. However, if women are to be denied access to IVF

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treatment on the basis of their chance of success, additional indices of ovarian senescence should be considered. Indeed, evidence is accumulating that, for a given patient, chronological age is a poor marker for ovarian ageing. In a recent prospective study of 120 women undergoing their first IVF cycle,44 the antral follicle count on cycle day 3 of an unstimulated cycle was found to be the best individual predictor of response to ovarian stimulation. Cycle day 3 follicle stimulating hormone (FSH) and inhibin B levels offered useful adjunctive information. The authors’ suggestion that such a combined approach should replace age in the prediction of response to IVF treatment would have major implications for patient selection in IVF but, if their findings are confirmed, we would support this approach. Ovarian ageing also affects livebirth rates by means of an increased risk of miscarriage. This may be two to three times more likely in women over 40 years45 and may exceed 80% above the age of 41.46 The increased risk of early pregnancy loss does not appear to be due to uterine ageing47 but seems to be related to a higher incidence of aneuploidy.48 Indeed, the primary impact of increasing age on oocyte and embryo quality may be mediated by an age related increase in aneuploidy. This points to a role for pre-implantation screening in selecting euploid embryos for transfer and allowing partial “correction” of the age factor.49 Duration of infertility: While duration of infertility has been shown to be associated with the chance of spontaneous pregnancy,21 its impact on the chance of success with IVF treatment has been less clear.50 Recently, Templeton et al2 were able to show in their analysis of factors affecting outcomes in IVF that there was a significant decrease in age adjusted livebirth rates with increasing duration of infertility. 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 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.14,51 Hydrosalpinx: One treatable factor which may affect the chances of success from IVF treatment is the presence of hydrosalpinges. Several retrospective studies have indicated that hydrosalpinges negatively influence the chance of success with IVF by decreasing implantation rates.52–54 A recent meta-analysis55 evaluated 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 0.64, 95% confidence interval 0.56 to 0.74). Implantation rates and delivery rates per embryo transfer in the hydrosalpinx group were approximately half of those of the nonhydrosalpinx group, whereas the incidence of early

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pregnancy loss was higher in the former group. While removing hydrosalpinges prior to IVF may be beneficial in some,55 the discussion of the potential risks and benefits should also highlight the potential effect of delaying IVF treatment, especially in older patients, particularly as the effects of transvaginal aspiration of hydrosalpinges or salpingectomy prior to commencing IVF has not been demonstrated in randomized controlled trials.

FUTURE DIRECTIONS As our knowledge of factors influencing outcome following fertility treatments increases, treatment will become more individualized, maximizing cost effectiveness and minimizing inconvenience and risk for the patient. Prognostic models based on individual factors will predominate over population cost effectiveness considerations when deciding, for example, who receives IUI rather than IVF for the treatment of unexplained infertility. In addition, the developments of minimal hyperstimulation IVF56,57 and the prospect of improving implantation rates by extended embryo culture58 and pre-implantation 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. Prediction models now enable us to optimize the individual FSH dosages for ovarian stimulation (Imani et al, unpublished data) and make it possible to determine the optimal number or embryos which should be transferred in a given cycle to a given patient to maximize the chance of a singleton pregnancy (Hunault et al, unpublished data). The future also holds out the prospect of rendering the current means of obtaining oocytes for IVF obsolete. The surgical removal of ovarian tissue followed by in vitro maturation of oocytes will enable almost indefinite delay in childbearing.59 In addition to the promise these techniques hold out for maintaining fertility in patients undergoing gonadotoxic cancer treatment, their impact on IVF is likely to be far broader. Given the increasing tendency to delay childbearing and the severe impact of ageing on fecundity, the most important indication for IVF in the future will be socioeconomic.

REFERENCES 1 Steptoe PC, Edwards RG, Purdy JM. Clinical aspects of pregnancies established with cleaving embryos grown in vitro. Br J Obstet Gynaecol (1980); 87:757–68. 2 Templeton AA, Morris JK, Parslow W. Factors that affect the outcome of in-vitro fertilization treatment. Lancet (1996); 348:1402–6.

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3 Van Steirteghem A, Liebaers I, Devroey P. Assisted reproduction. In: Hillier SG, Kitchener HC, Neilson JP, eds. Scientific essentials of reproductive medicine. London: WB Saunders (1996); 230–41. 4 Van Uem JFHM, Acosta AA, Swanson RJ, et al. Male factor evaluation in in vitro fertilization: Norfolk experience. Fertil Steril (1985); 44:375–83. 5 Thatcher SS, De Cherney AH. A critical assessment of the indications for in-vitro fertilization and embryo transfer. Hum Reprod (1989); 4(Suppl):11–6. 6 Hamberger L, Lundin K, Sjögren A, Söderlund B. Indications for intracytoplasmic sperm injection. Hum Reprod (1998); 13 (Suppl 1): 128–33. 7 Mahadevan MM, Trounson AO, Leeton JF. The relationship of tubal blockage, infertility of unknown cause, suspected male infertility and endometriosis to success of in-vitro fertilization and embryo transfer. Fertil Steril (1983); 40:755–62. 8 Jones HW, Acosta AA, Andrews MC, et al. Three years of in vitro fertilization at Norfolk. Fertil Steril (1984); 42:826–34. 9 Wardle PG, McLaughlin EA, McDermott A, Mitchell JD, Ray BD, Hull MGR. Endometriosis and ovulatory disorder: reduced fertilization invitro compared with tubal and unexplained infertility. Lancet 2:236–9. 10 Mills MS, Eddowes HA, Cahil DJ, et al. A prospective controlled study of in-vitro fertilization, gamete intrafallopian transfer and intrauterine insemination combined with superovulation. Hum Reprod (1992); 7:490–4. 11 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. 12 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. 13 Dor J, Seidman DS, Ben-Shlomo I, Levran D, Ben-Rafael Z, Mashiach S. Cumulative pregnancy rate following invitro fertilization: the significance of age and infertility aetiology. Hum Reprod (1996); 11:425–8. 14 Bachelot A, Pouly JL, Renon C, Devacchi A, De-Mouzon J. Les antecedents de grossesse fiv. Contracept Fertil Sex (1997); 25:507–10. 15 Prietl G, Engelberts U, Maslanka M, van-der-Ven HH, Krebs D. Kumalative Schwangerschaftsraten der konventionellen In-Vitro Fertilisation in Abhängigheit der Diagnose und des Alters der Patientinnen: Ergebnisse des Bonner IVF-Programms. Geburtshilfe und Frauenheilkunde (1998); 58:433–9. 16 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 treatment algorithm as first-line therapy for couples with infertility. Fertil Steril (1999); 71:468–75.

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17 MacDougall MJ, Tan SL, Balen A, Jacobs HS. A controlled study comparing patients with and without polycystic ovaries undergoing invitro fertilization. Hum Reprod (1993); 8:233–7. 18 The ESHRE Capri Workshop Group. Optimal use of infertility diagnostic tests and treatments. Hum Reprod (2000); 15:723–32. 19 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. 20 Eimers JM, te Velde ER, Gerritse R, Vogelzang ET, Looman CW, Habbema JD. The prediction of the chance to conceive in subfertile couples. Fertil Steril (1994); 61:44–52. 21 Collins JA, Burrows EA, Willan AR. The prognosis for live birth among untreated infertile couples. Fertil Steril (1995); 64:22–8. 22 Imani B, Eijkemans MJC, te Velde ER, Habbema JD, Fauser BCJM. Predictors of chances to conceive in ovulatory patients during clomiphene citrate induction of ovulation in normogonadotropic oligoamenorrheic infertility. J Clin Endocrinol Metab (1999); 84:1617–22. 23 Hull MGR, Eddowes HA, Fahy U, et al. Expectations of assisted conception for infertility. BMJ (1992); 304:1465–9. 24 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. 25 Goverde AJ, McDonnell J, Vermeiden JPW, Schats R, Rutten FFH, Schoemaker J. Intrauterine insemination or invitro fertilization in ideopathic subfertility and male subfertility: a randomized trial and cost-effectiveness analysis. Lancet (2000); 355:13–8. 26 Te Velde ER. Ovarian ageing and postponement of childbearing. Maturitas (1998); 30:103–4. 27 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. 28 Tucker MJ, Morton PC, Wright G, Ingargiola PE, Jones AE, Sweitzer CL. Factors affecting success with intracytoplasmic sperm injection. Reprod Fertil Devel (1995); 7:229–36. 29 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 Gynaecol Reprod Biol (1996); 66:155–9. 30 Yie SM, Collins JA, Daya S, Hughes E, Sagle M, Younglie EV. Polyploidy and failed fertilization in in-vitro fertilization are related to patients age and gamete quality. Hum Reprod (1996); 11:614–7. 31 Cordiero I, Calhaz-Jorge C, Barata M, Leal F, Proenca H, Coelho AM. 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.

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32 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. 33 Sharif K, Elgendy M, Lashen H, Afnan M. Age and basal follicle stimulating hormone as predictors of in vitro fertilization outcome. Br J Obstet Gynaecol (1998); 105:107–12. 34 Templeton A, Morris JK. Reducing the risk of multiple births oftwo embryos after in-vitro fertilization. N Engl J Med (1998); 339:573–7. 35 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. 36 Widra EA, Botchan A, Amit A, Kogosowaki A, Yovel I, Lessing JB. Endometrial receptivity: the age-related decline in pregnancy rates and the effect of ovarian function. Fertil Steril (1996); 65:103–8. 37 Alrayyes S, Kaih H, Khan I. Effect of age and cycle responsiveness in patients undergoing intracytoplasmic sperm injection. Fertil Steril (1994); 61:257–61. 38 Abdalla HI, Burton G, Kirkland A, et al. Age, pregnancy and miscarriage: uterine versus ovarian factors. Hum Reprod (1993); 8:1512–7. 39 Al Shawaf T, Nolan A, Guirgis R, Harper J, Santis M, Craft I. The influence of ovarian response on gamete intrafallopian transfer outcome in older women. Hum Reprod (1992); 7:1106–10. 40 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); 60:314–8. 41 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. 42 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. 43 Zaadstra BM, Seidell JC, van Noord PAH, et al. Fat and female fecundity: prospective study of effect of body fat distribution on conception rates. BMJ (1993); 306:484–7. 44 Bancsi LFJ, Broekmans FJM, Eijkemans MJC, Looman CWN, Habbema JDF, te Velde ER. Predictors of poor ovarian response in IVF: a prospective study comparing basal markers of ovarian reserve. Fertil Steril (2000); in press. 45 Toner JP, Flood JT. Fertility after the age of 40. Obstet Gynaecol Clin N Am (1993); 20:261–72. 46 Harrison KE, Breen TM, HennesseyJF, et al. Patient age and success in a human IVF programme. Aust NZ J Obstet Gynaecol (1989); 29:326– 8.

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47 Guanes PP, Remohi J, Gallardo E, Valbuena D, Simon C, Pellicer A. Age does not affect uterine resistance to vascular flow in patients undergoing oocyte donation. Fertil Steril (1996); 66:265–70. 48 Angell R. First-meiotic-division nondysjunction in human oocytes. Am J Hum Genet (1997); 61:23–32. 49 Gianaroli L, Magli M, Munne S, Fortini D, Ferraretti A. Advantages of day 4 embryo transfer in patients undergoing preimplantation genetic diagnosis of aneuploidy. J Assist Reprod Genet (1999); 16:170–5. 50 Collins JA, Milner RA, Rowe TC. The effect of treatment on pregnancy among couples with unexplained infertility. Int J Fertil (1991); 36:145–52. 51 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. 52 Barmat Ll, Rauch E, Spandorfer S, et al. The effect of hydrosalpinges on IVF-ET outcome. J Assist Reprod Genet (1999); 16:350–4. 53 Cohen MA, Lindheim SR, Sauer MV. Hydrosalpinges adversely affect implantation in donor oocyte cycles. Hum Reprod (1999); 14:1087–9. 54 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. 55 Aboulghar MA, Mansour RT, Serour GI. Controversies in the modern management of hydrosalpinx. Hum Reprod Update (1998); 4:882–90. 56 Fauser BC, Devroey P, Yen SS. Minimal ovarian stimulation for IVF: appraisal of potential benefits and drawbacks. Hum Reprod (1999); 14:2681–6. 57 de Jong D, Macklon NS, Fauser BCJM. A pilot study involving minimal ovarian stimulation for IVF by extending the “follicle stimulating hormone (FSH) window” with late follicular phase administration of the gonadotropicreleasing hormone antagonist cetrorelix and without luteal support. Fertil Steril (2000); 73:1051–4. 58 Huisman GJ, Fauser BCJM, Eijkemans MJC, Pieters MHEC. Implantation rates after IVF and transfer of a maximum of two embryos that have undergone 3 to 5 days of culture. Fertil Steril (2000); 73:117–22. 59 Oktay K, Newton H, Aubard Y, Salha O, Gosden RG. Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology? Fertil Steril (1998); 69:1–7.

33 Initial investigation of the patient (female and male) Bulent Gulekli, Tim J Child, Seang Lin Tan

INTRODUCTION Infertility affects one in six to 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 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

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investigations and/or treatment. Endometriosis may be suggested by a history of pelvic pain, and polycystic ovarian syndrome (PCOS) by oligomenorrhoea 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 18.5–25kg/m2. The association between obesity and ovulatory disturbances is well documented2,3 and there is a correlation between the amount of gonadotropins needed to stimulate the ovaries and the weight of the 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 into 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 disturbances, or a history of genitourinary infection may not be volunteered and should be specifically enquired 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 post-pubertal 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

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noted as it may have interfered with semen production causing an abnormal semen analysis result. Some prescription drugs affect male fertility either through 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 gonadotrophin drive to the testes and 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 patients’ 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 anatomical cause of primary amenorrhoea), imperforate hymen, vaginal septa (either transverse or longitudinal), or double cervixes can easily be detected. On speculum examination, the appearance of the cervix should be noted. A microbiological 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

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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 one month of immunization. All women presenting with infertility in our clinic are advised to take 0.4mg 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 and whether or not there is discomfort. The adnexal and parametrial structures are then 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 focussed 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.

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 criteria 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

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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 spermatoza. 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 mucous may be receptive only for a day or two. Because of this, we have largely abandoned the use of this test in our centre. 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 to 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 is 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 mid-luteal 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 18nmol/l to confirm ovulation as this represents the 2.5th centile in their large population study,10 whereas values >16nmol/l for a minimum five days or a single value exceeding 32nmol/l is advocated by ESHRE.19 If the results are equivocal, the test 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 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

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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 day 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 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 33.1), ovarian cysts, endometriomas, and polycystic ovaries (Fig 33.2). 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 Polycystic ovaries are commonly found in apparently normal women, with a prevalence in one study of 22%; in fertility patient populations the prevalence is increased to around 33%.29,30 Though many women are asymptomatic, polycystic ovaries are the commonest cause of anovulatory infertility. The polycystic

Fig 33.1 Hydrosalpinx adjacent to ovary. Color Doppler ultrasonography demonstrates the absence of vascularity within the structure.

Fig 33.2 Polycystic ovary. Increased stromal volume and numerous small circumferential cysts.

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Fig 33.3 Endometrial cavity polyp diagnosed during saline instillation. The patient was noted to have a thickened endometrium at baseline scan. 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 to also 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. Excessively thick endometrium at baseline scan may suggest the presence of a polyp or submucosal fibroid. Uterine cavity distension with saline instillation during vaginal ultrasonography assists in the differentiation of these pathologies (Fig 33.3). Lack of distension 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 polycystic ovaries have a higher ovarian stromal blood flow velocity not only at the baseline scan but also during

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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 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 33.4) on implantation rates, measured after pituitary suppression,36 on the day of hCG

Fig 33.4 Color and Pulsed Doppler ultrasonography of the uterine artery.

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administration37 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 2-D 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 2-D images by a 3-D system is required (Fig 33.5). 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 3-D examination, without the need for a hysterosalpingogram.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 3-D frontal view of the uterus the presence or absence of a septum can be seen (Fig 33.6). The frontal uterine view also allows for accurate examination of fibroids and polyps, and their degree of interference with the endometrial cavity. We previously showed that 3-D calculation of endometrial and ovarian volumes was associated with a low intra- and inter-observer variability.45 Low (<2ml) 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 3-D scanning on the first day of stimulation in an IVF program has been correlated with treatment outcome.47 Recently, the use of power Doppler 3-D 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 3-D reconstructed volume. A power Doppler 3-D image of the media can be reconstructed to demonstrate tubal filling and shape, and fimbrial spill.

Fig 33.5 3D multiplanar view of a normal uterus. The frontal view, demonstrating uterine cavity shape, is at the lower left.

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Fig 33.6 3D frontal view of a bicornuate uterus. The fundal myometrial indentation differentiates this from a septate uterus. 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 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 reduces inflammatory reactions, especially granulomatous inflammation, and the risk of oil embolism.52 In a metaanalysis, 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 suggested that using HSG as a screening test in a low risk infertile population and defering laparoscopy does 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 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

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salpingoscopy.56 Both approaches are essentially only of research interest at the present time. Complete assessment of the pelvis requires laparoscopy. Visualization of the pelvic cavity by laparoscopy is not only necessary to ascertain tubal patency but also determine if 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 abdomino-pelvic 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 post-operative infection and injury to bowel or blood vessels, and mortality of eight per 100000.57 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 two to five days’ abstinence. The WHO criteria for normal semen values are the ones normally 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 ICSI may be discovered without needing to proceed to testicular biopsy for sperm retrieval. The seminal vesicles contribute approximately 70%, the vasa

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deferentia 10%, and the prostate 20% of the ejaculate volume. A low volume (<1ml), acidic (pH<7.5), azoospermic ejaculate may be associated with 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 (>1ml, 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 warrants further investigation.61 If the basic semen analysis is abnormal on repeated testing, further tests are indicated. Vitality stains are used to distinguish live from dead sperm. Viable sperm have a plasma membrane which 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 which 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 computer assisted 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 (ASA) 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 ASA’s 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 oligoospermia should have their serum levels of FSH, LH, testosterone, and prolactin concentrations 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

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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. Hypogonadotrophic 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 north European descent is one in 25. The phenotypic expression varies depending on the combination of mutations inherited. At its most severe, men will manifest the full picture of cystic fibrosis along with bilateral vasal agenesis. A less severe manifestation is congenital bilateral absence of the vas deferens (CBAVD) in which the 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 low volume 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

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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 labelled as “unexplained infertility”. These couples either have a subtle cause of infertility not diagnosed by conventional infertility investigations or they 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 in vitro fertilization should be offered. For women with polycystic ovaries, an alternative treatment would be in vitro maturation of oocytes.66,67

REFERENCES 1 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 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 Gynecologists. 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 E, 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 Gynecol (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.

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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 WHO manual for standardized investigation and diagnosis of the infertile couple. Cambridge, New York, Melbourne: 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 Endocrinologica (1980); 94:89–98. 19 ESHRE. Guidelines to the prevalence, diagnosis, treatment and management of infertility, 1996. 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 Gynecol (1992); 89:985–8. 22 Noyes RW, Hertig AT, Rock J. Dating the endometrial biposy. 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 citrate-sensitive and clomiphene citrateresistant 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); ii: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); ii:870–2.

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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 hormone levels and its effect on stimulation quality in in vitro fertilization. Fertil Steril (1990); 54:297– 302. 36 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. 37 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. 38 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. 39 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. 40 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. 41 Dickey RP, Hower JF, Matulich EM, Brown GT. Effect of standing on non-pregnant uterine blood flow. Ultrasound Obstet Gynecol (1994); 4:480–7. 42 Cacciatore B, Tiitinen A. Transdermal nitroglycerin administration improves uterine blood flow in infertile women. J Assist Reprod Genetics (1997); 14:suppl PP20–451.

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43 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. 44 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. 45 Kyei-Mensah A, Maconochie N, Zaidi J, et al. Transvaginal threedimensional ultrasound: reproducibility of ovarian and endometrial volume measurements. Fertil Steril (1996); 66:718–22. 46 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. 47 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. 48 Sladkevicius P. Three-dimensional power Doppler imaging of the Fallopian tube. Ultrasound Obstet Gynecol (1999); 13:287. 49 Campbell S, Bourne TH, Tan SL. Hysterosalpingo contrast sonography (HyCoSy) and its future within the investigation of infertility in Europe. Ultrasound in 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 ed. 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 metaanalysis. 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. Journal of the Society of Obstetricians and Gynecologists of Canada (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 Ph, et al. Salpingoscopy: a new preoperative diagnostic tool in tubal infertility. Br J Obstet Gynecol (1987); 94:768–73.

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57 Chamberlain G, Brown JC. Gynecological laparoscopy—the report of the working party of the confidential enquiry into gynecological laparoscopy. London: Royal College of Obstetrics and Gynaecology, 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. Infer Med Reprod Clin N Am (1999); 10:435–70. 62 Kim ED, Lipshultz LI. Evaluation and imaging of the infertile male. Infer Med Reprod Clin N 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 Gates RD. The genetics of male reproduction. Infer Med Reprod Clin N 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.

34 Drug used for controlled ovarian stimulation: clomiphene citrate and gonadotropins Zeev Shoham

INTRODUCTION Infertility treatment became available thanks to developments in the characterization and purification of hormones. Treatment with gonadotropins became available in 1960 along with clomiphene citrate therapy. Since treatment with these drugs is still widely used it is the purpose of this chapter to overview the development, structure and mode of action of these drugs.

CLOMIPHENE CITRATE Clomiphene citrate (CC) was synthesized in 1956, and a real therapeutic breakthrough occurred in 1961 when Greenblatt and his group made the discovery that a non-steroidal analog of estradiol, clomiphene citrate, exerts a stimulatory effect on ovarian function in women with anovulatory infertility. It was approved by the United States Food and Drug Administration (FDA) for treatment 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, the importance of which to the action of 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. Clomiphene is a triarylethylene compound (1-pdiethyl aminoethoxyphenyl- 1,2-diphenyl-2-chloroethylene citrate) chemically related to chlorotrianisene (TACE), which is a weak estrogen. Structurally, it is related to the potent synthetic estrogen diethylstilbestrol. Although this compound is not a steroid but a triphenylchloroethylene, its steric configuration shows a remarkable structural similarity to estradiol, and consequently allows the binding to estrogen receptors.

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Clomiphene citrate is available as a racemic mixture of two stereochemical isomers referred to as (cis) Zu-clomiphene or the (trans) En-clomiphene configuration (Fig 34.1), the former being significantly more potent. In the commercially available preparations, the isomers are in the ratio of 38% Zu- and 62% En-clomiphene. In some parts of the world, the drug is available in its Zu form as a 10 mg tablet, which is reportedly equipotent with the 50mg tablet sold in the United States and Europe. Limited experience suggests that the clinical utility of CC may indeed be due to its cis isomer.1,2 It remains uncertain, however, whether cis-clomiphene citrate is more effective than clomiphene citrate proper in terms of ovulation and conception rates.3–6 Following the development of a reverse phase high pressure liquid chromatography (HPLC) assay that could distinguish the CC isomers, a comparison of the pharmacokinetic disposition of the Zu and En isomers of CC was performed.7 It is apparent that each isomer exhibits its own

Figure 34.1 Clomiphene citrate is available as a racemic mixture of two stereochemical isomers referred to as (cis) Zu-clomiphene or the (trans) Enclomiphene 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. characteristic pharmacokinetic profile, the En isomer being absorbed faster and eliminated more completely than the Zu isomer. Although CC tablets consist to 62% of the En isomer and 38% of the Zu isomer, the observed plasma concentrations of the Zu isomer were much higher than

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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 estrogen receptor. Tracer studies of CC with radioactive carbon labeling have shown that the main route of excretion is by the feces, although small amount are also excreted in urine. There is strong evidence that CC is concentrated in the bile and carried by it into the gut. Reabsorption takes place from the gut, so that CC is to some extent sequestered in the enterohepatic circulation, from which it leaks out slowly. After administration of CC for five consecutive days at a dose of 100mg daily, the drug could be detected in serum for up to 30 days. Although the precise mechanism of action remains largely unknown, administration of clomiphene citrate is followed in short sequence by enhanced release of pituitary gonadotropins, resulting in follicular recruitment, selection, assertion of dominance, and rupture. The drug interacts with estrogen receptor-binding proteins not unlike native estrogens and behaves as a competitive estrogen receptor antagonist.8,9 Of importance, CC does not display progestational, corticotropic, androgenic, or antiandrogenic properties. Side effects may include production of dry cervical mucus, hot flushes, and other symptoms like premenstrual discomfort, migraines, and breast soreness, visual difficulty such as spots, flashes, and blurry vision. The principal mechanism of action of CC is a reduction in the negative feedback of endogenous estrogens owing to prolonged depletion of hypothalamic and pituitary estrogen receptors,10,11 consequently leading to an increase in the release of gonadotropin releasing hormone (GnRH) from the hypothalamus into the hypothalamic pituitary portal circulation leading to an increase in the release of pituitary gonadotropins. A marked increase in serum concentrations of luteinizing hormone (LH) over that of FSH may sometimes occur,12 and this temporary change in the ratio of LH to FSH seems to bring about some impairment of follicular maturation, resulting in delayed ovulation. Following discontinuation of CC of shortly thereafter, both gonadotropins gradually decline to the preovulatory nadir, only to surge again at midcycle. In order to obtain good results, CC therapy should be carefully monitored. Obviously, serial measurements of LH, FSH, estradiol, and progesterone, and ultrasound measurements provide the most detailed information on the patient’s response to treatment. The Cochrane review of the clinical data regarding the use of CC for unexplained subfertility in women, based on five randomized trials of CC (doses of 50mg to 250mg per day for up to 10 days) compared with placebo or no treatment showed that the odds ratio for pregnancy per patient was 2.38 (95% confidence interval 1.22 to 4.62). The odds ratio for pregnancy per cycle was 2.5 (1.35 to 4.62).The conclusion of this review was that CC appears to modestly improve pregnancy rates in

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women with unexplained subfertility. However, adverse effects include a possible risk of ovarian cancer and risk of multiple pregnancy.13

HUMAN CHORIONIC GONADOTROPIN—THE LH SURROGATE Owing to inconsistency of the spontaneous LH surge in controlled ovarian stimulation—in any of its forms—and its inefficacy in patients who are being treated with GnRHagonists, 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 progesterone (P) synthesis, resumption of meiosis and occyte 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 thanks to the degree of homology between the two hormones. Both LH and hCG are glycoproteins with a molecular weight of some 30kD, both have nearly identical alpha subunits and a high cystine content. Most importantly, they have the same natural function—to cause lutinization and support lutein cells. Major differences include the sequence of the beta subunit, the regulation of secretion of the two hormones, and the pharmacokinetics of clearance of hCG as opposed to LH. Human chorionic gonadotropin has a slower plasma metabolic clearance rate than LH, which consists of a rapid disappearance phase in the first 5–9 hours after intramuscular injection, and a slower clearance rate in the 1–1.3 days after administration (Fig 34.2). Calculated half life of hCG was 36 hours, as opposed to around 12 hours for LH as determined after IV administration of rec-hLH (estimated to be one14 to three to five hours15,16).

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Fig 34.2 Pharmacokinetics of serum beta-hCG in two hypogonadotropic women: (a) the first woman, (b) the second woman. Three regimens of hCG injections were applied in each woman: 10000IU administered subcutaneously, or intramuscularly, and 5000 IU administered intramuscularly. (Modified from Weissman A, Lurie S, Zalel Y, Goldschmit R, Shoham Z. Human chorionic gonadotropin: pharmacokinetics of subcutaneous administration. Gynecol Endocrinol 1996:10; 273–6.) By day 10 after administration, less than 10% of the originally administered hCG was measurable.17 Some authors have advocated the presence of a serum factor directed against hCG preparations, which significantly prolongs the half life of administered hCG in women who have received repeated courses of gonadotropins.18 Others have not found such a correlation.17 It is interesting to note that hCG does not inhibit the subsequent spontaneous LH surge by the intact pituitary, confirming that an ultra-short loop feedback of LH (here hCG) on its own secretion is not functional.19–21 Increased P levels immediately after hCG administration,

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have been found to subsequently induce endogenous pituitary LH surges in CC/hMG cycles.19 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. Another result of hCG administration is the sustained luteotropic effect, development of multiple corpora lutea, and supraphysiological levels of estradiol and P synthesis. Sustained high level stimulation of the corpora lutea may lead to the ovarian hyperstimulation syndrome, a major complication of gonadotropin therapy.22 Administration of hCG results in an increase in LH-like activity, but does not reconstitute the midcycle physiological FSH surge. Another disadvantage of hCG over the physiological LH surge is the higher luteal phase levels of estradiol and P induced by supraphysiological 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.23,24 In a study by Imoedemhe et al,25 preembryo quality was better when a physiological LH/FSH surge preceded oocyte aspiration and fertilization, than when hCG was used, although this difference did not reach statistical significance. Follicular fluid P and inhibin concentrations were also higher in IVF cycles that utilized hCG as opposed to physiological pituitary LH/FSH surges in the final stage of ovarian stimulation,26 the significance of these findings is not clear yet, but they might be associated with the more common finding of luteinized unruptured follicles after hMG/hCG treatment cycles.27 Another possible disadvantage of the prolonged activity of hCG is delayed ovulation of small follicles, leading to multiple pregnancies. Nearly universal use of GnRH agonists and pituitary desensitization protocols has made the fear of untimely LH surges almost obsolete, hence the timing of the LH-like stimulus, hCG, has been given greater flexibility. Tan et al28 actually showed that there was no difference in cycle outcome with random timing of hCG administration over a period of three days.28 Unfortunately, invalidation of the pituitary mechanism freeing us from inappropriate LH surge also has made us completely dependent on hCG—with all its inherent problems—for the final stage of ovulation triggering.

GONADOTROPINS HISTORICAL OVERVIEW In 1927, Aschheim and Zondek discovered a substance in the urine of pregnant women with the same action as the gonadotrophic factor in the anterior pituitary.29 They called this substance gondadotrophin or Prolan. Moreover, they believed that there are two distinct hormones, Prolan A

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and Prolan B. They subsequently made use of their findings to develop the pregnancy test that carries their name. In 1930, Zondek reported that gonadotropins were also present in the urine of postmenopausal women,30 and in the same year, Cole and Hart found gonadotropins in the serum of pregnant mares.31 This hormone (PMSG) was found to have a potent gonadotropic effect in animals. However, only during 1937 Cartland and Nelson were able to produce a purified extract of this hormone.32 It was not until 1948 that, as a result of the work of Stewart, Sano, and Montgomery, gonadotropins in the urine of pregnant women were shown to come from the chorionic villi of the placenta rather than the pituitary.33 It was subsequently designated “chorionic gonadotropin”.34 Gradually, following years of experiments, it became apparent that the pituitary factor was needed for the production of mature follicles, and that chorionic gonadotropin could induce ovulation only once mature follicles were present.35 Within years, it became apparent that the use of

Table 34.1. Milestones of development in infertility treatment. 1927 The discovery of pituitary hormone controlling ovarian function 1959 Purification and clinical use of pituitary and urine gonadotropins 1960 Clinical use of clomiphene citrate 1966 Use of clomiphene citrate and gonadotropin becomes a common practice 1970 Development of radioimmunoassay for measuring hormone levels 1978 Ultrasound imaging of ovarian follicles 1984 Use of GnRH agonists in infertility treatment 1985 Further purification of urinary gonadotropins 1990 Use of recombinant gonadotropins gonadotropins extracts from non-primate 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 human menopausal gonadotropin (hMG) from postmenopausal urine. This purification method was based on a publication by Katzman et al published in 1943.36 The first urine extract of gonadotropin, which was 15-fold, contained LH and FSH and was given the name Pergonal, inspired by the Italian words “per gonadi” (for the gonads).37 The approval to sell Pergonal was first given by the Italian Authorities in 1950 (Table 34.1). Only in 1961, the first pregnancy was achieved using Pergonal in a patient with secondary amenorrhea. This pregnancy resulted in the first normal baby girl born in Israel in 1962.38 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 can be removed. Metrodin has a specific activity of 100–200IU of

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FSH per mg of protein, while Metrodin-HP has an activity of about 9000IU per mg of protein. FSH AND LH FSH and LH are the two anterior pituitary hormones that control gonadal function. Both hormones are synthesized and secreted by the same pituitary cells39,40 and a single hypothalamic hormone, GnRH, has been shown to influence both. Each hormone contains two subunits. The structure of the alpha subunits of all pituitary glycoproteins is identical. However, the beta chains are unique and after linkage to the alpha chain determine specific hormone function.41 Gonadotropins are glycoproteins with molecular weights of about 30000 daltons and contain fructose, mannose, galactose, acetylglucosamine, and N-acetylneuraminic acid as carbohydrates moieties.42 The sialic acid content varies widely among the glycoprotein hormones, from 20 residues in hCG and five in FSH to only one or two in human luteinizing hormone (hLH). These differences are largely responsible for the variations in the isoelectric points of gonadotropins, leading to the differences in molecular weight and biological activities of the hormones isolated from various sources. The higher the sialic acid content, the longer the biological half life, thus its increased amount in urinary gonadotropins is responsible for its significantly longer half life in comparison with that of the pituitary LH or FSH. The gonadotropic hormones consist of two hydrophobic noncovalently associated alpha and beta subunits. The three dimensional structure of each subunit is maintained by internally crosslinked disulfide bonds. Gonadotropic hormones can be dissociated into few component subunits by denaturing agents.43 The subunits are practically without biological activity, but the hormonal activity is regenerated by recombination of the subunits. All the gonadotropins share a common alpha subunit of 92 amino acid residues in the same sequence with five disulfide bonds as well as two carbohydrate moieties. The beta subunits (of FSH, LH, and hCG) are unique to each hormone and determine their biological specificity. They have amino acid chains of variable lengths (116–147 amino acid residues) and contain six disulfide bonds. FOLLICLE STIMULATING HORMONE FSH is a globular glycosylated protein with a molecular weight of 28000– 30000 daltons, consisting of an alpha and beta subunit. It shares a common alpha subunit with LH, CG, and TSH. Neither subunit has any biological activity individually. The four alpha-subunits have significant amino acid homology with one another and probably evolved from a common precursor. Both LH and FSH beta subunits are found in the same cells within the anterior pituitary.44 The amino acid sequence

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determination for the alpha-subunit was reviewed by Sairam.45 It consists of 92 amino acids with a molecular weight of 14600 daltons and it is stabilized by five disulfide bonds. The FSH beta subunit contains six disulfide bonds. Like the alpha subunit, oligosaccharides are N-linked at 2 asparagine residues. Neuraminic acid is always the terminal oligosaccharide, and there are no N-acetylgalactosamine residues. Once synthesized and secreted, the FSH molecule has a plasma half life averaging 149 minutes (ranging from 95 to 250 minutes), which is about five times longer than the approximately 30 minutes for LH.46,47 Removal of sialic (neuraminic) acid residues from the proteins reduces their plasma half lives because the liver binds asialoglycoproteins and removes them from the circulation.48 Metabolic clearance is decreased by only one-third following bilateral nephrectomy,49 further indicating that the liver is the major site of clearance. The implication of the slow clearance rate of serum concentrations of FSH in vivo is that they can neither increase nor decrease as rapidly as those of LH. It takes one half life for the equilibrium concentration of a molecule to be attained after its secretion rate has increased or decreased abruptly. This difference between LH and FSH clearance rate in vivo may explain why GnRH stimulation is incapable of eliciting pulses of FSH in vivo, while it does stimulate FSH pulses in vitro. In the ovary FSH binds to receptors located on granulosa cells and acts via the cAMPdependent protein kinase pathway. FSH binding enhances early follicle cell development.50 The mechanisms of FSH action have begun to be understood more recently with the development of molecular techniques.51 FSH is important in the development of preovulatory follicles. FSH binding to antral and preovulatory follicles increases aromatase activity52 subsequent to LH-stimulated increases in follicular 17ahydroxylase cytochrome P450 activity and mRNA content that leads to the production of androgens, which are then rapidly aromatized to estradiol by aromatase cytochrome P450.51 Estrogen and FSH then synergize to further increase aromatase activity, and P450 mRNA content is greatly enhanced in the corpora lutea of pregnancy.51 These processes require relatively constant stimulation rather than rapid fluctuations in serum levels of regulatory hormones. FSH, with its slow metabolic clearance rate and, consequently, long serum transients, provides this kind of stimulation. As a result, FSH does not need to be regulated from minute to minute. The effects of GnRH on FSH appear to be primarily on synthesis of the peptide and on its glycosylation rather than on secretion. LUTEINIZING HORMONE Lutropin or luteinizing hormone (LH) is a heterodimer with a molecular weight of about 29400 daltons that consists of two non-covalently linked

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alpha and beta subunits. The alpha subunit consists of 92 amino acids with a molecular weight of 14 600 daltons. The beta subunit has 114 amino acids and a molecular weight of 14 800 daltons. LH is a glycoprotein with considerable heterogeneity in the carbohydrate moieties, which can result in variations in immunological and biological activity. LH is synthesized by the gonadotrophs in the anterior pituitary gland. The liver and kidney are involved in the clearance and excretion of LH. Based on LH metabolic clearance rates, the pituitary content of LH is turned over once or twice per day. Therefore, rapid biosynthesis of LH must occur to account for the large amount of LH excreted. LH pulses, which are more frequent and of greater amplitude than FSH, are dependent on the degree of glycosylation. LH contains fewer sialic acid residues than FSH, which results in a more rapid clearance from the circulation and leads to more LH pulses. Pulsatile release of LH and FSH from the pituitary is necessary for normal reproductive function. LH binds to specific membrane receptors on the theca cells of the ovary and Leydig cells in the testes. Binding of LH to its receptors in both the ovary and the testes is rapid and reversible. LH stimulates the enzyme adenylate cyclase, resulting in an increased synthesis of cyclic AMP which provides the stimulus for steroidogenesis. It has been shown that both pituitary FSH and LH exist in several different forms (isohormones) which exhibit charge heterogeneity and may thus be separated by isoelectric focusing. The various FSH and LH species differ from each other not only in their isoelectric point but also in their relative abundance, receptor bindinactivity, biological activity and plasma half life.53 HUMAN MENOPAUSAL GONADOTROPIN Human menopausal gonadotropin contains an equivalent amount of 75IU of FSH and 75IU of LH in vivo bioactivity. It was shown by Cook et al that hMG preparations also consist of up to five different FSH isohormones and up to nine LH species.54 These differences may cause differences in patients’ response observed sometimes when using various lots of the same preparation. FSH which is the major active agent accounts for <5% of the local protein content in the extracted urinary gonadotropin products.55 The specific activity of these products does not usually exceed 150IU/mg protein. The different proteins found in various hMG preparations include tumor necrosis factor binding protein I, transferin, urokinase, TammHorsfall glycoprotein, epidermal growth factor, and immunoglobulin related proteins.56 Although hMG preparations have been effective and relatively safe, there have been documented cases of local side effects, such as pain and allergic reactions, that were possibly attributed to immune reactions, related to non-gonadotropin proteins.57

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The information regarding the metabolism of gonadotropin hormones is scarce. It has been shown that purified preparations of hFSH, hLH, and hCG injected intravenously into humans had serum half lives (as determined by bioassays) of 180–240 minutes, 38–60 minutes and six to eight hours respectively. Measuring levels of gonadotropins by in vivo bioassays serves to compare biological effects of gonadotropin preparations in a quantitative manner in animals. In the widely used Steelman-Pohley assay,58 21 day old female Sprague-Dawley rats are injected subcutaneously for three 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, means that an ampule of hMG which pertains to have 75IU of FSH can actually contain anywhere between 60 and 90IU. However, the ability to take metabolic half life of the hormone into account is an important advantage of the in vivo test. Therefore, biopotencies of commercially available urinary gonadotropin preparations are still measured using the Steelman-Pohley assay. In-vitro bioassays were introduced in order to improve sensitivity and to develop easier tests with improved reproducibility. Various in vitro cultures, using different animal or human target tissues and different end points, have been suggested.59–61 HALF-LIFE AND CLEARANCE Circulating levels of the gonadotropins measured at any given moment represent the balance between pituitary release and metabolic clearance. After intravenous injection, the initial half life of urinary FSH was demonstrated to be approximately two hours,62 and the true terminal (elimination) half life appears to be 17±5 hours. Following intramuscular injection of urinary FSH preparations, the half life was estimated to be approximately 35 hours.63 PURIFIED FSH (pFSH[METRODINR) Further purification of hMG substantially decreased LH-like activity, leading to a commercial pFSH preparation. MetrodinR was introduced in the mid-1980 and is a product from the

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Figure 34.3 Schematic presentation of the production of hMG and purification of uFSH and HPFSH. same source as hMG, but the LH component has been removed by immuno-affinity chromatography (Fig 34.3). Besides 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 can increase folliculogenesis.64 Furthermore, it was speculated that LH in gonadotropin preparations could be responsible for the high incidence of treatment complications in patients with elevated serum LH levels.65,66 However, other studies67,68 have indicated that the effectiveness of gonadotropin preparations and the occurrence of OHSS were not dependent on the LH/FSH ratio,63 even though the administration of pFSH did result in decreased LH levels as compared to hMG in PCOS patients.70 The desirable goal of having an FSH preparation of high purity led to the development of an immuno-purified product (Metrodin-HP) of >95% purity.71

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GONADOTROPIN-RELEASING HORMONE INTRODUCTION GnRH is the primary hypothalamic regulator of reproductive function. It 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.72,73 GnRH is a decapeptide which, like 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 the male and the female. The maintenance of gonadotropin secretion requires exposure of the pituitary to a pulsatile pattern of GnRH secretion, as was demonstrated in the classic series of experiments by Knobil and Hotchkiss.74 GnRH pulses occur in association with electrical activities within the arcuate nucleus, an area of functionally interconnected GnRH neurons also known as the “GnRH pulse generator.” Since GnRH is released directly into the portal circulation, its short half-life, in the range of several minutes, does not allow for measurement and correlation between serum GnRH levels and its release pattern. GNRH ANALOGS: MECHANISM OF ACTION Although the exact cellular basis for desensitization of the gonadotroph has not been fully delineated, the extensive use of agonistic analogs of GnRH in research allowed for an explosive increase in information and knowledge. Upon acute administration, all GnRH agonistic analogs increase gonadotropin secretion (the flare-up effect) and usually require seven to 14 days to achieve a state of pituitary suppression. Prolonged administration of a GnRH agonistic analog leads to downregulation of GnRH receptors. The agonist bound receptor is internalized via receptor mediated endocytosis,75 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.76 Antagonistic analogs of GnRH have a direct inhibitory effect on gonadotropin secretion. Antagonist 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 since receptor loss does not occur, it requires a constant supply of antagonist to the gonadotroph so that all GnRH receptors are continuously occupied. Consequently, compared to agonistic

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analogs, a higher dose range of antagonists is required for effective pituitary suppression (µg v. mg, respectively). BIOSYNTHESIS OF GnRH ANALOGS The elucidation of the structure, function, and metabolic pathways of native GnRH has prompted an intense effort by research laboratories and the pharmaceutical industry to synthesize potent and longer acting agonistic and antagonistic analogs. During the past three decades, thousands of analogs of GnRH, both agonists and antagonists, have been synthesized. Only few of the agonistic analogs have become approved and clinically used drugs. The first major step in increasing the potency of GnRH was made with substitutions of the 10th glycine at the C-terminus. While 90% of the biologic activity is lost with splitting of the 10th glycine, most of it is restored with the attachment of NH-ethylamide to the Pro9.77 The second major modification was the replacement of the glycine residue at position 6 by D-amino acids, which decreases enzymatic degradation. The combination of these two modifications was found to have synergistic biological activity and agonistic analogs with D-amino acids at position 6 and NH-ethylamide substituting the Gly10-amide not only are better protected against enzymatic degradation, but also have a higher receptorbinding affinity. The affinity can be further increased by introduction of larger, hydrophobic, and more lipophilic D-amino acids at the 6th amino acid position. The increased lipophilicity of the agonist is associated with 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.77 Thus, all the clinically available GnRHagonists contain a D-amino acid at position 6 (Table 34.2). The agonists leuprolide

Table 34.2. The structure of GnRH and GnRH agonistic analogs. Compound 6th position 10th position Amino Acid (No) 1 2 3 4 5 6 7 8 9 10 Native GnRH Glu His Trp Ser Tyr Gly Leu Arg Pro Gly-NH2 Nonapeptides Leuprolide Leu NHEt NHEt Buserelin Ser(OtBu) Goserelin Ser(OtBu) AzaGlyNH2 Histrelin DHis(Bzl) AzaGlyNH2 Decapeptides Nafarelin 2Nal GlyNH2 Triptorelin Trp GlyNH2

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Table 34.3. Trade names, plasmatic half life, relative potency, route of administration, and recommended dose for the clinically available GnRH-a. Generic Trade name Half Relative Administration Recommended name life potency route dose Native GnRH 1 IV,SC Nonapeptides Leuprolide Lupron 90 50–80 SC 500–1000µg/day min 20–30 IM dept 3.75–7.5 mg/month Buserelin Superfact, 80 20–40 SC, 200–500µg/day Supercur min Intranasal 300–400×3– 4/day Histrelin Supprelin <60 100 SC 100µg/day min Goserelin Zoladex 4.5 h 50–100 SC implant 3.6mg/month Decapeptides Nafarelin Synarel 3–4 h 200 Intranasal 200–400×2/day Triptorelin Decapeptyl 3–4.2 36–144 SC, 100–500µg/day h IM depot 3.75mg/month [D-Leu6,Pro9-Net] and buserelin [D-Ser(OtBu)6,Pro9-NEt] contain an ethylamide, and goserelin [D-Ser(OtBu)6,Pro9-AzaglyNH2] and histrelin [Nt-Bzl-D-His6,Pro9-AzaglyNH2] 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. The available data usually describes the relative potency of a certain GnRH-a compared to native GnRH (Table 34.3). Direct comparison between the clinically available GnRH-a under identical conditions has never been undertaken. Therefore, translation of data from these models to humans should be done with caution. All GnRH agonistic analogs are small polypeptide molecules that need to be administered parenterally since they would be otherwise susceptible to gastrointestinal proteolysis. The oral and rectal administration of analogs is associated with very low biopotency (0.0% to 1% v. parenteral administration). Since the first report on the use of the combination of the GnRH agonist buserelin and gonadotropins for ovarian stimulation for IVF back in 1984,78 numerous studies have shown the efficacy of this concept.

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Subsequently, the use of GnRH agonists has gained widespread popularity, and an increasing number of assisted reproductive technology (ART) programs use this approach nowadays as the predominant method of ovarian stimulation. The major advantage initially offered by the agonists was the efficient abolishment of the spontaneous LH surge. The first generation of antagonistic analogs had marked systemic histamine releasemediated side effects that slowed their further clinical development. At present, two third generation antagonists with fewer side effects were approved in the USA and Europe.

REFERENCES 1 Charles D, Klein T, Lunn SF, Loraine JA. Clinical and endocrinological studies with the isomeric components of clomiphene citrate. J Obstet Gynaecol Br Commun (1969); 76:1100–10. 2 Pandya G, Cohen MR. The effect of cis-isomer of clomiphene citrate (cis-clomiphene) on cervical mucus and vaginal cytology. J Reprod Med (1972); 8:133–8. 3 MacLeod SC, Mitton DM, Parker AS, Tupper WR. Experience with induction of ovulation. Am J Obstet Gynecol (1970); 108:814–24. 4 Murthy YS, Parakh MC, Arronet GH. Experience with clomiphene and cisclomphrene. Int J Fertil (1971); 16:66–74. 5 Van Campenhout J, Borreman E, Wyman H, Antaki A. Induction of ovulation with cis clomiphene. Am J Obstet Gynecol (1973); 115:321– 7. 6 Connaughton JF Jr, Garcia CR, Wallach EE. Induction of ovulation with cisclomiphene and a placebo. Obstet Gynecol (1974); 43:697–701. 7 Mikkelson TJ, Kroboth PD, Cameron WJ, Dittert LW, Chungi V, Manberg PJ. Single-dose pharmacokinetics of clomiphene citrate in normal volunteers. Fertil Steril (1986) 46:392–6. 8 Clark JH, Markaverich BM. The agonistic-antiagonistic properties of clomiphene: Review. Pharmacol Ther (1981); 15:467–519. 9 Clark JH, Peck EJ Ir. Anderson JN. Oestrogen receptors and antagonism of steroid hormone action. Nature (1974); 251:446–8. 10 Kahwanago I, Heinrichs WL, Hermann WL. Estradiol “receptors” in hypothalamus and anterior pituitary gland: inhibition of estradiol binding by SH-group blocking agents and clomiphene citrate. Endocrinology (1970); 86:1319–26. 11 Etgen AM. Antiestrogens: effects of tamoxifen, nafoxidine, and CI628 on behavior, cytoplasmic receptors, and nuclear binding of estrogen. Horm Behav (1979); 13:97–112. 12 Vandenberg G, Yen SS. Effect of anti-estrogenic action of clomiphene during the menstrual cycle: evidence for a change in the feedback sensitivity. J Clin Endocrinol Metab (1973); 37:356–65.

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13 Hughes E, Collins J, Vandekerckhove P. Clomiphene citrate for unexplained subfertility in women (Cochrane Review). In: The Cochrane Library. Issue 2, 2000. Oxford: Update Software. 14 Yen SSC, Llenera G, Little B, Pearson O. Disappearance rate of endogenous luteinizing hormone and chorionic gonadotropin in man . J Clin Endocrinol Metab (1968); 2:1763–7. 15 Fritz MA, McLachlan RI, Cohen NL, Dahl KD, Bremner WJ, Soules MR. Onset and characteristics of the midcycle surge in bioactive and immunoactive luteinizing hormone secretion in normal women: Influence of physiological variations in periovulatory ovarian steroid hormone secretion. J Clin Endocrinol Metab (1992); 75:489–93. 16 Deckfalusy E, Harlin J. Clinical-pharmacological studies on human menopausal gonadotropin. Hum Reprod (1988); 3:21–7. 17 Damewood MD, Shen W, Zacur H, Schlaff WD, Rock JA, Wallach EE. Disappearance of exogenously administered human chorionic gonadotropin. Fertil Steril (1989); 52:398–400. 18 Braunstein GD, Bloch SK, Rasor J, Winikoff J. Characterization of antihuman chorionic gonadotropin serum antibody appearing after ovulation induction. J Clin Endocrinol Metab (1983); 57:1164–72. 19 Kyle CV, Griffin J, Jarrett A, Odell WD. Inability to demonstrate an ultrashort loop feedback mechanism for luteinizing hormone in humans. J Clin Endocrinol Metab (1989); 69:170–6. 20 Nader S, Berkowitz AS. Endogenous luteinizing hormone surges following administration of human chorionic gonadotropin: further evidence for lack of loop feedback in humans. J Assisted Reprod Genetics (1992) 9:124–7. 21 Demoulin A, Dubois M, Gerday C, Gillain D, Lambotte R, Franchimont P. Variations of luteinizing hormone serum concentrations after exogenous human chorionic gonadotropin administration during ovarian hyperstimulation. Fertil Steril (1991); 55:797–804. 22 Golan A, Ron-El R, Herman A, Soffer Y, Weinraub Z, Caspi E. Ovarian hyperstimulation syndrome: an update review. Obstet Gynecol Surv (1989); 44:430–40. 23 Gidley-Baird AA, O’Neill C, Sinosich MJ, Porter RN, Pike IL, Saunders DM. Failure of implantation in human in vitro fertilization and embryo transfer patients: the effects of altered progesterone/estrogen ratios in human and mice. Fertil Steril (1986); 45:69–74. 24 Forman R, Fries N, Testart J, Belaisch-Allrt J, Hazout A, Frydman R. Evidence for an adverse effect of elevated serum estradiol concentrations on embryo implantation. Fertil Steril (1988); 49:118– 22. 25 Imoedemhe DA, Sigue AB, Pacpaco ELA, Olazo AB. Stimulation of endogenous surge of luteinizing hormone with gonadotropin-releasing

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hormone analog after ovarian stimulation for in vitro fertilization. Fertil Steril (1991); 55:238–32. 26 Yding Anderson C, Forsdahl F. Endocrine composition of follicular fluid comparing human chorionic gonadotropin to a gonadotropinrelehormone agonist for ovulation induction. Hum Reprod (1993); 8:840–3. 27 Check JH, Nazari A, Barnea ER, Weiss W, Vetter BH. The efficacy of short-term gonadotropin-releasing hormone agonists versus human chorionic gonadotropin to enable oocyte release in gonadotropin stimulation cycles. Hum Reprod (1993); 8:568–71. 28 Tan SL, Balen AH, Hussein E, Mills C, Campbell S, Yovich J. A prospective randomized study of the optimum timing of human chorionic gonadotropin administration after pituitary desensitization in in vitro fertilization. Fertil Steril (1992); 576:1259–64. 29 Aschheim S, Zondek B. Klin Wochenschr (1928); 7:8–9. 30 Zondek B, Klin Wochenschr (1930); 9:393–6. 31 Cole HH, Hart GH. Am J Physiol (1931); 93:57–68. 32 Cartland GF, Nelson JW. J Biol Chem (1937); 199:59–67. 33 Stewart HL, Sano ME, Montgomery TL. J Clin Endocrinol (1948); 8:175–88. 34 Kotz HL, Herrmann W. A review of the endocrine induction of human ovulation. Fertil Steril (1961); 12:375–94. 35 Knobil E, Kostyo J, Greep R. Fed Proc (1958): 17:88 36 Katzman PA, Godfrid M, Cain CK, Doisy EA. J Biol Chem (1943); 148:501–7. 37 Donini P, Montezemolo R. Rassegna di Clinca, Terapia e Scienze Affini. A publication of the Biologic Laboratories of the Instituto Serono (1949); 48:3–28. 38 Lunenfeld B, Sulimovici S, Rabau E, Eshkol A. L’Induction de l’ovulation dans les amenorhees hypophysaires par un traitment combine de gonadotrophines urinaries menopausiques et de gonadotrophines chorioniques. C R Soc Francaise de Gynecol (1962); 5:1–6. 39 Herbert DC, Localization of antisera to LH-beta and FSH-beta in the rat pituitary gland. Am J Anat (1975); 144:379–85. 40 Herbert DC. Immunocytochemical evidence that luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are present in the same cell type in the rhesus monkey pituitary gland. Endocrinology (1976); 98:1554–7. 41 Pauser BCJM, Hsueh AJW. Bioassays of gonadotropins: Background and clinical applications. In: Pellet D, Bidart JM, eds. Serono Symposia. Structure-function relationship of gonadotropins, vol 65. New York: Raven Press (1989):133–6. 42 Butt WR, Kennedy JF. Structure-activity relationships of protein and polypeptide hormones. In: Margoulis M, Greenwood PC. Protein and polypeptide hormones. Amsterdam: Excerpta Medica (1971):115.

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43 De La Llosa P, Jutisz M. Protein and polypeptide hormones. In: Margoulis M, ed. Protein and polypeptide hormones. Amsterdam: Exerpta Medica (1969):298–34. 44 Denef C. Paracrine interactions in the anterior pituitary. Clin Endocrinol Metab (1986); 15:1–32. 45 Sairam MR. Gonadotropic hormones: relationship between structure and function with emphasis on antagonists. In: Li CH, ed. Hormonal proteins and peptides: gonadotropic hormones. New York: Academic Press (1983); 11:1–79. 46 Bogdanove EM, Gay VL. Studies on the disappearance of LH and FSH in the rat: a quantitative approach to adenohypophysial secretory kinetics. Endocrinology (1969); 84:1118–31. 47 Bogdanove EM, Nolin JM, Campbell GT. Qualitative and quantitative gonad pituitary feedback. Recent Frog Horm Res (1975); 31:567–626. 48 Morrell AG, Gregoriadis G, Scheinberg IH, Hickman J, Ashwell G. The role of sialic acid in determining the survival of glycoproteins in the circulation. J Biol Chem (1971); 246:1461–7. 49 Gay VL. Decreased metabolism and increased serum concentrations of LH and FSH following nephrectomy of the rat: absence of short-loop regulatory mechanisms. Endocrinology (1974); 95:1582–8. 50 Lunenfeld B, Kaiem Z, Eshkol A. The function of the growing follicle. J Reprod Fertil (1975); 45:567–74. 51 Richards JS, Hedin L. Molecular aspects of hormone action in ovarian follicular development, ovulation, and luteinization. Ann Rev Physiol (1988); 50:441–63. 52 Dorrington JH, Moon YS, Armstrong DT Estradiol-17B biosynthesis in culture granulosa cells from hypophysectomized immature rats. Endocrinology (1975); 97:1328–31. 53 Ulloa-Aguirre A, Espinoza R, Damian-Matsumura P, Chappel SC. Immunulogical and biological potencies of the different molecules species of gonadotropins. Hum Reprod (1988); 3:491–501. 54 Cook AS, Webster BW, Terranova PF, Keel BA. Variation in the biologic and biochemical characteristics of human menopausal gonadotropin. Fertil Steril (1988); 49:704–12. 55 Howles CM, Loumate E, Giroud D, Luyet G. Multiple follicular development and steroidogenesis following subcutaneous administration of a highly purified urinary FSH preparation in pituitary desensitized women undergoing IVF: a multicenter European phase III study . Hum Reprod (1994); 9:424–30. 56 Giudice E, Crisci C, Eshkol A, Papoian R. Composition of commercial gonadotrophin preparations extracted from human post-menopausal urine: Characterization of non-gonadotrophin proteins. Hum Reprod (1994); 9:2291–9. 57 Li TC, Hindle JE. Adverse local reaction to intramuscular injections of urinary-derived gonadotropin. Hum Reprod (1993); 8:1835–6.

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58 Steelman SL, Pohley FM. Assay of the follicle stimulating hormone based on the augmentation with human chorionic gonadotropin. Endocrinology (1953); 53:604–11. 59 Van Damme MP, Robertson DM, Diczfalusy E. An improved in vitro bioassay method for measuring luteinizing hormone (LH) activity using mouse Leydig cell preparations. Acta Endocrinol (1974); 77:655–60. 60 Padmanabhan V, Chappel SC, Beitins IZ. An improved in-vitro bioassay for follicle stimulating hormone (FSH): suitable for measurement of FSH in unextracted human serum. Endocrinology (1987); 121:1089–109. 61 Dahl KD, Jia XC, Hsueh AJW. Granulosa cell aromatase bioassay for follicle-stimulating hormone. Methods Enzymol (1989); 168:414–22. 62 Le Cotonnec JV, Porchet HC, Beltrami V, Khan A, Toon S, Rowland M. Clinical pharmacology of recombinant human follicle-stimulating hormone (FSH). I Comparative pharmacokinetics with urinary FSH. Fertil Steril (1994); 61:649–78. 63 Diczfalusy E, Harlin J. Clinical-pharmacological studies on human menopausal gonadotrophin. Hum Reprod (1988): 3:21–7. 64 Shoemaker J, Wentz CA, Jones GS, Dubin NH, Sapp K. Stimulation of follicular growth with “pure” FSH in a patient with anovulation and elevated LH levels. Obstet Gynecol (1978); 51:270–6. 65 Raj SG, Berger MJ, Grimes EM, Taymor ML. The use of gonadotropins for the induction of ovulation in women with polycystic ovarian disease. Fertil Steril (1977); 28:1280–4. 66 McFaul PB, Traub AI, Thompson W. Treatment of clomiphene citrateresistant polycystic ovary syndrome with pure follicle stimulating hormone or human menopausal gonadotropin. Fertil Steril (1990): 53:792–7. 67 Jacobson A, Marshall JR. Ovulation response rate with human menopausal gonadotropins of varying FSH/LH ratios. Fertil Steril (1969); 20:171–5. 68 Louwerens B. The clinical significance of FSH/LH ratio in gonadotropin preparations of human origin. Acta Obstet Gynecol Scand (1969); 48:31–6. 69 Lanzone A, Fulghesu AM, Spina MA, et al. Successful induction of ovulation and conception with combined gonadotropin-releasing hormone agonist plus purified follicle stimulating hormone in patients with polycystic ovarian disease. J Clin Endocrinol Metab (1987); 65:1253–8. 70 Anderson RE, Cragun JM, Chang RJ, Stanczyk FZ, Lobo RA. A pharmacodynamic comparison of human urinary follicle stimulating hormone and human menopausal gonadotropin in normal women and polycystic ovary syndrome. Fertil Steril (1989); 52:216–20. 71 Le Cotonnec JY, Porchet HC, Beltrami VC, Howles CM. Comparative pharmacokinetics of two urinary follicle stimulating hormone

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preparations in healthy female and male volunteers. Hum Reprod (1993); 8:1604–11. 72 Schally AV, Arimura A, Kastin AJ. Gonadotropin-releasing hormone: one polypeptide regulates secretion of luteinizing hormone and folliclestimulating hormones. Science (1971); 173:1036–8. 73 Amoss M, Burgus R, Blackwell R, Vale W, Fellows R, Guillemin R. Purification, amino acid composition and Nterminus of the hypothalamic luteinizing hormone releasing factor (LRD) of ovine origin. Biochem Biophys Res Commun (1971); 44:205–10. 74 Knobil E, Hotchkiss J. The Menstrual cycle and its neuroendocrine control. In: Knobil E, Neill J, eds. The physiology of reproduction, 1971–94. Raven Press: New York (1988). 75 Suarez-Quian CA, Wynn PC, Catt KJ. Receptor-mediated endocytosis of GnRH analogs: differential processing of gold-labeled agonist and antagonist derivatives. J Steroid Biochem (1986); 24:183–92. 76 Schvartz I, Hazum E. Internalization and recycling of receptor-bound gonadotropin-releasing hormone agonist in pituitary gonadotropes. J Biol Chem (1987); 262:17046–50. 77 Karten JM, Rivier JE. Gonadotropin-releasing hormone analog design. Structure-function studies toward the development of agonists and antagonists: rationale and perspective. Endocr Rev (1986); 7:44–66. 78 Porter RN, Smith W, Craft IL, Abdulwahid NA, Jacobs HS. Induction of ovulation for in vitro fertilization using buserelin and gonadotrophins. Lancet (1984); ii:1284–5.

35 Inducing follicular development in anovulatory patients and normally ovulating women: current concepts and the role of recombinant gonadotropins Juan Balasch

INTRODUCTION AND OVERVIEW The ovary has two essential physiological responsibilities: the periodic release of oocytes and the production of the steroid hormones, estradiol, and progesterone. Both activities are integrated in the continuous repetitive process of follicle growth and maturation, ovulation, and corpus luteum formation and regression, which integrates 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. Human menopausal gonadotropin (hMG) has been used effectively for ovulation induction, and for years this has been the only urinary gonadotropin available for clinical use. The FSH and LH content of hMG is theoretically equal (75IU of FSH and 75IU 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 15 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 FSH highly purified (FSH-HP) in 1993. FSH-HP, contained <0.1% LH contamination and was the first highly pure biological extract (~4% impurity) and as a result of this, it could be injected subcutaneously (s.c.), unlike the earlier preparations which had to be administered intramuscularly (i.m.). More recently, recombinant human FSH (rhFSH), which is completely devoid of both LH activity and non-specific urinary proteins, has become

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available for clinical use; this represents the final transition to a true drug, where the starting material and complete manufacture are under rigorous control.1 Two rhFSH preparations have recently been registered as follitropin alpha, which was marketed first in 1995 (Gonal-F®, Serono International, Geneva, Switzerland), and follitropin beta (Puregon®, NV Organon, Oss, the Netherlands). Both follitropins are structurally identical to native FSH and each comprises the alpha and beta subunits which compose this gonadotropin; the nomenclature for these recombinant products does not refer to those subunits but is

Fig 35.1 Schematic representation of potential clinical advantages of using recombinant gonadotropins over urinary gonadotropins. merely a means of distinguishing chronologically one from another.1 Like rhFSH, recombinant human LH (rhLH) (LHadi®, Serono International) and recombinant human chorionic gonadotropin (rhCG) (Ovidrel®, Serono International) are produced under the most stringent manufacturing conditions and are being assessed successfully for clinical use.2,3 The three gonadotropins now produced in vitro, by recombinant DNA technology share several advantages over urinary products: a) a fully controlled production process from bulk to finished product b) high purity and specific activity, c) unlimited supply with batch-to-batch consistency, and d) complete absence of contamination by the other gonadotropins.1,3

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This leads to a circumvention of adverse immune reactions owing to contaminant urinary proteins, the possibility of s.c. self-administration, and prevention of the variability in the ovarian response to gonadotropin administration observed cycle to cycle in the same patient. Most importantly, these highly specific monohormonal products have permited 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 (Fig 35.1).4,5 At present, rhFSH is commercially available, and rhLH and rhCG will be marketed in the near future.

GONADOTROPIC CONTROL OF FOLLICULAR GROWTH AND FUNCTION As stated above, recent studies with rhFSH and rhLH which are absolutely monohormonal products, allowed a clear definition of the individual roles of FSH and LH on follicular development in humans. Although the physiological 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 better define the specific spectrum of both FSH and LH actions. This subject has recently been reviewed elsewhere5–8 and is summarized here. FOLLICLE STIMULATING HORMONE (FSH) At birth, the human ovaries contain ~2000000 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 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 ~0.25mm in diameter (the so-called gonadotropin-regulated growth phase) (Fig 35.2). The selective rise in serum FSH beyond a critical “threshold” level that occurs during the luteal-follicular transition is a potent stimulus for

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follicle recruitment (Fig 35.3) 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,

Fig 35.2 Gonadotropin-dependent preovulatory follicular

stages in development.

“Recruitment”: at the beginning of each adult menstrual cycle, plasma FSH levels rise sufficiently to stimulate proliferation and functional differentiation of granulosa cells in multiple follicles between <5mm and ~10mm in diameter, including induction of LH receptors and aromatase activity. Tonic stimulation by LH maintains thecal androgen synthesis in these follicles. The next follicle that will ovulate emerges as the one that is most responsive to FSH (has the lowest FSH “threshold”). “Dominance”: by the mid-follicular phase, the preovulatory (Graafian) follicle becomes recognizable as the largest (≥10mm diameter) healthy follicle in either ovary, containing granulosa cells expressing LH receptors coupled to aromatase. Since this follicle is uniquely responsive to both FSH and LH, it continues to grow and secrete estradiol in the face of declining plasma levels of FSH. Development-dependent paracrine signals maintain the dominance of this follicle, amplifying its responsiveness to FSH and LH. (From Zeleznik and Hillier7 with permission.

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Fig 35.3 Gonadotropin-ovarian interaction. Diagram of gonadotropin-mediated follicular recruitment, selection, and maturation of the dominant follicle by the rise in FSH secretion at the beginning of the menstrual cycle. (From SSC Yen, The human menstrual cycle, in: SSC Yen and RB Jaffe, eds, Reproductive Endocrinology, 2nd ed., WB Saunders: Philadelphia, 1986, with permission.) however, is eventually “selected” to ovulate while the others become atretic. Selection of the dominant follicle can be explained by developmentrelated changes in responsiveness to FSH and LH, which occur in granulosa and thecal 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 non-ovulatory and undergo atresia, while the dominant follicle continues to mature and secrete estrogen.

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LUTEINIZING HORMONE (LH) At the mid-follicular phase, the dominant follicle reaches ≥10mm 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 socalled two cell, two gonadotropin model). In addition, a major development-related response of granulosa cells to FSH is their increased expression of LH receptors functionally coupled to steroid synthesis. LH receptors are constitutively present on thecal 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 the corpus luteum) to respond directly to LH. In fact, LH seems to be able to sustain preovulatory follicular endocrine activity previously induced by FSH,5,9 but definite in vivo evidence in the human is still lacking. In addition, both experimental and clinical evidence clearly indicate that while follicular growth may not require LH, LH plays a primary part in complete maturation of the follicle and oocyte competence.9–12 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 will be 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.8 Up 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 to 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.13 THE LH “CEILING” HYPOTHESIS 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 normal 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.5,14,15 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

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a “ceiling” within which it should be stimulated by LH, unless further normal development be terminated (Table 35.1).5,16 During the second half of the follicular phase as plasma FSH concentrations decline, the LH-dependent phase of preovulatory follicular development only proceeds normally if LH is present at concentrations beneath this ceiling value. When the ceiling is exceeded at the mid-cycle surge of LH, further division of granulosa cells ceases as luteinization proceeds. 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 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 assisted reproduction techniques (ART). The therapeutic aim in each group, however, is quite different. In the former, it is desirable to stimulate monovulation with a view to conception occuring 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. This is discussed below.

OVULATION INDUCTION IN THE ANOVULATORY PATIENT Ovulatory disturbances are present in about 15–25% of couples presenting for an infertility evaluation.17,18 Most infertile anovulatory patients fall into the WHO group II (normogonadotropic anovulation)19 category and the great majority of these women are diagnosed as having polycystic ovary syndrome (PCOS).18,20 These women are well estrogenized and have normal FSH levels but LH may be elevated. On the contrary, WHO group I anovulation or hypogonadotropic hypogonadism (HH), is a much less frequent

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Table 35.1. The LH “ceiling” hypothesis.26 • 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. 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 gonadotropin releasing hormone (GnRH) secretion, all of which are incompatible with normal folliculogenesis and subsequent ovulation.21 Both groups of patients have different gonadotropin requirements for ovulation induction and will be discussed separately. The most important principle in ovulation induction is to provide as close as possible physiological restoration of cyclical 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 which are very sensitive to gonadotropin stimulation.22 Excessive follicular development (>35–40 follicles) usually associated with very high estradiol levels (>4,000–5,000pg/ml) may lead to two important iatrogenic complications: the ovarian hyperstimulation syndrome (OHSS) and multifetal pregnancy. OHSS is a potentially lethal condition, the pathophysiological hallmark of which is marked hemodynamic derangement caused by peripheral arterial vasodilation and vascular leakage of fluid from the intravascular space into peritoneum causing ascites and hemoconcentration.23,24 Multifetal pregnancies are associated with considerable maternal-fetal morbidity and mortality and, contrarily to a common belief, around 75% of iatrogenic multifetal pregnancies are due to ovulation induction in anovulatory women while only the remaining 25% are the product of ART.25–28 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,29,30 and early studies both in vitro31 and in

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vivo32 provided evidence that the self-perpetuating state of biochemical imbalance so characteristic of PCOS could be interrupted in a physiological 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,33 it is accepted that when endogenous LH is already elevated (for example, in PCOS) FSH alone is conceptually better.5,6,30 In addition, a meta-analysis from the Cochrane Library on clinical trials comparing urinary FSH and hMG for ovulation induction in women with clomiphene-resistant PCOS, concluded that no significant benefit could be demonstrated from urinary FSH versus hMG in terms of pregnancy rate, but a significant reduction in OHSS associated with FSH was observed.34 According to experimental data, this could be explained by the reciprocal paracrine signalling between LH-stimulated thecal cells and FSH-stimulated granulosa cells which could bring about follicular hypersensitivity to FSH.35 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 ampoules of hMG (~150IU 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 35.2). 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 preovulatory development as discussed above.4,5 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. The three low dose regimens are focused to fulfill the two essential requirements for successful ovulation induction in PCOS patients: to allow FSH to rise slowly to just above FSH threshold level (which is increased in PCOS patients—as evidenced by normal endogenous FSH concentrations—but having great interindividual variability) while

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Table 35.2. Results of conventional step-up protocol (starting dose 2 ampoules hMG/day) for gonadotropin ovulation induction in WHO group II infertile patients*. • No. of patients 1047 • No. of cycles >2500 • Ovulatory cycles (%) 62–98 • Conceptions (per ovulatory cycle) (%) 10–20 • Multiple pregnancy rate (%) 15–36 • Abortion rate (%) 24–42 • Hyperstimulation rate (%) 1.1–14 *Summary of 6 series. (Adapted from Fauser and Van Heusden).29 avoiding an “explosive” ovarian response because of the exquisite sensitivity of polycystic ovaries to exogenous gonadotropins (Fig 35.4). The chronic low dose step up regimen for gonadotropin induction of ovulation has been the preferred method of ovarian stimulation in PCOS patients since 1990.22,29,33 This regimen is based on the threshold concept suggested by Brown et al36,37 and amplified by Zeleznik38 which argues that the development of multiple follicles results from 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.37 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.39 At present this step-up approach is characterized by a low initial daily FSH dose, usually 75IU, and the dose is increased gradually by small amounts (37.5IU per 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 75IU/day or OHSS in previous treatment cycles, the starting dose may be lower (1/2 to 2/3 ampoule 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

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Fig 35.4 The threshold theory. When the FSH level is above threshold a follicle will be “rescued” (continue to growth). response on ultrasound (Fig 35.5). 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 35.3).33,40–42 Also, two recent comparative prospective studies of the conventional regimen with the chronic low-dose step-up protocol using urinary FSH43 or rhFSH44 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. The step-down protocol applies decremental doses of gonadotropins once ovarian response is established but starting dose is higher than in the step-up approach (Fig 35.6). The aim is to approximate physiological circumstances mimiking the natural intercycle FSH elevation and the subsequent decreasing dependence of the dominant follicle with respect to FSH.29 According to this “threshold/window” concept the duration, rather than magnitude, of FSH increase affects follicle development.45 Monitoring of follicular growth is, however, more stringent than with the step-up approach. In addition, the long half-life 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.22 Notwithstanding the above, results from a pioneering team suggest the

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step-down protocol is an effective approach for FSH administration in PCOS patients.42,46

Fig 35.5 The chronic low dose step up protocol for ovulation induction with FSH in PCOS patients.

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Fig 35.6 The step down protocol for ovulation induction with FSH in PCOS patients according to van Santbrink et al.42 Clinical results are similar to those obtained with the step-up approach (Table 35.3). 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 step-down regimen after follicular selection (leading follicle ≥14mm). In a comparative study with the standard low dose step up regimen, both approaches resulted safe and effective.47 WHAT IS THE CONTRIBUTION OF RECOMBINANT GONADOTROPINS TO OVULATION INDUCTION IN PCOS PATIENTS? Recombinant human FSH (rhFSH). Three recent reports48–50 have compared FSH-HP48,50 or pFSH49 with rhFSH for ovulation induction in WHO group II (PCOS) patients according to a low dose step up48,49 or a sequential protocol.50 These reports show that rhFSH is more effective than urinary FSH (both pFSH and FSH-HP) in inducing follicular development in patients with chronic anovulation (WHO group II), as demonstrated by a significantly lower total dose needed in a shorter

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treatment period and also a lower threshold dose. This implies a higher activity and in vivo biopotency of rhFSH compared with urinary FSH, which is evidenced in a situation such as PCOS where intraovarian action of FSH is disrupted. Favoring this postulate is the fact that the three studies point in the same

Table 35.3. Results of low-dose regimens for gonadotropin ovulation induction in WHO group II infertile women in four large series. Parameter Balen et al White et al Balasch et al van Santbrink et (1994)40 (1996)33 (1996)41 al (1995)42 Low-dose regimen Step-up Step-up Step-up Step-down No. of patients 103 225 234 82 No. of cycles 603 934 534 234 Ovulatory cycles (%) 68 72 78 91 Cycle fecundity (per 14 11 17 16 started cycle) (%) Multiple pregnancy 18 6 15 12 rate (%) Abortion rate (%) 16 28 11 19 Severe OHSS 0.5 0 0 0 direction. In addition, in one of them,48 patients were used as their own controls and the same treatment protocol was applied to the two different gonadotropin drugs in the same patient. This is a remarkable feature considering that PCOS is an heterogeneous condition where disturbed intraovarian regulation of FSH action makes response to exogenous FSH different from normal.29 Hence, the presence or absence of ovarian abnormalities in patients may influence treatment outcome after exogenously administered gonadotropins. This may explain major differences in the FSH threshold and duration of stimulation needed to induce preovulatory follicle development in these patients.29 There is no definite explanation for an increased bioactivity of rhFSH compared with urinary FSH, but several hypotheses may be raised. First, calibration and assignment of gonadotropins’ biopotency is based on an in vivo bioassay (the Steelman-Pohley bioassay), which per se is rather insensitive and imprecise (inherent variation of 40%) and has a poor correlation with clinical response in humans.6,51 Gonadotropin preparations obtained with recombinant techniques and having constant isoform composition may be controlled using physico-chemical technique thus leading to release in mass units (mcg).1,6 This would lead to more precise unitage assignment and to predictably lower individual variability with respect to the therapeutical response. On the contrary, minor differences of FSH isohormone profiles and considerable batch-to-batch

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variability in urinary preparations may result in a differential regulation of steroidogenesis and variations of circulating bioactive FSH actually stimulating the ovaries.29,52 Finally, other factors postulated to explain the higher biopotency of rhFSH compared with urinary FSH include subtle differences in glycoform profile or the presence of proteinaceous contaminants in the urinary product inhibiting FSH action.51,53,54 In accord with the above evidence, it should be stressed that monitoring follicular development must be adjusted to FSH preparations with different potency. Thus, close ultrasonic control and not too early increases in the daily dose may be needed to compensate for the apparently higher bioactivity and terminal half life of rhFSH (the maximal effect of a given dose cannot be observed before 3 to 4 days)55,56 in PCOS patients. It is of note, however, that rhFSH has proved to be effective and safe for ovulation induction in PCOS patients with history of severe OHSS.57 On the other hand, from the large studies performed to date using rhFSH, a starting dose of 75IU has been demonstrated to yield satisfactory results, in spite of the higher potency of rhFSH.58 An attempt to use a lower starting dose (50IU rhFSH) was less effective; in a small series of 11 PCOS patients four of them (36%) had their cycles cancelled because an overresponse and the duration of treatment (mean 20.3 days) was unusually long in a low dose step up protocol.59 This study also evidenced that the incremental dose increase and its timing are as important as the starting dose; the incremental dose increase was 100% after 7 days.59 However, the use of half the current starting dose—37.5IU follitropin alpha—with strict adherence to the principles of chronic low dose therapy regarding duration of the initial dose, may contribute to further refinement of protocols for ovulation induction in some PCOS patients in order to identify the threshold for stimulation leading to monofollicular development.60 Thus, there are no data supporting the better safety or efficacy of an approach implying universal lower starting doses when using rhFSH. This means that, at present, while the ideal regimen has still to be formulated, one should adhere to the principle of the classic chronic low dose step up regimen as discussed above which is to employ a 75IU FSH starting dose for 14 days and then use small incremental dose rises (usually 37.5IU) when necessary, at intervals of not less than 7 days, until follicular development is initiated.58 Recently, a new presentation containing 37.5IU follitropin alpha (Serono International) has been made commercially available and this should further facilitate the clinicians ability to fine tune dosing according to ovarian response. RECOMBINANT hCG (rhCG) Although rhFSH has been the major advance with respect to recombinant gonadotropins for the induction of ovulation in anovulatory infertility associated to PCOS, rhCG has also been successfully used in such

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conditions to trigger ovulation when used instead of urinary hCG.3 hCG may also be used to support the luteal phase after ovulation induction. Although this is a controversial topic in PCOS patients,33,41,43 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.61 In a large multicenter study,41 we found a 10% of spontaneous miscarriage which contrast sharply with spontaneous abortion rates >25–30% reported by others.33,43 OVULATION INDUCTION IN HYPOGONADOTROPIC HYPOGONADISM (HH) (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 ovulatory and pregnancy rates of 75% and 18%, respectively.62 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.63,64 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.11,12,65–68 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 as 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 5 years, however, a number of case reports have suggested that rhLH is effective and safe when administered in association with rhFSH in WHO group I anovulatory patients.11,69–71 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.5,16 WHICH REGIMEN OF GONADOTROPIN ADMINISTRATION? A chronic step-up regimen is the usual gonadotropin treatment approach.40,64,66 However, because these patients usually have longstanding HH associated with extremely low concentrations of FSH/LH

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and estradiol serum levels, the following facts should be considered when inducing ovulation: 1 Pretreatment with sequential estrogen-progestin combination for 1 or 2 cycles “primes” the endometrium and cervical glands and may result in a better response to gonadotropins. 2 Both initial dose (currently 2×75IU ampoules of hMG) and dose increments (usually 1 ampoule per day) 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 step-approach where patients receive two to three ampoules daily of hMG (according to patient’s BMI) on stimulation days 1 and 2, and 1 ampoule on days 3 to 7. From day 8 onward hMG is administered on an individual basis according to the ovarian response and the principles of the step-up regimen (Fig 35.7). Once rhLH becomes commercially available, it will be possible to stimulate with 2×75 IU rhFSH coupled with 75 IU rhLH (see below). 3 Because tonic ovarian stimulation by pituitary gonadotropin is absent in these patients, luteal phase support, preferentially with hCG, is indicated. WHAT IS THE CONTRIBUTION OF RECOMBINANT GONADOTROPINS TO OVULATION INDUCTION IN WHO GROUP I ANOVULATORY PATIENTS? 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

Fig 35.7 The combined (step down and step up) protocol for gonadotropin therapy in WHO group I anovulatory patients. rhLH for supporting rhFSH-induced follicular development in these LHand FSH-deficient anovulatory patients. This was done in a recently

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published multicenter dose-finding study where patients were randomized to receive rhLH (0, 25, 75, or 225IU/day) in addition to a fixed dose of rhFSH (150IU/day).72 The study concluded that rhLH was found to: 1 Promote dose-related increases in estradiol and androstendione secretion by rhFSHinduced follicles. 2 Increase ovarian sensitivity to FSH, as demonstrated by the proportion of patients who developed follicles after the administration of a fixed dose of rhFSH. 3 Enhance the ability of these follicles to luteinize when exposed to hCG. In the study,72 it was shown that a daily dose of 75IU rhLH was effective in most women in promoting optimal follicular development, but a minority of patients may require up to 225IU/day. Therefore, this pioneering study confirms 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.72 The use of hMG containing fixed proportions of FSH and LH for ovulation induction in HH women, has been linked to high prevalence of multiple folliculogenesis which is considered as a major drawback to its use.62,64 Further refinement of 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.4,73

SUPEROVULATION OR STIMULATION OF MULTIPLE FOLLICULAR DEVELOPMENT (MFD) While the goal of induction of ovulation in anovulatory infertile women for conception in vivo is to approach to normal menstrual cycle as closely as possible, the aim of 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 super threshold level during the time of follicle recruitment thus overriding ovarian mechanisms of follicle selection (Fig 35.8). In addition, most ART patients are normally ovulating women. Therefore, as previously stressed,29 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 nondown-regulated cycles,74,75 at present, most patients undergoing IVF or intracytoplasmic sperm injection (ICSI) also receive concomitant GnRH analogues to prevent spontaneous LH surges and improving follicular

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response. The low endogenous LH levels achieved with GnRH analogues in some cases may amplify the differences, if any, in treatment outcome seen with the use of hMG

Fig 35.8 Stimulation of multiple follicular development for ART. Maintenance of a super threshold FSH level during the time of multiple follicular recruitment. 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. 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.14 However, there seems to be a range of LH concentrations obtained in patients treated with GnRH agonists, and these can be maintained for considerable duration; with the new urinary FSH preparations containing negligible LH activity it is possible that there may be a subgroup of patients with low LH concentrations in which ovarian responses are influenced.35,76 This can become specially relevant considering the following: a) such women cannot be identified in advance by measuring LH levels after downregulation;77 b) oocyte maturity and fertilization rate in ART are influenced by the particular hormonal stimulation that preceded oocyte

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retrieval;78 c) the recent availability of both recombinant human FSH (rhFSH) preparations, which are fully devoid of LH activity, and potent GnRH antagonists suppressor of pituitary gonadotropin secretion. A recent study reported that “too much” pituitary suppression induced by GnRH antagonist in women stimulated with rhFSH markedly reduced follicular phase and preovulatory estradiol levels and was associated with poor pregnancy rates and elevated miscarriages rates.79 On this evidence, 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.14,80 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 down-regulated women (reviewed in references8,80–83). A number of individual studies did not identify any substantial differences in the use of different types of exogenous gonadotropins; other reports suggested that the use of FSHonly preparations may be clinically advantageous; and, finally, some studies suggest that preparations that contain hMG may be superior to those that contain only FSH. According to epidemiologists, this controversy is due to underpowered studies dealing with this topic and a solution to this problem is to perform a systematic review with metaanalysis of the data, so that the role of treatment can be ascertained and any factors leading to heterogeneity in the effect of treatment can be explored.80,81 Thus, in a recent review, Daya80 summarized the evidence from a variety of sources, including his own experience, wherein hMG was compared with urinary FSH, as follows. First, from the large database of IVF treatment cycles in France (FIVNAT), it was found that FSH use was associated with higher pregnancy rates (Fig 35.9). Second, the largest randomized trial so far published comparing the two gonadotropins which was also done by that researcher, also demonstrated higher clinical pregnancy rates with FSH administration, an effect that was verified by a meta-analysis of 10 randomized trials (Fig 35.10). Interestingly, higher pregnancy rates were also observed with urinary FSH v hMG irrespective of using GnRH agonists or not and regardless the type of GnRH agonist protocol used (Fig 35.11). Third, a cumulative meta-analysis indicated that, for IVF treatment, no further comparative study of the two gonadotropins was necessary. The common odds ratio appears to have stabilized at around 1.7 (Fig 35.12). Therefore, the addition of further studies does not appear to be necessary because the effect will be only to improve the precision of

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this effect but not its magnitude. Finally, although no major differences in oocyte quality were observed, complete fertilization failure was more likely with hMG use. Thus, in view of these findings, it is not clear whether an absolute LH deficiency really exists following GnRH agonist down-regulation. In any case, only a very small population (<6% of patients), if any patients, seems that it would benefit from exogenous LH administration to turn on the estradiol synthesis during the follicular phase.77 WHICH REGIMEN OF GONADOTROPIN ADMINISTRATION? 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 pituitarysuppression, wherein the highest dose of FSH is

Fig 35.9 FIVNAT clinical pregnancy rate with urinary FSH and hMG for ovarian stimulation in IVF using various GnRHagonist protocols. (Data are from women <35 years, with normal ovulatory status, undergoing their first oocyte retrieval procedure). (From Daya77 with permission).

Fig 35.10 Meta-analysis of randomized controlled trials comparing urinary FSH with hMG for ovarian stimulation in IVF. Odds ratio for clinical pregnancy per cycle initiated. Breslow-Day test c2=6.12; P=0.728. (From Daya77 with permission).

Fig 35.11 Meta-analysis of randomized controlled trials comparing urinary FSH with hMG for ovarian stimulation in IVF. Predicted clinical pregnancy rate after adjusting for GnRH-agonist use and type of protocol (From Daya77 with permission).

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Fig 35.12 Cumulative meta-analysis for clinical pregnancy per cycle initiated in controlled trials comparing urinary FSH and hMG for ovarian stimulation in IVF. Breslow-Day test c2=6.12; P=0.728. (From Day a77 with permission). given on stimulation days 1 and 2 (4–6 75IU FSH ampoules) and is then reduced to two ampoules daily once follicular recruitment has been achieved. This regimen has proved to be clinically efficacious84,85 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 physiological level to maximimally stimulate follicle recruitment in the primary cohort.86,87 Second, follicles recruited by exogenous FSH require an FSH threshold concentration that is higher than that in the natural cycle.86 Third, marked interindividual variation exists in FSH thresholds as well as in FSH metabolic clearance and ovarian sensitivity to FSH.88–90 Remarkably, in clinical studies, such a threshold level was reached with a single injection of six ampoules of FSH 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 ampoules.86 Finally, studies in primates have shown that the step down regimen leads to greater synchronization of follicular maturation when compared with conventional step up stimulation.91

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WHAT IS THE CONTRIBUTION OF RECOMBINANT GONADOTROPINS TO OVARIAN STIMULATION IN ART PATIENTS? Recombinant human FSH (rhFSH). Once the use of FSH-only preparations has been selected (see above), the next step is to explore whether rhFSH is more effective and may yield higher pregnancy rates in down-regulated ART patients than urinary FSH. Very recently, a further meta-analysis on this topic has been presented.92 Five studies fulfilled the inclusion criteria, which were as follows: randomized clinical trial, direct comparison of rhFSH versus pFSH or FSH-HP in IVF cycles, using the long protocol of GnRH agonist administration. The five studies reported on a total of 865 patients treated with rhFSH and 627 patients treated with urinary FSH. The clinical pregnancy rates varied between 20% and 44.53% with rhFSH and between 15.87% and 36.87% in women treated with urinary FSH. The number of ampoules was 29.2 for rhFSH and 30.3 for urinary FSH. Separately, all studies showed improved pregnancy rate when rhFSH was used, but all Cl included 1 and were therefore not significant. The combined odds ratio was 1.36 (95% Cl 1.06–1.74) (Table 35.4). Patients treated with rhFSH were 1.36 times more likely to achieve clinical pregnancy than those treated with urinary FSH, and the 95% Cl suggests that this is significant. The number needed to be treated with rhFSH on average, is 16 and ranges between 6 and 25 patients for an extra clinical pregnancy to be achieved.92 The higher clinical pregnancy rate per cycle started with rhFSH as compared with urinary FSH (OR 1.20, 95% Cl 1.02–1.42) in assisted reproduction was confirmed in another metaanalysis including 12 comparative trials reporting on a total of 2875 patients, of which 1556 were allocated to rhFSH and 1319 to urinary FSH.93 Overall, both meta-analyses indicate that rhFSH is more effective than urinary FSH for inducing multiple follicular development in downregulated women for ART. At present, however, the point is whether rhFSH is better than FSH-HP which is now the only marketed urinary FSH preparation and a product with a specific activity that approaches rhFSH which can also be injected subcutaneously.1 Two recent studies deal with this topic94,95 and both concluded that the mean number of oocytes recovered was significantly higher in the rhFSH group although treatment duration and number of 75IU ampoules were both significantly less in this treatment group. However, in both studies a 11% treatment difference with respect to the number of patients receiving HCG was observed in favor of rhFSH.94,95 This was due to a high incidence of cancelled cycles because of low ovarian response in the u-FSH-HP group where also the collection of only one or two oocytes at retrieval was higher than expected.94 Accordingly, a higher proportion of patients had only one embryo replaced in the u-FSH-HP group. Despite this fact,

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however, there were no significant differences between the rhFSH and uFSH-HP groups in the pregnancy rate per started cycle either in the study by Bergh et al94 or Frydman et al.95 Any ART general population, however, is rather heterogeneous with respect to a woman’s age and ovarian reserve as well as to indication for ART, which in its turn may also affect ovarian response to gonadotropin stimulation. In fact, pharmacokinetic and pharmacodynamic studies have concluded that the large interindividual variability in the responses to rhFSH in women pretreated with GnRH agonists, is mainly attributable to interindividual diversities in ovarian sensitivity to FSH rather than to differences in pharmacokinetics.96 Therefore, the use of the same treatment protocol applied to different gonadotropin drugs in the same patient seems the more appropriate study design when ovarian performance and hormonal levels are the objectives to be compared. This was done in a recent study by us where characteristics of consecutive IVF/ICSI cycles among patients treated with FSH-HP (1st ART treatment cycle) versus rhFSH (2nd ART treatment cycle) were compared.97 In this way, patients served as their own controls and there was

Table 35.4. Pregnancy rates per started cycle in IVF trials comparing rhFSH (follitropin alpha or beta) and urinary FSH (pFSH or FSH-HP) in down-regulated women*. Author, year Type of gonadotropin, pregnancy rate rhFSH group Control group rhFSH Study Group, 1995 α, 20% pFSH, 16% Bergh et al, 1997 α, 45% FSH-HP, 37% Manassiev et al, 1997 α, 43% FSH-HP, 24% Out et al, 1995 β, 22% pFSH, 18% Hedon et al, 1995 β, 35% pFSH, 27% Meta-analysis OR=1.36 (95% Cl=1.06–1.74) *Adapted from Mannasiev et al.92 no interindividual variability. In both treatment cycles the same step-down method of gonadotropin administration (see above) was used and the FSH dose during days 1 to 5 of gonadotropin stimulation was identical in both treatment cycles. That study demonstrated that rhFSH is more efficacious than FSH-HP when used in the same patient in inducing multiple follicular development in down-regulated cycles as indicated by ovarian performance and oocyte maturity obtained using the same amount of FSH.97 In addition, the implantation rate was significantly higher in rhFSH treated cycles than among a control group of women undergoing their second IVF/ICSI attempt following ovarian stimulation with FSH-HP as done in their first ART treatment cycle.97 This could be related to better follicular dynamics

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and oocyte competence (which are both ovarian markers of implantation potential in ART98) obtained with rhFSH. Apparent discrepancies between our study97 and previous reports94,95 indicating that rhFSH is more efficient than FSH-HP as assessed by the total amount of FSH required in ART cycles, can be explained on the basis of regimens of gonadotropin administration used in the three studies and current concepts on gonadotropic control of folliculogenesis as discussed above. As previously discussed, during the stimulation process, both the timing and dose of FSH administered determine the number of follicles recruited and selected. Follicular recruitment takes place in the early follicular phase and each follicle in the cohort has a “threshold” beyond which it is stimulated by FSH to start pre-ovulatory development, otherwise it becomes atretic.86 Thus, the initial loading dose used in our study is probably optimal to stimulate the development of the follicle cohort in different patients types, although it will be of more advantage to some patients than others and, overall, the number of ampoules used was no different between rhFSH and FSH-HP.97 In contrast, in those previously reported studies94,95 the starting dose for both gonadotropin preparations was 150 IU (2 ampoules) daily during the first 6 days. This relatively modest starting dose allowed one to maximize any apparent difference between the two FSH preparations in favor of a higher biopotency for rhFSH in comparison with FSH-HP. Finally, with respect to rhFSH in ART, recent clinical trials suggest clinical efficacy equivalence between follitropin alpha and follitropin beta in terms of oocytes obtained and cumulative FSH dose.99–101 The apparent differences favoring follitropin alpha with regard to local tolerance,99,102 morphological quality of embryos,103 and pregnancy rate99 deserve further study. rhLH. Preliminary results of a multicenter study comparing rhLH (Luveris, Serono International, Geneva, Switzerland) with urinary hCG,104 have demonstrated the clinical efficacy of rhLH in inducing follicular maturation and early luteinization in women undergoing superovulation with rhFSH for IVF. An important finding of this study is that its results support the hypothesis that the use of rhLH as shorter lasting and therefore more physiological surrogate surge would be beneficial in terms of reducing the risk of OHSS. A single dose around 20,000 IU rhLH provides the best efficacy/safety ratio. On the other hand, a recent pilot study comparing FSH-HP alone or in combination with rhLH in ART reported that there were more cancellations for poor ovarian response among FSH-HP+rhLH patients than among FSH-HP patients while mean implantation rate and clinical pregnancy results were improved in the latter group of women (differences not reaching statistical significance because of limited sampling).105 A comparative multicenter clinical trial was designed to determine the benefit of augmentating rhFSH treatment with exogenous rhLH in patients

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undergoing ICSI who have been down-regulated with low-dose luteal GnRH agonist.106 Preliminary results indicated that rhFSH is efficacious as a single therapeutic agent regardless of circulating levels of LH and regardless of the addition of exogenous LH. Potential deleterious effect (significantly reduced implantation rate) attributed to the addition of exogenous rhLH in some patient population (those women having serum FSH levels >1.5IU/I on the day down-regulation was achieved) need to be confirmed with an analysis of a higher number of patients. Overall, both studies105,106 favor the postulate that appropriate endogenous LH concentrations exist despite pituitary suppression, thereby obviating the need for supplemental LH administration in a general ART population. RechCG (rhCG). The empty follicle syndrome is a frustrating condition causing expense and inconvenience. It is characterized by the lack of retrieved oocytes from follicles after ovulation induction and apparently normal follicular development for IVF, despite repeated aspiration and flushing. The syndrome has been “cured” in the same cycle when oocytes were not obtained from follicles in one ovary and a second injection of hCG from a totally different batch yielded retrieved oocytes from the other ovary 36 hours later.107 This would imply that empty follicle syndrome is, in many cases, a drug-related problem rather than a clinical dysfunction. Recently, the first case of recurrent empty follicle syndrome, despite the use of three different batches of commercially available urinary hCG, and its successful treatment using rhCG (Ovidrel, Serono International, Geneva, Switzerland has been reported.108 This may be related to marked differences in the manufacturing process of urinary and hCG. Therefore, it is plausible to postulate that rhCG may prove to be a more reliable ovulation inducing agent than urinary hCG.

CONCLUSIONS AND FUTURE DIRECTIONS All three human gonadotropins (FSH, LH, and hCG) are now produced by recombinant DNA technology which results in reliable supply, high batch-to-batch consistency, high purity, absence of contaminating human proteins, the likelihood of reducing the risk of infectious particles, and the elimination of drugs co-extracted from urine, which is collected from numerous donors. Even more important than such advantages as therapeutic products, is clinical relevance and efficacy of these new drugs. rhFSH has proved to be more efficacious than urinary FSH for ovulation induction in PCOS patients and inducing multiple follicular development in pituitary suppressed women undergoing ART. rhFSH in combination with rhLH has also resulted in effective stimulation of follicular growth in WHO group I anovulation. Interestingly, preliminary data indicate that rhLH used for induction of follicular maturation and early luteinization in

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women undergoing superovulation with rhFSH for ART, is a more physiological surrogate surge than urinary hCG and thus would be beneficial in terms of reducing the risk of OHSS. Finally, rhCG is a safe and effective agent in triggering ovulation and it may prove to be a more reliable ovulation inducing agent than urinary hCG. For the future, a number of questions remain. While the higher efficiency of rhFSH justifies its preferential use in the chronic low dose protocols in PCOS patients, the higher biopotency of that drug in comparison with urinary FSH, and the exquisite sensitivity of polycystic ovaries to gonadotropin stimulation, could make recommendable closer monitoring follicular development and further refinements in low-dose FSH treatment. Future perspectives in the use of recombinant gonadotropins may also contribute to avoid multiple follicular development so frequently seen in PCOS. Thus, preliminary data suggest that high dose rhLH, in association with rhFSH, administered in the late follicular phase, may induce atresia of secondary follicles while supporting the growth of a dominant follicle to pre-ovulatory conditions. In hypogonadotropic hypogonadism, the availability of preparations containing varying ratios of rhFSH and rhLH could introduce effective treatments for patients with differing hormonal needs. This can also apply to down-regulated women undergoing ART depending on the level of pituitary suppression achieved and whether an agonist or an antagonist of GnRH is being used for this purpose. Finally, by controlling the degree of glycosylation during the production of rFSH, and so producing a range of preparations with different half-lives, it may be possible to devise a more “physiological regimen” of induction of ovulation. To simulate the changes in concentration of FSH which occur during the normal follicular phase with the aim of producing a single ovulation, it may be necessary not only to reduce the amount of FSH administered but also to change the molecular form which would facilitate the adjustment of doses required to reproduce the fall in FSH concentration which occurs in the mid- and late follicular phase of the cycle.109 As this chapter was in proof a new meta-analysis comparing urinary FSH and hMG for ovarian stimulation in IVF concluded that both preparations have similar efficacy when used in the long GnRH agonist protocol for pituitary desensitization.110 However, the lack of comprehensiveness of search for comparative trials, questionable inclusion and exclusion criteria applied, and other discrepancies, seriously undermine the integrity of that meta-analysis.111

REFERENCES 1 Howles CM, Wikland M. The use of recombinant human FSH in in vitro fertilization. In: Shoham Z, Howles CM, Jacobs HS, eds. Female

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Infertility Therapy: Current Practice. Martin Dunitz: London, 1999; 103–114. 2 Eshkol A. Recombinant gonadotrophins: an introduction. Hum Reprod (1996); 11 (suppl.1):89–94. 3 Loumaye P, Martineau I, Piazzi A et al. Clinical assessment of human gonadotrophins produced by recombinant technology. Hum Reprod (1996); 11 (suppl.1):95–107. 4 Hillier SG. Ovarian manipulation with pure gonadotropins. J Endocrinol (1990); 127:1–4. 5 Hillier SG. Current concepts of the roles of follicle stimulating hormone and luteinizing hormone in folliculogenesis. Hum Reprod (1994); 9:188–91. 6 Simoni M, Nieschlag E. FSH in therapy: physiological basis, new preparations and clinical use. Reprod Med Rev (1995); 4:163–77. 7 Zeleznik AJ, Hillier SG. The ovary: endocrine function. In: Hillier SG, Kitchener HC, Neilson JP, eds, Scientific Essentials of Reproductive Medicine. WB Saunders: London, 1996; 133–46. 8 Filicori M. The role of luteinizing hormone in folliculogenesis and ovulation induction. Fertil Steril (1999); 71:405–14. 9 Sullivan MW, Stewart-Akers A, Krasnow JS, Berga SL, Zeleznik AJ. Ovarian responses in women to recombinant follicle-stimulating hormone and luteinizing hormone (LH): A role for LH in the final stages of follicular maturation. J Clin Endocrinol Metab (1999); 84:228–32. 10 Cortvrindt R, Hu Y, Smitz J. Recombinant luteinizing hormone as a survival and differentiation factor increases oocyte maturation in recombinant follicle stimulating hormone-supplemented mouse preantral follicle culture. Hum Reprod (1998); 13:1292–302. 11 Balasch J, Miró 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. 12 Fox R, Keroma A, Wardle P. Ovarian response to purified FSH in infertile women with long-standing hypogonadotropic hypogonadism. Aust NZ J Obstet Gynaecol (1997); 37:92–4. 13 Peinado JA, Molina I, Pla M, Tresguerres JAF, Romeu A. Recombinant-human luteinizing hormone (rh-LH) as ovulatory stimulus in superovulated does. J Assist Reprod Genet (1995); 12:61– 4. 14 Chappel SC, Howles C. Re-evaluation of the roles of luteinizing hormone and follicle-stimulating hormone in the ovulatory process. Hum Reprod (1991); 6:1206–12. 15 Jacobs HS. The LH hypothesis. In: Shaw RW, ed. Polycystic Ovaries—A Disorder or a Symptom? Advances in Reproductive Endocrinology, vol. 3. Parthenon: Carnforth, UK, 1991; 91–8.

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16 Hillier SG. Ovarian stimulation with recombinant gonadotrophins: LH as an adjunct to FSH. In: Jacobs HS, ed. The New Frontier in Ovulation Induction. Parthenon: Carnforth, UK, 1993; 39–47. 17 Hull MGR. Epidemiology of infertility and polycystic ovarian disease: endocrinological and demographic studies. Gynecol Endocrinol (1987); 1:235–45. 18 Speroff L, Glass RH, Kase NG. Clinical Endocrinology and Infertility. 5th ed. Williams and Wilkins: Baltimore, 1994. 19 WHO Scientific Group Report. Agents stimulating gonadal function in the human. WHO Techn Rep Ser (1973); 514:1–28. 20 Hill GA. The ovulatory factor and ovulation induction. In: Wentz AC, Herbert III CM, Hill GA (eds), Gynecologic Endocrinology and Infertility. Williams and Wilkins: Baltimore, 1988; 147–60. 21 Santoro N, Filicori M, Crowley Jr WF. Hypogonadotropic disorders in men and women: diagnosis and therapy with pulsatile gonadotropinreleasing hormone. Endocrin Rev (1986); 7:11–23. 22 Franks S, Gilling-Smith C. Advances in induction of ovulation. Curr Opin Obstet Gynecol (1994); 6:136–40. 23 Balasch J, Fábregues F, Arroyo V. Peripheral arterial vasodilation hypothesis: a new insight into the pathogenesis of ovarian hyperstimulation syndrome. Hum Reprod (1998); 13:2718–30. 24 Elchalal U, Schenker JG. The pathophysiology of ovarian hyperstimulation syndrome-views and ideas. Hum Reprod (1997); 12:1129–37. 25 Levene MI, Wild J, Steer P. Higher multiple births and the modern management of infertility in Britain. Br J Obstet Gynaecol (1992); 99:607–13. 26 Hecht BR. Iatrogenic multifetal pregnancy. Assist Reprod Rev (1993); 3:75–87. 27 Evans MI, Littmann L, Louis LSt et al. Evolving patterns of iatrogenic multifetal pregnancy generation: Implications for aggressiveness of infertility treatments. Am J Obstet Gynecol (1995); 172:1750–5. 28 Corchia C, Mastroiacovo P, Lanni R, Nannazzu R, Curro V, Sabris C. What proportion of multiple births are due to ovulation induction? A register-based study in Italy. Am J Public Health (1996); 86:851–4. 29 Fauser BCJM, Van Heusden AMV. Manipulation of human ovarian function: physiological concepts and clinical consequences. Endocrine Rev (1997); 18:71–106. 30 Taymor ML. The regulation of follicle growth: some clinical implications in reproductive endocrinology. Fertil Steril (1996); 65:235–47. 31 Erickson GF, Hsueh AJW, Quigley ME, Rebar RW, Yen SSC. Functional studies of aromatase activity in human granulosa cells from normal and polycystic ovaries. J Clin Endocrinol Metab (1979); 49:514–9.

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32 Seibel MM, Kamreva MM, MacArdle C, Taymor ML. Treatment of polycystic ovary disease with chronic low-dose follicle stimulating hormone: Biochemical changes and ultrasound correlation. Int J Fertil (1984); 29:39–43. 33 White DM, Polson DW, Kiddy D, et al. Induction of ovulation with low-dose gonadotropins in polycystic ovary syndrome: an analysis of 109 pregnancies in 225 women. J Clin Endocrinol Metab (1996); 81:3821–4. 34 Hughes E, Collins J, Vandekerckhove P. Ovulation induction with urinary follicle stimulating hormone vs human menopausal gonadotropin for clomiphene-resistant polycystic ovary syndrome. In: Lilford R, Hughes E, Vandekerckhove P. Subfertility Module of The Cochrane Database of Systematic Reviews, [updated 03 March 1997]. 35 Smyth CD, Miró F, Howles CM, Hillier SG. Effect of luteinizing hormone on follicle stimulating hormoneactivated paracrine signalling in rat ovary. Hum Reprod (1995); 10:33–9. 36 Brown JB. Pituitary control of ovarian function-concepts derived from gonadotrophin therapy. Aust NZ J Obstet Gynaecol (1978); 18:47–54. 37 Brown JB, Evans JH, Adey FD, Taft HP, Townsend L. Factors involved in the induction of fertile ovulation with human gonadotrophins. J Obstet Gynaecol Br Commonw (1969); 76:289–306. 38 Zeleznik AJ. Gonadotropic control of folliculogenesis: the threshold theory. In: Filicori M, Falmigni C, eds. Ovulation induction: basic science and clinical advances. Excerpta Medica: Amsterdam, 1994; 37–46. 39 Franks S, Hamilton-Fairley D. Ovulation induction: Gonadotropins. In: Adashi EY, Rock JA, Rosenwaks Z, eds. Reproductive Endocrinology, Surgery, and Technology. Lippincott-Raven: Philadelphia, 1996; 1207–23. 40 Balen AH, Braat DD, West C, Patel A, Jacobs HS. Cumulative conception and live birth rates after the treatment of anovulatory infertility: safety and efficacy of ovulation induction in 200 patients. Hum Reprod (1994); 9:1563–70. 41 Balasch J, Tur R, Peinado JA. The safety and effectiveness of stepwise and low-dose administration of follicle stimulating hormone in WHO group II anovulatory infertile women: evidence from a large multicenter study in Spain. J Assist Reprod Genet (1996); 13:551–6. 42 van Santbrink EJP, Donderwinkel PFJ, van Dessel TJHM, Fauser BCJM. Gonadotrophin induction of ovulation using a step-down dose regimen: single-centre clinical experience in 82 patients. Hum Reprod (1995); 10:1048–53. 43 Homburg R, Levy T, Ben-Rafael Z. A comparative prospective study of conventional regimen with chronic lowdose administration of follicle-stimulating hormone for anovulation associated with polycystic ovary syndrome. Fertil Steril (1995); 63:729–33.

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44 Hedon B, Hugues JN, Emperaire JC, et al. A comparative prospective study of a chronic low dose versus a conventional ovulation stimulation regimen using recombinant follicle stimulating hormone in anovulatory infertile women. Hum Reprod (1998); 13:2688–92. 45 Schipper I, Hop WCJ, Fauser BCJM. The follicle-stimulating hormone (FSH) threshold/window concept examined by different interventions with exogenous FSH during the follicular phase of the normal menstrual cycle: duration, rather than magnitude, of FSH increase affects follicle development. J Clin Endocrinol Metab (1998); 83:1292–8. 46 van Santbrink EJP, Fauser BCJM. Urinary follicle-stimulating hormone for normogonadotropic clomipheneresistant anovulatory infertility: prospective, randomized comparison between low dose stepup and stepdown dose regimens. J Clin Endocrinol Metab (1997); 82:3597–602. 47 Hugues JN, Cédrin-Durnerin I, Avril C, Bulwa, Hervé F, Uzan M. Sequential step-up and step-down dose regimen: an alternative method for ovulation induction with follicle-stimulating hormone in polycystic ovarian syndrome. Hum Reprod (1996); 11:2581–4. 48 Balasch J, Fábregues F, Peñarrubia J, et al. Follicular development and hormonal levels following highly purified or recombinant folliclestimulating hormone administration in ovulatory women and WHO group II anovulatory infertile patients. J Assist Reprod Genet (1998); 15:552–9. 49 Coelingh-Bennink HJT, Fauser BCJM, Out HJ. Recombinant folliclestimulating hormone (FSH; Puregon) is more efficient than urinary FSH (Metrodin) in women with clomiphene citrate-resistant, normogonadotropic, chronic anovulation: a prospective, multicenter, assessor-blind, randomized, clinical trial. Fertil Steril (1998); 69:19– 25. 50 Hugues JN, Cedrin-Durnerin I, Bstandig B, Blasquez M, Hervé F, Uzan M. Comparison of recombinant and urinary follicle stimulating hormone preparations efficiency for achievement of follicular selection in patients with chronic anovulation (World Health Organization group II). Hum Reprod (1999); 14(Abstract book 1):127–8. 51 Bergh C, Howles CM, 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. Hum Reprod (1997); 12:2133–9. 52 Mason HD, Mannaerts B, de Leeuw R, Willis DS, Franks S. Effects of recombinant human follicle stimulating hormone on cultured human granulosa cells: comparison with urinary gonadotrophins and actions in preovulatory follicles . Hum Reprod (1993); 8:1823–7. 53 Out HJ, Mannaerts BMJL, Driessen SGAJ, Coelingh Bennink HJT. A prospective, randomized, assessor-blind, multicentre study comparing

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recombinant and urinary follicle stimulating hormone (Puregon versus Metrodin) in in-vitro fertilization. Hum Reprod (1995); 10:2534–40. 54 Lambert A, Rodgers M, Mitchell R, et al. In-vitro biopotency and glycoform distribution of recombinant human follicle stimulating hormone (Org 32489), Metrodin and Metrodin-HP. Hum Reprod (1995); 10:1928–35. 55 Le Cotonnec JY, Porchet HC, Beltrami V, Khan A, Toon S, Rowland M. Clinical pharmacology of recombinant human follicle-stimulating hormone. II. Single doses and steady state pharmacokinetics. Fertil Steril (1994); 61:679–86. 56 Shoham Z, Insler V. Recombinant technique and gonadotropins production: new era in reproductive medicine. Fertil Steril (1996); 66:187–201. 57 Aboulghar MA, Mansour RT, Serour GI, Amin YM, Sattar MA, El Attar E. Recombinant follicle-stimulating hormone in the treatment of patients with history of severe ovarian hyperstimulation syndrome. Fertil Steril (1996); 66:757–60. 58 Homburg R, Howles CM. Low-dose FSH therapy for anovulatory infertility associated with polycystic ovary syndrome: rationale, results, reflections and refinements. Hum Reprod Update (1999); 5:493–9. 59 Hayden CJ, Rutherford AJ, Balen AH. Induction of ovulation with the use of a starting dose of 50 units of recombinant human folliclestimulating hormone (Puregon*). Fertil Steril (1999); 71:106–8. 60 Balasch J, unpublished observations. 61 Blumenfeld Z, Nahhas F. Luteal dysfunction in ovulation induction: The role of repetitive human chorionic gonadotropin supplementation during the luteal phase. Fertil Steril (1988); 50:403–7. 62 Filicori M, Flamigni C, Dellai P, et al. Treatment of anovulation with pulsatile gonadotropin-releasing hormone: prognostic factors and clinical results in 600 cycles. J Clin Endocrinol Metab (1994); 79:1215–20. 63 Filicori M, Flamigni C. Ovulation induction regimens: is a consensus possible? In: Filicori M, Flamigni C, eds. Ovulation induction: basic science and clinical advances. Elsevier Science: Amsterdam, 1994; 371–87. 64 Martin KA, Hall JE. Pulsatile GnRH in hypogonadotropic hypogonadism. In: Filicori M, Flamigni C, eds. Ovulation induction. Update ’98. The Parthenon Publishing Group: New York, 1998; 47– 54. 65 Couzinet B, Lestrat N, Brailly S, Forest M, Schaison G. Stimulation of ovarian follicular maturation with pure follicle-stimulating hormone in women with gonadotropin deficiency. J Clin Endocrinol Metab (1988); 66:552–6. 66 Shoham Z, Balen A, Patel A, Jacobs HS. Results of ovulation induction using human menopausal gonadotropin or purified follicle-

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stimulating hormone in hypogonadotropic hypogonadism patients. Fertil Steril (1991); 56:1048–53. 67 Schoot DC, Coelingh-Bennik H JT, Mannaerts BM, Lamberts SW, Bouchard P, Fauser BC. 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. 68 Schoot DC, Harlin J, Shoham Z, et al. Recombinant human folliclestimulating hormone and ovarian response in gonadotropic-deficient women. Hum Reprod (1994); 9:1237–42. 69 Hull M, Corrigan E, Piazzi A, Loumaye E. Recombinant human luteinizing hormone: an effective new gonadotropin preparation. Lancet (1994); 344:334–5. 70 Kousta E, White DM, Piazzi A, Loumaye E, Franks S. Successful induction of ovulation and completed pregnancy using recombinant luteinizing hormone and follicle stimulating hormone in a woman with Kallman’s syndrome. Hum Reprod (1996); 11:70–1. 71 Agrawal R, West C, Conway GS, Page ML, Jacobs HS. Pregnancy after treatment with three recombinant gonadotropins. Lancet (1997); 349:29–30. 72 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- and FSH-deficient anovulatory women: a dose-finding study. J Clin Endocrinol Metab (1998); 83:1507–14. 73 Hillier SG. Ovarian stimulation with recombinant gonadotropin: LH as an adjunct to FSH. In: Jacobs HS, ed. The new frontier in ovulation induction. The Parthenon Publishing Group: Carnforth, UK, 1993; 39– 47. 74 Jansen CAM, Van Os MC. Puregon without analogs: an oxymoron. Gynecol Endocrinol (1996); 10 (suppl. 1):34. 75 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 nondown-regulated cycles. Hum Reprod (1995); 10:3097–101. 76 Fleming R, Chung CC, Yates RWS, Coutts JRT. 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. 77 Loumaye E, Engrand P, Howles CM, O’Dea L. Assessment of the role of serum luteinizing hormone and estradiol response to folliclestimulating hormone on in vitro fertilization outcome. Fertil Steril (1997); 67:889–99.

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78 Pieters MHEC, Dumoulin JCM, Engelhart CM, Bras M, Evers JHL, Geraedts JPM. Immaturity and aneuploidy in human oocytes after different stimulation protocols. Fertil Steril (1991); 56:306–10. 79 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. 80 Daya S. hMG versus FSH: is there any difference? In: Filicori M, Flamigni C, eds. Ovulation induction: Update ’98. The Parthenon Publishing Group: New York, 1998; 183–92. 81 Daya S. The role of meta-analysis in determining which gonadotropin to use for ovarian stimulation. In: Shoham Z, Howles CM, Jacobs HS, eds. Female infertility therapy: current practice. Martin Dunitz: London, 1999; 177–88. 82 Hull MGR, Armatage RJ, McDermott A. Use of follicle-stimulating hormone alone (urofollitropin*) to stimulate the ovaries for assisted conception after pituitary desensitization. Fertil Steril (1994); 62:997– 1003. 83 Söderström-Anttila V. Clinical outcome of ovulation induction: highly purified FSH versus hMG. In: Filicori M, Flamigni C, eds. Ovulation induction: update ’98. Parthenon Publishing: New York; 193–200. 84 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. 85 Davis OK, Rosenwaks Z. In vitro fertilization. In: Adashi EY, Rock JA, Rosenwaks Z, eds. Reproductive endocrinology, surgery, and technology, vol. 2. Lippincott-Raven: Philadelphia, 1996; 2319–34. 86 Lolis DE, Tsolas O, Messinis IE. The follicle-stimulating hormone threshold level for follicle maturation in superovulated cycles. Fertil Steril (1995); 63:1272–7. 87 Messinis IE, Templeton AA. The importance of follicle-stimulating hormone increase for folliculogenesis. Hum Reprod (1990); 5:153–6. 88 Porchet HC, Le Cotonnec JY, Loumaye E. Clinical pharmacology studies of recombinant human folliclestimulating hormone. III. Pharmacokinetic-pharmacodynamic modeling after repeated subcutaneous administration. Fertil Steril (1994); 61:687–95. 89 Ben-Rafael Z, Levy T, Schoemaker J. Pharmacokinetics of follicle stimulating hormone: clinical significance. Fertil Steril (1995); 63:689–700. 90 van Santbrink EJP, Hop WC, van Dessel TJHM, de Jong FH, Fauser BCJM. Decremental follicle-stimulating hormone and dominant follicle development during the normal menstrual cycle. Fertil Steril (1995); 64:37–43.

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91 Abbasi R, Kenigsberg D, Danforth D, Falk JR, Hodgen GD. Cumulative ovulation rate in human menopausal/human chorionic gonadotropin-treated monkeys: “step-up” versus “step-down” dose regimens. Fertil Steril (1987); 47:1019–24. 92 Manassiev NA, Tenekedjier KI, Collins J. Does the use of recombinant follicle-stimulating hormone instead of urinary follicle-stimulating hormone lead to higher pregnancy rates in in vitro fertilization-embryo transfer cycles? Assist Reprod (1999); 9:7–12. 93 Daya S, Gunby J. Recombinant versus urinary follicle stimulating hormone for ovarian stimulation in assisted reproduction. Hum Reprod (1999); 14:2207–15. 94 Bergh C, Howles CM, 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. Hum Reprod (1997); 12:2133–9. 95 Frydman R, Avril C, Camier B, et al. A double-blind, randomized study comparing the efficacy of recombinant human follicle stimulating hormone (rhFSH-Gonal F) and highly purified urinary FSH (uhFSH/Metrodin HP) in inducing superovulation in women undergoing assisted reproductive techniques. Hum Reprod (1998); 13 (Abstract Book 1):94. 96 Porchet HC, le Cotonnec JY. Pharmacokinetic and pharmacodynamic characteristics of recombinant human follicle-stimulating hormone. Assist Reprod Rev (1994); 4:110–7. 97 Balasch J, Fábregues F, Creus M, et al. Follicular development and hormonal levels following highly purified or recombinant-follicle stimulating hormone administration in ovulatory women undergoing ovarian stimulation after pituitary suppression for in vitro fertilization. Implications for implantation potential. J Assist Reprod Genet (2000); 17:20–7. 98 Gregory L. Ovarian markers of implantation potential in assisted reproduction. Hum Reprod (1998); 13 (Suppl.4):117–32. 99 Brinsden P, Akagbosu F, Gibbons L, et al. Gonal-F® versus Puregon®: results of a randomized, assessor-blind, comparative study in women undergoing assisted reproductive technologies. Hum Reprod (1998); 13 (Abstract book 1):70. 100 Brami C, Heluin G, Briot P, Testart J. Inductions d’ovulation par FSH recombinant (r-FSH) dans un programme de FIV: Analyse des résultats comparant les traitments par Gonal(F-75) et Puregon(100). Contracept Fertil Sex (1998); 26:620. 101 Fried G, Harlin J, Wramsby H. Recombinant FSH-Clinical experience with two different preparations. Fertil Steril (1998); 70 (Suppl.1):S112.

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102 Afnan MA, Kennefick A. Recombinant gonadotrophins: is there a difference in the tolerability of these products? Hum Reprod (1999); 14 (Abstract book 1):62–3. 103 Phillips E, Page M, Fleming SD. A prospective comparison of two different recombinant FSH preparations, 11th World Congress on In Vitro Fertilization and Human Reproductive Genetics, Sydney, Australia. Abstract book (O053), p. 88. 104 Loumaye E, Engrand P, Piazzi A, Arguinzoniz M. Use of recombinant human luteinizing hormone to reduce the risk of ovarian hyperstimulation syndrome. Presented at the 11th World Congress on In Vitro Fertilization and Human Reproductive Genetics, 9–14 May 1999, Sydney, Australia. 105 Sills ES, Levy DP, Moomjy M, McGee M, Rosenwaks Z. A prospective, randomized comparison of ovulation induction using highly purified follicle-stimulating hormone alone and with recombinant human luteinizing hormone in in-vitro fertilization. Hum Reprod (1999); 14:2230–5. 106 Kelly EE, Nebiolo L. Recombinant FSH therapy alone versus combination therapy with recombinant LH therapy in patients downregulated with a low-dose luteal GnRH agonist protocol: preliminary results. In: Jansen R, Mortimer D, eds. Towards Reproductive Certainty: Fertility & genetics beyond 1999. Parthenon Publishing: New York, 1999; 200–4. 107 Ndukwe G, Thornton S, Fishel S, et al. “Curing” empty follicle syndrome. Hum Reprod (1997); 12:21–3. 108 Peñarrubia J, Balasch J, Fábregues F, Creus M, Civico S, Vanrell JA. Recurrent empty follicle syndrome successfully treated with recombinant human chorionic gonadotrophin. Hum Reprod (1999); 14:1703–6. 109 Baird DT. Pharmacokinetics of follicle-stimulating hormone and a new “physiological” regimen for ovulation induction. In: Howles CM, ed. Gonadotrophins, gonadotrophin-releasing hormone analogues and growth factors in infertility: Future Perspectives. Alden Press: Oxford, 1991; 43–50. 110 Agrawal R, Holmes J, Jacobs HS. Follicle-stimulating hormone or human menopausal gonadotropin for ovarian stimulation in in vitro fertilization cycles: a meta-analysis. Fertil Steril (2000); 73:338–43. 111 Daya S. Re. Agrawal et al. Fertil Steril (2000); 74:420–2.

36 Use of recombinant DNA technology in ART Colin M Howles

INTRODUCTION Although research into the purification of human gonadotrophins for clinical use began in the late 1940s, it was not until the early 1960s that human menopausal gonadotrophin (hMG), a urinary extract comprising a mixture of FSH and LH, was first made available to physicians from the laboratories of Serono. Subsequently, considerable improvements have facilitated separation of follicle stimulating hormone (FSH) from human luteinizing hormone (hLH), and then 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 FSH, and the need for posttranslational modification of the molecule by protein folding and glycosylation, made functional protein production impossible in prokaryotes. Thus, a eukaryotic cell culture system was employed with functional molecules being produced in Chinese hamster ovary (CHO) cells. The world’s first recombinant human FSH (rhFSH) preparation for clinical use was produced by Serono laboratories in 1988, and was licensed for marketing as Gonal-F® in 1995 (Fig 36.1). An rhFSH product was also licensed by Organon laboratories in 1996.

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Fig 36.1 Computer model hormone FSH.

of

the

glycoprotein

Since then, the genes for the other gonadotrophins have been transfected into CHO cells, and recombinant human luteinizing hormone (rhLH) and human chorionic gonadotrophin (rhCG) are currently undergoing clinical trials; both will be commercially available in 2001 (rhLH as Luvems, Serono International; rhCG as Ovidrelle, Serono International). hFSH, hLH and hCG are members of the same glycoprotein hormone family and each of them is a heterodimer, sharing a common α-subunit but having different, hormone specific, β-subunits.1,2 Therefore, the production of these three hormones by recombinant technology required isolation and cloning of four genes: the common αsubunit gene and three specific β-subunit genes. ISOLATION AND CLONING OF THE α-SUBUNIT GENE COMMON TO hFSH, hLH AND hCG The principles of construction of a DNA library are outlined in Fig 36.2. Sequence information from the α-subunit of hCG was used to construct probes for the common αsubunit for use in the screening of a human fetal liver DNA library in bacteriophage λ Charon 4A.3 A 17kb clone was isolated, containing the whole of the α-subunit sequence (Fig 36.3). The endogenous gene promoter was removed by cleavage at the unique BamH1 site in exon 1, giving a resulting BamH1-EcoR1 fragment of approximately 11kb in length. This fragment contained part of exon I (without the first 35 nucleotides of the 5′ untranslated region of the mRNA), the coding exons II, III and IV, the intervening sequences, and

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about 2kb of the 3′ flanking sequences including the polyadenylation signal. The termini of the 11kb fragment were converted to recognition sites for the restriction endonuclease Sall before insertion into the unique Xhol restriction endonuclease site of the CLH3AXSV2DHFR plasmid.3 In this construct, transcription of the α-subunit gene was directed by the mouse metallothionein-l promotor. The endogenous polyadenylation signal of the α-subunit was used for 3′ processing of the mRNA. The expression vector also contained the mouse dihydrofolate reductase (DHFR) gene, which was used for selection and amplification of the gene when inserted into mammalian cells (see below).

Fig 36.2 The principles of construction of DNA and cDNA libraries.

Fig 36.3 Structural organization of the transfection plasmid containing the genomic DNA fragment containing the FSH gene.

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ISOLATION AND CLONING OF THE β-SUBUNIT GENE FOR hFSH The DNA library described above was screened using two probes incorporating sequences from the partially sequenced β-hFSH subunit. One of these probes allowed isolation of one recombinant bacteriophage containing a 13kb insert (Fig 36.4). A 6.8kb fragment of this clone produced by BamHI-EcoRI cleavage and containing the β-hFSH gene was subcloned. A 2kb fragment containing the complete protein coding region was then obtained by cleavage with Ddel-Sau3AI; this fragment contained the last 35 base pairs of the first intron, exon II, intron II and exon III of the β-hFSH gene.3 Sites for the recognition of Sall were again engineered at the extremities of the fragment before it was inserted into the Xhol site of the plasmid expression vector pCLH3AXSV2ODC.3 In the resulting recombinant plasmid, pHFSHβODC the β-subunit gene was under the control of the mouse metallothionein-I promotor. As the endogenous βhFSH polyadenylation signal had been removed during plasmid engineering, the SV40 early polyadenylation signal supplied by the expression vector was used for 3′ processing of the subunit transcript.

Fig 36.4 Structural organization of the transaction plasmid containing the genomic DNA fragment containing the beta-FSH gene.

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ISOLATION AND CLONING OF THE ß-SUBUNIT GENE FOR hLH Two clones containing portions of the coding sequence of the hLH βsubunit were isolated from a human pituitary cDNA library.3,4 Neither clone contained the complete sequence, but a combination of the two encoded 15 amino acids of the signal peptide, the whole of the mature peptide and the complete 3′ untranslated region of the mRNA.5 The first four amino acids of the signal peptide, including the start codon, were not present in either of the clones. To produce a functional expression construct a small fragment from the 5′ end of the hCG β-subunit cDNA was added to the 5′ end of the LH composite clone. The resulting hybrid molecule contained a start codon and encoded the complete signal peptide. The resulting peptide had one amino acid difference from the wild-type LH β-subunit—a substitution of phenylalanine in the recombinant peptide for leucine in the natural LH at amino-acid four of the signal peptide. However, this secretory signal peptide is removed from the protein when it is translocated across the endoplasmic reticulum, giving a recombinant β-hLH with an identical sequence to the natural hormone. The 5′ end of the subunit fragment was further modified by fusing about 670 base pairs of the first intron of the mouse immunoglobulin κ chain upstream of the expression construct to enhance mRNA accumulation. The complete engineered fragment was then inserted into the same plasmid expression vector as used for the hFSH β-subunit. Again, expression of the fragment was under the direction of the mouse metallothionein-1 promoter and the vector SV40 early polyadenylation signal was used for 5′ processing. ISOLATION AND CLONING OF THE β-SUBUNIT GENE FOR hCG A full length cDNA encoding the hCG β-subunit was isolated from a human placenta library cloned into pBR322.5 The fragment included the 5′ untranslated leader sequence and the polyadenylation signal. Following modification of the fragment the cDNA fragment was then inserted into an expression vector pCLH3AXSV2ODChαIVSA. This vector contains a portion of the first intron of the genomic human CG αsubunit (hαIVSA), which acts to enhance expression of the β-subunit. Expression of hCG was under the control of the mouse metallothioneinI promoter and its own polyadenylation signal.

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EXPRESSION OF THE hFSH GENES IN MAMMALIAN CELLS The principles underlying production of recombinant FSH are outlined in Fig 36.5. Expression vectors containing the α- and β-subunit genes were transfected into a DHFRdeficient CHO cell line. Such cells are sensitive to the tetrahydrofolate analog

Fig 36.5 The principles underlying recombinant hFSH production. methotrexate (MTX) which can, therefore, be used for selective amplification of cells containing the DHFR gene from plasmid vectors. The CHO cell line DUKX-B116 was maintained in minimal essential medium (αMEM). The α- and β-subunit expression vectors were cotransfected into the CHO cells in equimolar amounts, using a modification of the calcium phosphate precipitation procedure.7 The cells were cultured for about 2 weeks in a selection medium of αMEM minus ribonucleosides and deoxyribonucleosides, and 0.02µM MTX. After this time, individual colonies were visible and were transferred to establish isolated cultures (Fig 36.6).

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After expansion into T-flasks, gene amplification was promoted by exposure of the individual isolates to increasing concentrations of MTX (0.02–5µM). This led to coamplification of both the α and β subunit expression constructs. The culture supernatants were assayed for FSH productivity per cell; an increase in production indicated successful gene amplification. A particular cell line (cell line 39) was selected as the base candidate for subcloning, on the basis of its high productivity (6.14 pg/cell/24 h), the stability of its FSH secretion over extended duration of culture, and the quality of the FSH protein it produced. Cell line 39 was then cloned by limiting dilution, and the single-cell clonal cell line 39-A2 was chosen for use in bio-production, based on its high FSH productivity of 15.1 pg/cell/24 h and growth characteristics that were suitable for largescale production. A flowchart summarizing the process for bulk production of r-hFSH is shown in Fig 36.7. In situ hybridization using fluorescent FSH α- and β-subunit probes indicated that the two expression constructs were co-integrated and co-amplified in closely related chromosomal locations in the CHO genome.

EXPRESSION OF THE hLH GENES IN MAMMALIAN CELLS The α- and β-subunit plasmid expression vectors were transfected into DHFR-deficient CHO cells in a ratio of either 1:1 or 1:3 (α:β), using the calcium precipitation method. Gene expression was again selected for and amplified using successive MTX titrations. Of the most promising cell lines (those with high LH productivity in the presence of MTX, and good growth and stability characteristics), the one with the highest rate of LH production

Fig 36.6 Expression of rhFSH in CHO cells.

Fig 36.7 A flow chart summarizing the processes used in the bulk production of rhFSH. in the absence of MTX—line 111—was selected for further cloning by limiting dilution. The VIIC6 subclone, subcloned in the absence of MTX, was selected for use in large scale hLH production using techniques similar to those summarized in Figs 36.6 and 36.7 for rhFSH. Using α-

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and β-subunit DNA probes, fluorescence in situ hybridization analysis was used to assess the physical state of the transfected plasmids. The chromosomal position of the fluorescent signal for all probes was consistent and indicated that the two transfection plasmids had cointegrated and co-amplified at the same location in the VIIC6 genome.

EXPRESSION OF THE hCG GENES IN MAMMALIAN CELLS CHO DUKX-B11 cells were co-transfected, using the calcium precipitation method, with the αhCG and β-hCG expression vectors. Gene expression amplification was as described for hFSH and hLH. At each stage, hCG productivity was measured and poorly expressing cell lines were discarded. Viable cell lines producing hCG in media containing concentrations of MTX of 5mM were selected, and hCG productivity in the absence of MTX was assessed. One cell line was expanded by serial passage under non-selective conditions and, based on morphology, growth rate and hCG expression, 10 subclones were selected. Of these, a single clone was selected for use in the bio-production of rhCG, the processes for which are broadly similar to those outlined in Figs 36.6 and 36.7 for rhFSH.

PURIFICATION OF hFSH FROM CELL CULTURE SUPERNATANT The crude culture supernatant was processed using five chromatographic purification steps and ultrafiltration (Fig 36.8).3,4,8 The first two chromatographic steps were designed to remove bulk impurities, and the further three steps removed remaining contaminants that were present in trace amounts. The culture supernatant was first filtered then adsorbed onto an ion-exchange column. Semi-purified FSH was recovered in the unbound fraction of the supernatant. This was then loaded onto an immunoaffinity column primed with murine-derived anti-FSH monoclonal antibody. This primary purification step produced bulk highly purified FSH with only trace amounts of contaminants and is unique to rhFSH (Gonal-F, Serono International). These were removed using ionexchange, reverse-phase and size-exclusion chromatography. Characterization of the resulting protein, using specific immunoassays and silver-stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis, showed that it was at least 95% pure. The elution profile of rhFSH on reverse-phase HPLC matched that of urinary hFSH, with discrete peaks for the α- and β-subunits (Fig 36.9).

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Fig 36.8 A flow chart summarizing the stages involved in the purification of rhFSH (Gonal F, Serono International).

PURIFICATION OF hLH AND hCG FROM CELL CULTURE SUPERNATANTS Production of active rhCG from cell culture supernatant involves a fivestep purification process (Fig 36.10).4 After clarification, the supernatant was concentrated using C4 silica chromatography and ultrafiltration. The crude extract then underwent two rounds of ionexchange chromatography, followed by reverse-phase high performance liquid chromatography (HPLC) and size exclusion HPLC. The purity of the final product, assessed using SDS-PAGE followed by silver staining, was at least 95%. Active rhLH of high purity was obtained from cell culture supernatant using a similar purification process. Electron profiles of r-LH and r-CG matched those of naturally occurring proteins.

CHARACTERIZATION OF THE rhFSH PROTEIN The complete amino-acid sequences of the α- and β-subunits of rhFSH were determined by automated sequencing. Both subunits were found to have sequences corresponding

Fig 36.9 The elution profile of a sample of purified rhFSH on reverse-phase HPLC compared with that of urinary hFSH.

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Fig 36.10 A flow chart summarizing the stages involved in the purification of rhLH and rhCG. to those predicted from their DNA sequences, and matching those of urinary hFSH. The N-terminal-truncated form of the β-subunit, which comprised about 50% of the protein, was identical to that found in pituitary and urinary hFSH, as it had lost two N-terminal residues.9,10 The N-terminal-truncated species of the α-subunit was present in only trace amounts (1–2%) and differed from the pituitary and urinary form, as it was lacking two Nterminal amino acids, rather than three. Glycosylation of the FSH molecule is essential for its efficacy, and plays a strong part in determining its biological activity. In particular, capping of carbohydrate chains with sialic acid prevents hepatic clearance of the molecule by the asialoglycoprotein receptor, thereby reducing its rate of clearance and prolonging its duration of action.11 It was found that in rhFSH, α-subunit Asn residues at positions 52 and 78, and β-subunit Asn residues at positions 7 and 24 were glycosylated. These were the only sites of glycosylation, and corresponded to the sites on the native hFSH molecule.12,13 There were no O-linked sites of glycosylation. The monosaccharide composition of the molecule was determined by highperformance anion-exchange chromatography. This was compared with those of urinary and pituitary hFSH, and the compositions of the three molecules were found to be similar (Table 36.1). The structure of the carbohydrate chains was identified by glycan mapping. The carbohydrate species of rhFSH were less heterogeneous than those of urinary hFSH or pituitary hFSH.12,13 Less heavily sialylated carbohydrates were present in rhFSH compared with urinary FSH. This probably reflects a selection process, by which the longer lived species of FSH are more likely to survive metabolism and concentrate in the urine. Changes in glycosylation of the FSH molecule are reflected in the isoelectric focusing profile of the product. The profile of the rhFSH was similar to that of pituitary hFSH, and slightly less acidic than that of urinary FSH.

Table 36.1. Monosaccharide compositions of naturally occurring and recombinant FSH compared. Reproduced with permission.9 Mannose Fucose N-acetyl-glucosamine Galactose Sialic acid Pituitary hFSH 3.0 0.7 4.4 2.8 2.8 Urinary hFSH 3.0 0.6 5.2 3.0 2.9 r-hFSH 3.0 0.5 4.6 2.8 2.2

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CHARACTERIZATION OF THE rhLH PROTEIN The α- and β-subunits of the molecule were completely sequenced. The asubunit aminoacid sequence was as expected, however, several different β-subunit sequences of different lengths were identified. These resulted from C-terminal heterogeneity, as is found in pituitary hLH.14,15 The monosaccharide content of the N-linked carbohydrate chains was determined. All the oligosaccharide chains on both subunits of the rhLH molecule contained a terminal sialic acid and an isoelectric focusing profile of the recombinant molecule proved broadly similar to that of purified human pituitary LH.16

CHARACTERIZATION OF rhCG PROTEIN The primary sequence of the α- and β-subunits of rhCG was determined by automated Edman degradation of tryptic peptides obtained from the separated subunits. The characteristics of the α-subunit were as described for rhFSH. A minor species of β-subunit was detected with two additional amino acids (β+2; tryptophan and alanine).9 This corresponds to cleavage at an alternative site. A single, full-length C-terminal sequence was detected, corresponding with that of urinary-hCG. Two sites of N-linked glycosylation and four sites of O-linked glycosylation were identified in the β-subunit sequence (Asn13 and Asn30; and Ser121, Ser127, Ser132 and Ser138, respectively). These sites are conserved with respect to the urinary-hCG sequence. Mass spectrometry showed the most commonly occurring oligosaccharide of rhCG to be of the N-linked, biantennary, complex type. The extent of sialylation varied.

CONCLUSION Recombinant technology has allowed the production of highly purified hormones that are free from contaminating human proteins and with excellent consistency from batch to batch. The rhFSH molecule has already demonstrated its worth in clinical use in assisted reproductive techniques, and hLH and hCG have shown great promise in early clinical trials. Because of their exceptional purity, all three products are easily self-administered by subcutaneous injection, making their use simple and convenient. In addition to the cloning, production and purification of recombinant gonadotrophins, gonadotrophinreceptor interactions at the molecular level are being investigated.17,18 Such studies may lead to further therapeutic improvements in ART regimens through the production of molecules with the same biological activities as the

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gonadotrophins but that can be taken orally—the subject of considerable interest within Serono.

ACKNOWLEDGMENT The author of this review wishes to respectfully acknowledge the dedication and commitment of all the many Serono scientists who have contributed to the development and production of recombinant gonadotrophins over the past two decades.

REFERENCES 1 Pierce JD, Parsons TF. Glycoprotein hormones: structure and functions. Ann Rev Biochem (1981) 50:465–95. 2 Gray CJ. Glycoprotein gonadotrophins. Structure and synthesis. Acta Endocrinol (1988); 288:20–7. 3 Recombinant Human FSH Product Development Group, Recombinant follicle stimulating hormone: development of the first biotechnology product for the treatment of infertility. Hum Reprod Update (1998); 4:862–81. 4 Chappel SC, Kelton C, Nugent N. Expression of human gonadotropins by recombinant DNA methods. In: Genazzaini AR, Petrahlia F, eds. Hormones in gynecologic endocrinology. Parthenon Publishers: London, 1992; 179–84. 5 Chappel SC, Kelton C, Nugent N. Basic knowledge about recombinant gonadotropic hormone production. In: Shoham Z, Howles CM, Jacobs HS, eds. Female infertility therapy: current practice. Martin Dunitz: London, 1999; 95–102. 6 Urlaub G, Chasin LA. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity. Proc Natl Acad Sci USA (1980); 77:4216–20. 7 Wigler M, Silverstein S, Lee L-S, et al. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell (1977); 11:223–32. 8 Howles CM. Genetic engineering of human FSH (Gonal-F®). Human Reproduction Update (1996); 2:172–91. 9 Rathnam P, Saxena BB. Primary amino acid sequence of folliclestimulating hormone from human pituitary glands. J Biol Chem (1975); 250:6735–46. 10 Saxena BB, Rathnam P. Amino acid sequence of the β-subunit of follicle-stimulating hormone from human pituitary glands. J Biol Chem (1976); 251:993–1005. 11 Chappel SC. Heterogeneity of follicle stimulating hormone: control and physiological function. Hum Reprod Update (1995); 1:479–87.

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12 Green ED, Baenziger JU. Asparagine-linked oligosaccharides on Lutropin, Follitropin and Thyrotropin. I. Structural elucidation of the sulphated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J Biol Chem (1988); 263:25–35. 13 Green ED, Baenziger JU. Asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin. II. Distributions of sulphated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J Biol Chem (1988); 263:36–44. 14 Shome B, Parlow AF. The primary structure of the hormone specific beta subunit of human pituitary luteinizing hormone (hLH). J Clin Endocrinol Metab (1973); 6:618–21. 15 Sairam MR, Li CH. Human pituitary lutrophin, isolation, properties and complete amino acid sequence of the beta subunit. Biochim Biophys Acta (1975); 412:70–81. 16 Snyder PJ, Bashey HM, Montecinos A, et al. Secretion of multiple forms of human luteinizing hormone by cultured fetal human pituitary cells. J Clin Endocrinol Metab (1989); 68:1033–8. 17 el Tayar N. Advances in the molecular understanding of gonadotrophins-receptors interactions. Mol Cell Endocrinol (1996); 125:65–70. 18 Jiang X, Dreano M, Buckler DR, et al. Structural predictions for the ligand-binding region of glycoprotein hormone receptors and the nature of hormone-receptor interactions. Structure (1995); 3:1341–53.

37 Endocrine characteristics of ART cycles Jean Noel Hugues, Isabelle Cedrin-Durnerin

INTRODUCTION The hormonal control of ovarian function by gonadotrophins plays a key part in the physiological process of follicular growth and differentiation. Over the last decade, the respective contribution of follicle stimulating hormone (FSH) and luteinizing hormone (LH) to follicular development has been better defined mainly through clinical data obtained from assisted reproductive techniques (ART) cycles performed with gonadotropin releasing 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 role of FSH and LH. In every situation, measurements of plasma concentrations of FSH and LH were used to evaluate the endocrine environment of the follicle. While it is clear that hormonal assays from blood sampling cannot adequately reflect the biological activity of gonadotrophins, 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 gonadotrophins 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 gonadotrophins while the determination of androgen production is only performed in a few clinical studies. In this chapter, we will consider how the therapeutical agents currently used in ART cycles (GnRH analogs, exogenous gonadotrophins) 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.

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GONADOTROPHIN PROFILES DURING OVARIAN STIMULATION FOR ART CYCLES According to the two cell-two gonadotrophin model, both FSH and LH are required for promoting follicular growth and differentiation.1 We will be considering their respective contribution in stimulation regimens separately. FSH It is well documented that FSH plays a crucial part in the recruitment, selection and dominance processes during the whole follicular phase.2 On 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 activity, inhibin and the LH receptor whose expression clearly reflects follicle differentiation. From outstanding clinical studies performed by Brown in the late sixties,3 it has become clear that a certain amount of FSH secretion defined as the “FSH threshold” is required to induce a 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 37.1). 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 follicles’ 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 concentrations above the threshold of the dominant follicle opens the window until the final stages of follicular development: a crucial component for controlled ovarian hyperstimulation (COH). These two concepts justify the assumption that FSH is the main therapeutical agent to control folliculogenesis in all situations except that of severe hypogonadotrophic hypogonadism. Indeed, in this latter case, an LH supply is also required to ensure adequate steroid production according to the two cell-two gonadotrophin model.5 Both gonadotrophin preparations and GnRH analogs are commonly used to achieve multifollicular development but the effects of each agent on FSH accumulation are quite different.

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• As far as gonadotrophin administration is concerned, it has been stated that, because of the long elimination half life (30–35 hours) of the FSH molecule,6 a plateau of plasma FSH is obtained after five 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 after the cessation of FSH administration.9 Furthermore, determination of plasma FSH levels after intramuscular or subcutaneous administration of FSH has shown that there is a modest and transient rise (of 4–8 hours) 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 daily adjusted according to the simultaneous evaluation of plasma FSH levels. In this way, the authors were able to surely 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 37.1 Follicular growth starts at the early follicular phase when plasma FSH concentration is above a threshold 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

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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 has got a LH receptor, which allows LH to contribute to ovarian steroidogenesis.

Fig 37.2 “Stable” FSH plasma 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 (Fig 37.2). Consequently, it does appear 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 on FSH secretion largely depend on the way of using these pharmaceutical agents. The initial flare up effect of the agonist at the pituitary level is associated with a significant increase in plasma concentrations of FSH, which participates in the follicular recruitment in the so called short protocol. Several studies have shown that the amplitude of the FSH response to GnRH-a is lower than that of LH.11,12,13 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

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hypophyseal control of gonadotrophin secretion. As a lower dose of GnRH-a than that usually recommended may induce a larger increase in the FSH response,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 on 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 also been 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 after administration of a GnRH antagonist provided similar conclusions. Indeed, the gonadotroph suppression was less marked for FSH than for LH, attesting once again for the relative GnRH dependence 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 gonadotrophins compared with gonadotrophins alone in order to compensate 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 gonadotrophin regimen in a proper manner. It therefore seems more appropriate to restrict this evaluation to clinical research studies. LH The role of LH on folliculogenesis varies according to the stage of follicular development. On 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 gonadotrophin theory, androgens play a key part as substrates for aromatase activity and contribute to the production of oestradiol 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 studies that, while LH induces a dose dependent protein synthesis (aromatase activity), its effect on cell proliferation is negative, at least in high concentrations.21 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 hypogonadotrophic hypogonadism. Indeed, in those patients deprived of hypophyseal gonadotroph production, substitution with recombinant FSH results in follicular growth but does not allow any concomittant steroid output. In contrast, addition of recombinant LH induces a dose-dependent increase in oestradiol production, a condition required to ensure endometrial preparation for

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embryo implantation.5 This observation underlines that a minimal amount of LH defined as the “LH threshold” is required for pregnancy. However, as discussed later on, the 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 concentrations are often associated with an increased incidence of infertility or miscarriages. Another study 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 than on the oocyte/conceptus itself.24 More recently, the concept of the “LH ceiling” has been proposed by Hillier on the basis of his own experiments showing an inhibitory effect of high LH doses on cell growth.25 Thus, LH, beyond a certain “ceiling” level, suppresses granulosa proliferation and initiates atresia of less mature follicles. Preliminary unpublished clinical data, performed in hypogonadotroph hypogonadic patients, tend to support this concept: 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. Let us consider now the plasma LH variations when using drugs for ART cycles. • Urinary hMG preparations have been commonly used with success over the last 30 years for ovulation induction. In regimens performed with gonadotrophins alone, it has been demonstrated that LH is rapidly cleared from circulation, owing to its relatively short half life.26 Thus, in contrast to FSH, there is slight evidence of plasma LH accumulation following a single injection of hMG (Fig 37.3). Furthermore, determination of plasma LH from a morning blood sample after an evening injection of gonadotrophins is not very informative for evaluating the actual consequences of the LH content of human menopausal gonadotrophin (hMG) preparations. Thus, during gonadotrophin treatment, 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 ultra short 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

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to be the best predictor of the ovarian sensitivity to gonadotrophins. Thus, determination of plasma LH does not appear relevant during the flare up period.

Fig 37.3 Pharmacodynamic of LH: per cent change in plasma LH after an intramuscular injection (arrow) of either hFSH or hMG (150IU) in normal ovulatory controls (upper panel) and PCOS patients (lower panel). A significant increase in plasma LH levels is only minimal and transient.26 • In contrast, measurements of plasma LH are routinely performed at the time of hypophyseal desensitization to make sure that gonadotrophin secretion is adequately down regulated 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.27 During this period and as long as

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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.28–30 It is also well documented that both intensity and duration of LH suppression are dose dependent.31,32 However, some unanswered questions remain regarding the state of hypophyseal desensitization. One of them is on to elucidate the reasons why there is an evident need for a higher amount of exogenous gonadotrophins to get an adequate ovarian reponse. It is commonly stated that the more profound the hypophyseal desensitization is, the worse the ovarian responsiveness to stimulation will be. This led to the proposal of using a lower dose of GnRH-a, especially for patients with a previous low response to gonadotrophins. 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 two-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 non-biologically active hormones (alpha subunits and/or molecules with modified glycosylation).33 It has also been shown that a daily GnRH-a administration leads to a partial release of measurable alpha32 and that stopping the daily agonist administration induces a sharp decrease in concentrations of both plasma dimeric LH and alpha subunits34,35 (Fig 37.4). Thus it must be stressed that the residual measurable LH secretion depends on the GnRH-a formulation and on the duration of administration.

Fig 37.4 Effects of the duration of GnRH agonist administration on plasma LH concentrations: plasma dimeric LH and alpha subunit concentrations in patients treated with GnRH agonist (decapeptyl 0.1mg daily) for 7 (o) 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 alpha subunit plasma concentrations. Thus, in contrast to GnRH antagonist, a daily agonist administration sustains a partial release of measurable alpha subunit and LH from the hypophysis.34

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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 gonadotrophin 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 get a high implantation rate.36,37 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 al38 and Fleming et al39 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 mid-follicular phase. Selecting a subgroup of patients whose residual plasma levels were lower than 0.5IU/1, 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 al 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.40 In this model, it was shown that only a small population (less than 6% of patients) might benefit from exogenous LH administration and that measuring plasma LH levels after down regulation is of no practical benefit to identify this subgroup of patient. 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 of LH in ART cycles. Acting as a competitor to endogenous GnRH at the receptor level, the GnRH antagonists induces a rapid and reversible reduction in LH secretion without any interference with the hypophyseal machinery. In that respect, the hormonal situation induced by antagonist is easier to assess than that induced by agonist: a parallel decrease in plasma dimeric and alpha subunit LH concentrations is elicited by GnRH antagonist administration41,42 and a rapid recovery of the pituitary gonadal axis is predictable after discontinuation of treatment. A dose finding study43 showed that plasma LH concentrations decrease in dose dependent manner following the administration of Org 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

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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 treatment with GnRH analogs (Fig 37.5), 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 table also emphasizes that assessment of LH requirement needs to take into account the joint effects of both gonadotrophins and GnRH analogs on plasma LH secretion.

STEROID PROFILES DURING OVARIAN STIMULATION FOR ART CYCLES In contrast with gonadotrophin, 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 ART cycles. Finally, plasma androgen levels are rarely determined except for clinical research. ESTRADIOL Estradiol plays a crucial endocrine part in the reproductive system, being involved in the production of cervical mucus, in the endometrial proliferation for embryo implantation and in the induction of the midcycle LH surge. In contrast, the autocrine role of estradiol

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Fig 37.5 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 affecting negatively follicular growth. Both gonadotrophins 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. in the follicle development, firstly 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 gonadotrophin doses in conjunction with data obtained by ultrasound. Indeed, it is well admitted that E2 synthesis is directly related to follicular size and that the contribution of mature follicle to the E2 output may be estimated at about 200pg/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 pattern (Fig 37.6).44 Similarly, in protocols using GnRH-a, it was suggested that an increase in plasma E2 concentrations for six consecutive days would be optimal for the success of the cycle.45 Owing to the extreme diversity of protocols

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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 values for more than three days is commonly associated with a poor outcome of the ART cycle. Conversely, measurements of plasma E2 is helpful to detect the risk of excessive ovarian response and to decide coasting of gonadotrophin administration, cancelling the cycle or the embryo transfer. For those reasons, it seems that plasma E2 determination must be included, to some extent, in the monitoring of ART cycle treatment, while 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 sensitve index of ovarian responsiveness to gonadotrophins and, to some extent, as a predictor of the outcome of the ART cycle. The administration of a fixed dose

Fig 37.6

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Plasma estradiol patterns: diagrammatic representation of the various E2 patterns of response to gonadotrophins alone. The number of cases in each pattern and the pregnancy rate by pattern are indicated.44 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.46 Other authors suggested that an early determination of plasma E2, after only a few days of gonadotrophin administration, may be useful to predict the subsequent ovarian response.47 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 gonadotrophin 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 COH and described four patterns of E2 variations with different prognosis for the cycle. In contrast to Padilla et al, Winslow et al,48 using the same agonist, correlated the relative increment of plasma estradiol from day 2 to day 3 (∆E2) with the ovarian response to stimulation. In a similar study using triptorelin as GnRH-a, we also showed 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 exist.49 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 gonadotrophin administration accordingly. To sum up these data, the predictive E2 values for each test are presented in Table 37.1. 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 50pg/ml to make sure that ovarian activity is actually suppressed, which usually occurs after two weeks of GnRH-a administration. Starting GnRH-a administration in the mid luteal phase50–51 or using a long-acting formulation of the agonist52–53 may allow to get desensitization more rapidly than when using short acting from the early follicular phase. However, it is still unclear whether there is any clinical advantage in achieving a prompt and profound desensitization. It was even suggested that a prompt desensitization would induce an ovarian refractory state to exogenous gonadotrophins.52 In every situation, it is recommended to start

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ovarian stimulation with FSH only when ovarian activity is suppressed whatever the duration of GnRH-a administration needed to get it. Finally, the recent availability of GnRH antagonist in ART cycles may lead to a reassessment of the usefulness of plasma E2 determination. 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 protocol 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 the ART cycles.

Table 37.1. Stimulation tests predictive of the ovarian response to gonadotrophins. Tests ∆E2 (pg/ml) Exogenous FSH (EFORT) >30 Lupron screening test >20 Triptorelin screening test >5 Different stimulation tests proposed to evaluate the ovarian sensitivity to gonadotrophins: hFSH (300IU IM) (EFORT test) or leuprolide acetate (1mg sc) or triptorelin (0.1mg sc 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 h later at day 3. ∆E2 represents differences between plasma E2 values. Figures indicate the E2 cut off values predictive of an adequate ovarian response. PROGESTERONE Before the introduction of GnRH analogs in ART cycles, detection of premature endogenous LH surge was a constant concern because LH surges usually occurred when follicular development was still uncompleted and had some deleterious effects on the oocyte quality and on the implantation rate. At that time, determination of plasma concentrations of progesterone (P) was considered as 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 GnH antagonists, two agents effective to prevent LH surges, have led to strictly limit the determination of plasma P to some periods of the ART cycles. It is usual to control plasma P values at the time of the hypophyseal desensitization. It seems worth while 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,

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if a 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 punction before FSH administration. It is recommended not to start ovarian stimulation in a hormonal environment that may be deleterious for the oocyte or the endometrium. In that respect, an increase in plasma P, which would overcome follicular phase values at the time of hypophyseal desensitization, is considered as deleterious for the subsequent ART cycle and requires to extend the administration of the GnRH-a alone and to postpone ovarian stimulation. With a similar concern, special attention has been paid to other situations where an increase in plasma P have been correlated with a potential risk of poor outcome for the ART cycle. The first one refers to the endocrine consequences of the flare up effect induced by GnRH-a in the so called short term protocols. As previously mentioned, the initial administration of the agonist induces a sharp increase in both gonadotrophin and steroid production and release but some reports55–57 outlined 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,58 it is only beyond a threshold of plasma P values that impairment of follicular development may be observed. Furthermore, we showed, in a prospective randomized study, that the outcome of the ART cycles were actually similar in 2 groups of patients pretreated or not by a progestogen which completely prevent any plasma P increase in the flare-up period.49 Thus, there is no evidence so far that any increase in plasma P may be detrimental at the very early follicular phase of the cycle and we do not advocate to perform this determination any longer during the flare-up of short-term GnRH-a protocol. Another circumstance where a plasma P determination must be considered is in the late phase of ovarian stimulation. Indeed, despite an effective suppression of endogenous gonadotrophins 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 authors59–61 reported a negative effect on the pregnancy rate through inadequate endometrial preparation while others62–65 could not find any significant relation. Furthermore, whether or not a P increase may be 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 gonadotrophins are not clearly demonstrated. Exposure to large doses of exogenous FSH seems to be associated with a higher incidence of high P plasma values66 but it is still unclear whether the P increase is related to some disruptions of ovarian steroidogenic pathway induced by high FSH doses or is the early expression of an occult ovarian failure as recently suggested.67 Furthermore, the specific

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contribution of the adrenal gland must be also considered because dexamethasone administration enables a partial reduction in plasma P levels.66 Nevertheless, it is likely that the impact of exogenous gonadotrophins on the ovary predominantly accounts for this process.69 Thus, additional studies are needed to conclude on this specific issue and, in clinical practice, plasma P cutoff values, as a means of making decision to cancel the fresh transfer, should be questioned. ANDROGENS Determination of plasma androgens, namely testosterone and ∆4 androstenedione, are 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 may be contributive for measuring a low LH bioactivity. This is partly related to the fact that both ovary and adrenal gland contribute to the androgen production in women with normal reproductive function. Conversely, excessive androgen production may be easily detected by plasma androgen measurements and, with the exception of partial enzymatic adrenal defects, are mainly related to ovarian hyperandrogenism with or without LH hypersecretion. During the last decade, it became more evident that assessement of ovarian morphology by transvaginal probes allows a more accurate evaluation of the polycystic ovaries syndrome than plasma androgen measurements. However, while androgen determination does not appear to be a contributive factor for the assessment of COH, some reports recently showed that androgens actually exert a stimulatory effect on granulosa cell proliferation in human beings and may be involved in the follicular recruitment.70 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 androgen. During ART cycles, one study mentioned that the flare up effect of the GnRH-a in short term protocols is associated with an increased androgen production, but there is no significant evidence that the oocyte quality may be consequently reduced.71 To sum up, it does seem that the determination of plasma androgens should not be systematically included in the monitoring of ART cycles but may be worth while in clinical research.

CONCLUSIONS It does seem from this review that the endocrine characteristics of ART cycles largely depend on the drugs used to achieve COH. It is clear that FSH treatment is mandatory in every stimulation but assessment of FSH plasma values is not enough predictive of the adequacy of FSH supply to

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be routinely determined. As far as plasma LH determinations are concerned, immunometric LH assays cannot properly reflect the bioactivity of the circulating residual LH after administration of GnRH analogs. Furthermore, there is no evidence that plasma LH measurements could be helpful to detect patients that might need some addition of LH during ART cycles. Consequently, plasma LH determinations may be restricted to the control of hypophyseal desensitization. Inversely, while there is a trend to minimize the cost of ART cycles 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, specially with the recent use of GnRH antagonists. Thus, more information is needed before a definitive conclusion can be given. 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 their measurement is worthwhile to indirectly assess the residual LH bioactivity in patients treated with GnRH analogs.

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 and FSH-deficient anovulatory women: a dose-finding study. J Clin Endocrinol Metab (1998); 83:1507–14. 6 Le Cotonnec JY, Porchet HC, Beltrami V, Khan A, Toon S, Rowland M. Clinical pharmacology of recombinant human follicle-stimulating hormone (FSH). I. Comparative pharmacokinetics with urinary human FSH. Fertil Steril (1994); 61:669–78. 7 Diczfalusy E, Harlin J. Clinical pharmacological studies on human menopausal gonadotrophin. Hum Reprod (1988); 3:21–7. 8 Ben-Rafael Z, Strauss JFd, Mastroianni L Jr, Flickinger GL. Differences in ovarian stimulation in human menopausal gonadotropin treated

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women may be related to follicle-stimulating hormone accumulation. Fertil Steril (1986); 46:586–92. 9 Mizunuma H, Takagi T, Yamada K, Andoh K, Ibuki Y, Igarashi M. Ovulation induction by step-down administration of purified urinary follicle-stimulating hormone in patients with polycystic ovarian syndrome. Fertil Steril (1991); 55:1195–6. 10 Van Weissenbruch MM, Schoemaker HC, Drexhage HA, Schoemaker J. Pharmaco-dynamics of human menopausal gonadotrophin (HMG) and follicle-stimulating hormone (FSH). The importance of the FSH concentration in initiating follicular growth in polycystic ovary-like disease. Hum Reprod (1993); 8:813–21. 11 Lemay A, Metha AE, Tolis G, Faure N, Labrie F, Fazekas AT. Gonadotropins and estradiol responses to single intranasal or subcutaneous administration of a luteinizing hormone-releasing hormone agonist in the early follicular phase. Fertil Steril (1983); 39:668–73. 12 Padilla SL, Smith RD, Garcia JE. The Lupron screening test: tailoring the use of leuprolide acetate in ovarian stimulation for in vitro fertilization [published erratum appears in Fertil Steril 1991 Dec;56(6):1210]. Fertil Steril (1991); 56:79–83. 13 Hugues JN, Attalah M, Herve F, Martin-Pont B, Kottler ML, Santarelli J. Effects of short-term GnRH agonist—human menopausal gonadotrophin stimulation in patients pre-treated with progestogen. Hum Reprod (1992); 7:1079–84. 14 Deaton JL, Bauguess P, Huffman CS, Miller KA. Pituitary response to early follicular-phase minidose gonadotropin releasing hormone agonist (GnRHa) therapy: evidence for a second flare. J Assist Reprod Genet (1996); 13:390–4. 15 Scott RT, Carey KD, Leland M, Navot D. Gonadotropin responsiveness to ultralow-dose leuprolide acetate administration in baboons. Fertil Steril (1993); 59:1124–8. 16 Bständig B, Cedrin-Durnerin I, Hugues JN. Effectiveness of low dose of gonadotropin releasing hormone agonist on hormonal flare-up, J Assist Reprod Genet (2000); 17:113–17. 17 Parinaud J, Oustry P, Perineau M, Reme JM, Monrozies X, Pontonnier G. Randomized trial of three luteinizing hormone-releasing hormone analogues used for ovarian stimulation in an in vitro fertilization program. Fertil Steril (1992); 57:1265–8. 18 Matikainen T, Ding YQ, Vergara M, Huhtaniemi I, Couzinet B, Schaison G. Differing responses of plasma bioactive and immunoreactive follicle-stimulating hormone and luteinizing hormone to gonadotropinreleasing hormone antagonist and agonist treatments in postmenopausal women. J Clin Endocrinol Metab (1992); 75:820–5. 19 Huhtaniemi IT, Dahl KD, Rannikko S, Hsueh AJ. Serum bioactive and immunoreactive follicle-stimulating hormone in prostatic cancer

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patients during gonadotropin-releasing hormone agonist treatment and after orchidectomy. J Clin Endocrinol Metab (1988); 66:308–13. 20 Gonzalez-Barcena D, Vadillo Buenfil M, Garcia Procel E, et al. Inhibition of luteinizing hormone, folliclestimulating hormone and sexsteroid levels in men and women with a potent antagonist analog of luteinizing hormone-releasing hormone, Cetrorelix (SB-75). Eur J Endocrinol (1994); 131:286–92. 21 Yong EL, Baird DT, Yates R, Reichert LE Jr, Hillier SG. Hormonal regulation of the growth and steroidogenic function of human granulosa cells. J Clin Endocrinol Metab (1992); 74:842–9. 22 Stanger JD, Yovich JL. Reduced in-vitro fertilization of human oocytes from patients with raised basal luteinizing hormone levels during the follicular phase. Br J Obstet Gynaecol (1985); 92:385–93. 23 Regan L, Owen EJ, Jacobs HS. Hypersecretion of luteinising hormone, infertility, and miscarriage. Lancet (1990); 336:1141–4. 24 Ashkenazi J, Farhi J, Orvieto R, et al. Polycystic ovary syndrome patients as oocyte donors: the effect of ovarian stimulation protocol on the implantation rate of the recipient. Fertil Steril (1995); 64:564–7. 25 Hillier SG. Ovarian stimulation with recombinant gonadotrophins: LH as an adjunct to FSH. In: Jacobs HS, ed. The New Frontier in Ovulation Induction. Parthenon: Carnforth, UK, 1993; 39–47. 26 Anderson RE, Cragun JM, Chang RJ, Stanczyk FZ, Lobo RA. A pharmacodynamic comparison of human urinary follicle-stimulating hormone and human menopausal gonadotropin in normal women and polycystic ovary syndrome. Fertil Steril (1989); 52:216–20. 27 Hugues JN, Cedrin-Durnerin I. Revisiting gonadotrophin-releasing hormone agonist protocols and management of poor ovarian responses to gonadotrophins. Hum Reprod update (1998); 4:83–101. 28 Fraser HM. Effect of oestrogen on gonadotrophin release in stumptailed monkeys (Macaca arctoides) treated chronically with an agonist analogue of luteinizing hormone releasing hormone, J Endocrinol (1981); 91:525–30. 29 Broekmans FJ, Bernardus RE, Broeders A, Berkhout G, Schoemaker J. Pituitary responsiveness after administration of a GnRH agonist depot formulation: Decapeptyl CR. Clin Endocrinol (Oxf) (1993); 38:579– 87. 30 Caraty A, Locatelli A, Delaleu B, Spitz IM, Schatz B, Bouchard P. Gonadotropin-releasing hormone (GnRH) agonists and GnRH antagonists do not alter endogenous GnRH secretion in short-term castrated rams. Endocrinology (1990); 127:2523–9. 31 Broekmans FJ, Hompes PG, Lambalk CB, Schoute E, Broeders A, Schoemaker J. Short term pituitary desensitization: effects of different doses of the gonadotrophin-releasing hormone agonist triptorelin. Hum Reprod (1996); 11:55–60. 32 Oppenheim DS, Bikkal H, Crowley WF Jr, Klibanski A. Effects of chronic GnRH analogue administration on gonadotrophin and alpha-

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subunit secretion in post-menopausal women. Clin Endocrinol (Oxf) (1992); 36:559–64. 33 Meldrum DR, Tsao Z, Monroe SE, et al. Stimulation of LH fragments with reduced bioactivity following GnRH agonist administration in women. J Clin Endocrinol Metab (1984); 58:755–7. 34 Cedrin-Durnerin I, Bidart JM, Robert P, Wolf JP, Uzan M, Hugues JN. Consequences on gonadotrophin secretion of an early discontinuation of gonadotrophin releasing hormone agonist administration in shortterm protocol for in-vitro fertilization . Hum Reprod (2000); 15:1009– 14. 35 Sungurtekin U, Jansen RP. Profound luteinizing hormone suppression after stopping the gonadotropin-releasing hormone-agonist leuprolide acetate. Fertil Steril (1995); 63:663–5. 36 Hedon B, Out HJ, Hugues JN, Camier B, et al. Efficacy and safety of recombinant follicle stimulating hormone (Puregon) in infertile women pituitary-suppressed with triptorelin undergoing in-vitro fertilization: a prospective, randomized, assessor-blind, multicentre trial. Hum Reprod (1995); 10:3102–6. 37 Bergh C, Howles CM, 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. Hum Reprod (1997); 12:2133–9. 38 Westergaard LG, Erb K, Laursen S, Rasmussen PE, Rex S. The effect of human menopausal gonadotrophin and highly purified, urinederived follicle stimulating hormone on the outcome of in-vitro fertilization in downregulated normogonadotrophic women. Hum Reprod (1996); 11:1209–13. 39 Fleming R, Lloyd F, Herbert M, Fenwick J, Griffiths T, Murdoch A. Effects of profound suppression of luteinizing hormone during ovarian stimulation on follicular activity, oocyte and embryo function in cycles stimulated with purified follicle stimulating hormone. Hum Reprod (1998); 13:1788–92. 40 Loumaye E, Engrand P, Howles CM, O’Dea L. Assessment of the role of serum luteinizing hormone and estradiol response to folliclestimulating hormone on in vitro fertilization treatment outcome. Fertil Steril (1997); 67:889–99. 41 Mortola JF, Sathanandan M, Pavlou S, et al. Suppression of bioactive and immunoreactive follicle-stimulating hormone and luteinizing hormone levels by a potent gonadotropin-releasing hormone antagonist: pharmacodynamic studies. Fertil Steril (1989); 51:957–63. 42 Fluker MR, Monroe SE, Marshall LA, Jaffe RB. Contrasting effects of a gonadotropin-releasing hormone agonist and antagonist on the secretion of free alpha subunit. Fertil Steril (1994); 61:573–5. 43 The ganirelix dose-finding study group. A double-blind, randomized, dose-finding study to assess the efficacy of the gonadotrophin-

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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. 44 Jones HW Jr, Acosta A, Andrews MC, et al. The importance of the follicular phase to success and failure in in vitro fertilization. Fertil Steril (1983); 40:317–21. 45 Levran D, Lopata A, Nayudu PL, et al. Analysis of the outcome of in vitro fertilization in relation to the timing of human chorionic gonadotrophin administration by the duration of oestradiol rise in stimulated cycles. Fertil Steril (1995); 44:335–41. 46 Fanchin R, de Ziegler D, Olivennes F, Taieb J, Dzik A, Frydman R. Exogenous follicle stimulating hormone ovarian reserve test (EFORT): a simple and reliable screening test for detecting ‘poor responders’ in in-vitro fertilization. Hum Reprod (1994); 9:1607–1. 47 Phelps JY, Levine AS, Hickman TN, Zacur HA, Wallach EE, Hinton EL. Day 4 estradiol levels predict pregnancy success in women undergoing controlled ovarian hyperstimulation for IVF. Fertil Steril (1998); 69:1015–9. 48 Winslow KL, Toner JP, Brzyski RG, Oehninger SC, Acosta AA, Muasher SJ. The gonadotrophin-releasing hormone agonist stimulation test—a sensitive predictor of performance in the flare-up in vitro fertilization cycle. Fertil Steril (1991); 56:711–7. 49 Cedrin-Durnerin I, Herve F, Huet-Pecqueux L, Kottler ML, Hugues JN. Progestogen pretreatment in the shortterm protocol does not affect the prognostic value of the oestradiol flare-up in response to a GnRH agonist. Hum Reprod (1995); 10:2904–8. 50 Urbancsek J, Witthaus E. Midluteal buserelin is superior to early follicular phase buserelin in combined gonadotrophin-releasing hormone analog and gonadotrophin stimulation in in vitro fertilization. Fertil Steril (1996); 65:966–71. 51 Ron-El R, Raziel A, Herman A, et al. Ovarian response in repetitive cycles induced by menotrophin alone or combined with gonadotrophin releasing hormone analogue. Hum Reprod (1990); 5:427–30. 52 Gonen Y, Dirnfeld M, Goldman S, Koifman M, Abramovici H. The use of long-acting gonadotropin-releasing hormone agonist (GnRH-a; decapeptyl) and gonadotropins versus short-acting GnRH-a (buserelin) and gonadotropins before and during ovarian stimulation for in vitro fertilization (IVF). J In Vitro Fert Embryo Transf (1991); 8:254–9. 53 Vauthier D, Lefebvre G. The use of gonadotropin-releasing hormone analogs for in vitro fertilization: comparison between the standard form and long-acting formulation of D-Trp-6-luteinizing hormone-releasing hormone. Fertil Steril (1989); 51:100–4. 54 Goswami SK, Chakravarty BN, Kabir SN. Significance of an abnormal response during pituitary desensitization in an in vitro fertilization and embryo transfer program. J Assist Reprod Genet (1996); 13:374–80.

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55 Loumaye E, Vankrieken L, Depreester S, Psalti I, de Cooman S, Thomas K. Hormonal changes induced by shortterm administration of gonadotropin-releasing hormone agonist during ovarian hyperstimulation for in vitro fertilization and their consequences for embryo development. Fertil Steril (1989); 51:105–11. 56 Brzyski RG, Muasher SJ, Droesch K, Simonetti S, Jones GS, Rosenwaks Z. Follicular atresia associated with concurrent initiation of gonadotropin-releasing hormone agonist and follicle-stimulating hormone for oocyte recruitment. Fertil Steril (1988); 50:917–21. 57 Antoine JM, Firmin C, Alvarez S. Hormonal levels in late follicular phase with long and short regimen of GnRH agonists. Contracept Fertil Sex (1988); 16:630–1. 58 Sims JA, Seltman HJ, Muasher SJ. Early follicular rise of serum progesterone concentration in response to a flareup effect of gonadotrophin-releasing hormone agonist impairs follicular recruitment for in-vitro fertilization. Hum Reprod (1994); 9:235–40. 59 Fanchin R, de Ziegler D, Taieb J, Hazout A, Frydman R. Premature elevation of plasma progesterone alters pregnancy rates of in vitro fertilization and embryo transfer. Fertil Steril (1993); 59:1090–4. 60 Schoolcraft W, Sinton E, Schlenker T, Huynh D, Hamilton F, Meldrum DR. Lower pregnancy rate with premature luteinization during pituitary suppression with leuprolide acetate. Fertil Steril (1991); 55:563–6. 61 Shulman A, Ghetler Y, Beyth Y, Ben-Nun I. The significance of an early (premature) rise of plasma progesterone in in vitro fertilization cycles induced by a “long protocol” of gonadotropin releasing hormone analogue and human menopausal gonadotropins. J Assist Reprod Genet (1996); 13:207–11. 62 Edelstein MC, Seltman HJ, Cox BJ, Robinson SM, Shaw RA, Muasher SJ. Progesterone levels on the day of human chorionic gonadotropin administration in cycles with gonadotropin-releasing hormone agonist suppression are not predictive of pregnancy outcome [see comments]. Fertil Steril (1990); 54:853–7. 63 Givens CR, Schriock ED, Dandekar PV, Martin MC. Elevated serum progesterone levels on the day of human chorionic gonadotropin administration do not predict outcome in assisted reproduction cycles. Fertil Steril (1994); 62:1011–7. 64 Abuzeid MI, Sasy MA. Elevated progesterone levels in the late follicular phase do not predict success of in vitro fertilization-embryo transfer. Fertil Steril (1996); 65:981–5. 65 Hofmann GE, Khoury J, Johnson CA, Thie J, Scott RT Jr. Premature luteinization during controlled ovarian hyperstimulation for in vitro fertilization-embryo transfer has no impact on pregnancy outcome. Fertil Steril (1996); 66:980–6.

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66 Fanchin R, de Ziegler D, Castracane VD, Taieb J, Olivennes F, Frydman R. Physiopathology of premature progesterone elevation. Fertil Steril (1995); 64:796–801. 67 Younis JS, Haddad S, Matisky M, Ben-Ami M. Premature luteinization: could it be an early manifestation of low ovarian reserve? Fertil Steril (1998); 69:461–5. 68 Eldar-Geva T, Margalioth EJ, Brooks B, et al. Elevated serum progesterone levels during pituitary suppression may signify adrenal hyperandrogenism. Fertil Steril (1997); 67:959–61. 69 Fanchin R, Righini C, Olivennes F, Taieb J, de Ziegler D, Frydman R. Premature plasma progesterone and androgen elevation are not prevented by adrenal suppression in in vitro fertilization. Fertil Steril (1997); 67:115–9. 70 Vendola KA, Zhou J, Adesanya OO, Weil SJ, Bondy CA. Androgens stimulate early stages of follicular growth in the primate ovary. J Clin Invest (1998); 101:2622–9. 71 San Roman GA, Surrey ES, Judd HL, Kerin JF. 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.

38 Gonadotropins-only based controlled ovarian hyperstimulation protocols: benefits and drawbacks Kees Jansen, Katherine E Tucker

INTRODUCTION Gonadotropin releasing hormone (GnRH) analog agonists have acquired an important place in in vitro fertilization (IVF) treatments. They owe their popularity on the one hand to their ability to down-regulate luteinizing hormone (LH), thereby preventing premature luteinization, and on the other hand to the fact that the effects of follicular dominance as such can be obviated in contrast to gonadotropin-only protocols. We should still remain cautious, however, with regard to the exclusive use of these compounds in ovarian stimulation, and one should wonder whether the features that make these compounds so popular are sufficient justification for the widespread use of them as a first choice. In about 15% of cycles, gonadotropin-only stimulation for controlled ovarian hyperstimulation (COH) is characterized by the appearance of a premature LH peak once the dominant follicle has reached about 17mm in size. This percentage increases, however, with the growth of the dominant follicle. This pronounced elevation of LH before the administration of hCG does not necessarily have to lead to inadvertent ovulation, but it does lead to luteinization of the follicle and will diminish results. Non-pulsatile administration of GnRH agonists suppresses LH levels by receptor desensitization—after an initial flare up response in gonadotropin release—and because of this feature these drugs have become standard in IVF treatments. It is with remarkable ease, however, that these substances have been introduced since, retrospectively, 85% of all patients might not have needed them. A meta-analysis of prospective randomized studies comparing downregulated and non-down-regulated treatments as first choice in IVF has revealed a significant difference in pregnancy rates between the two stimulation protocols.1 When all the data are combined, there is no doubt the results differ significantly in terms of results per cycle, cancellation rates, and results per follicle aspiration in favor of agonist treatment. The meta-analysis, however, just confines itself to stating the differences

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between both groups without fully discussing the causes. Thus, when one scrutinizes the data, it is not so much that the results in the downregulated cycles are superior, but that the results in non-down-regulated cycles are unusually low. Earlier studies, as well as some of the prospective randomised trials, have shown that results in the non downregulated cycles can be virtually identical to those in which agonists have been used. In addition, the metaanalysis does not take into consideration the other side of the coin, such as the increased risks (multiples and the ovarian hyperstimulation syndrome, OHSS). The authors specifically stipulate that further research is needed to investigate these aspects. Any introduction of a new medication scheme should be justifiable according to a number of criteria. The new scheme should be either more effective, have less side effects or both, or—when equally effective—it should have a better “cost/benefit” ratio. Agonists do not fulfill any of these requirements. As mentioned previously, the superiority of the agonists is based upon unacceptably low results in the control groups in those studies that are responsible for the differences. In this chapter, the published literature will be discussed, and a critical appraisal will be given of the use of these drugs. Although agonists do have a definite role to play in IVF, we can not rule out the importance and efficacy of the gonadotropin-only COH schemes as first choice medication scheme for IVF.

THE ROLE OF GnRH AGONISTS IN ART The goal of the use of agonists is to obtain better results in follicular stimulation. It is clear that these drugs exert their effect by the prevention of a premature or mature LH surge. If follicular monitoring is inaccurate, sooner or later all patients will exhibit a LH peak. Thus the incidence is dependent on the accuracy of follicular monitoring. Treating all patients with agonists essentially translates to an “overkill” of up to 85% in infertility centers with accurate monitoring. Agonists act as an “umbrella” against the “rainfall” of premature LH peaks. It seems a strange policy, however, to always use an umbrella when one is aware that it only rains in 15% of the time. There is no conclusive evidence that agonists act by synchronizing follicle growth, preventing dominance or are helpful for insufficient response. The purported synchronization can be related to the fact that it is possible to extend the stimulation period beyond the time the follicle has reached a size at which an endogenous LH peak would occur in gonadotropin-only cycles. This would, therefore, allow more subordinate follicles to achieve a sufficient size. On ultrasound monitoring this may seem to lead to more “synchronized” follicle growth, as the relative difference in size between the individual follicles will invariably decrease.

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THE OCCURRENCE OF AN UNTIMELY LH RISE In the epoch before the use of agonists, many centers adopted the policy to cancel a COH stimulation cycle in the event of a premature or mature LH surge. There is, however, little to no proper scientific evidence to support this decision. Pregnancies may occur in spite of the occurrence of a premature LH peak. In the early days, laparoscopy was necessary for follicular puncture. The patient had to be admitted to hospital and had to undergo general anaesthesia. Nowadays, however, transvaginal follicle aspiration is an outpatient procedure and the method of choice. The decision to cancel a cycle cannot properly be defended. Many studies based their policy on that of the Jones Centre of Norfolk. These authors refer in many articles to a chapter written by the Norfolk Group stating literally that “based on clinical impressions we have designed the following cancellation criteria…” Therefore, by cancelling, the cycles part of the differences between the groups may be the result of a “selffulfilling prophecy”, merely by the differences in cancellation percentage. In an early study, performed between 1985 and 1988 in COH with gonadotropins only, the occurrence of a premature LH peak was 14% (104/736 cycles). Cycles were allowed to proceed regardless of the presence of a peak. There was a 26% difference between both groups in terms of take home baby rates per started cycle (10/104 versus 82/632), although this was not significant (P=0.41).2 Thus, although results are lower, it is clear that even in the presence of a LH peak, births may occur at such a rate that cancellation is not justified. Although it is clear that in the 10 years since then, results have improved, this specific feature remains unaltered.

EFFICACY OF CYCLES WITH AND WITHOUT AGONISTS The infertility guild is notorious for the introduction of new protocols without a proper assessment of efficacy and effectivity and the only way these can truly be evaluated is the double blind, placebo controlled, randomized, prospective trial. For GnRH agonists no such trials have been performed in comparison to gonadotropin-only cycles. It is often said that the mechanism of action of agonists precludes such a study. As mentioned above, all prospective randomized studies performed are non-blinded, creating the possibility of cancelling a cycle in both arms of the study which may lead to the “selffulfilling prophecy” discussed previously.

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THE IVF CASCADE Some clinicians may believe that the option of the type of controlled study mentioned above is moot since the end result, one or more living children, is not dependent upon subjectivity. This is not correct. An IVF treatment comprises of a cascade of steps: cycles started, aspirations, IVF procedures, transfers, clinical and ongoing pregnancies and deliveries of one or more children. From the moment of puncture, subjectivity will play a minor part, but especially the first step may lend itself susceptible to subjective assessment; the investigator who knows the stimulation scheme, and who decides whether or not to proceed to puncture or cancel the cycle, may—knowingly or unknowingly—influence the results. Therefore, one should judge the data per cycle as well as per aspiration, especially when the cancellation rate differs greatly. For example, a study such as that of Gonen, that does not mention the cancellation rate, cannot be judged on its proper value.3

STUDIES Many studies only contain small numbers of patients. Some comparative studies that contain larger numbers of patients do not adhere to the most elementary requirements, especially because of the selection of patients for both groups.4,5 These studies have to be discarded. For example, Macnamee from Bourn Hall compared a group of patients stimulated without agonists with a group that were stimulated with agonists. However, all patients in the first group were allowed to start, whereas the patients in the second group were selected on the basis of several favorable factors, such as the absence of cysts and a very low plasma concentration of progesterone at the start of the cycle.4 Strangely, the author does not address this issue nor does he mention the number of patients who dropped out in the second group because of this additional selection criterion. In other studies the choice of the stimulation scheme (with or without agonists) was left to the investigator.6 In some studies, no randomization took place. For instance, stimulations with HMG alone took place in the beginning and those with GnRH down-regulated cycles at the end of the study, two years later. In addition, these authors do not mention the cancellation rate as well.7 It is clear that these studies cannot be used for comparison.

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PROSPECTIVE, RANDOMIZED STUDIES In a literature search only 11 studies adhered to the criteria of prospective randomization.8–18 One study only analysed low responders, and, as this was not a first choice medication scheme, it was not included.19 Another study was prospective, but not truly randomized; selection took place on alternate days. This study concerned IVF as well as GIFT; the results for IVF have been included.12 Three of the 11 studies found a significant difference in pregnancy rates per cycle, but they also find a significant difference in cancellation rates. As the only important parameter—the take home baby rate—often was not mentioned in the articles, this rate was obtained separately, either directly or from related publications by the same investigators. The only author that finds a significant difference in take home baby rate is Neveu.8 This study, however, included only 10 patients per arm: 1 in 10 in the HMG group, 6 in 10 in the down-regulated group. The delivery rate for the latter group (60% deliveries per started cycle), however, cannot be considered representative. A meta-analysis is published that confines itself to description of the facts rather than analysis of the differences.1 When the 11 studies are analysed together, there is a significant difference in results, which, incidentally, is largely the result of the difference in cancellation rate. Fig 38.1 depicts the reported or calculated take home baby rate per cycle initiated in such studies with agonists as a first choice. In terms of delivery rate per aspiration the difference is 13 versus 16% (P<0.03). The most striking finding in all studies, however, is not so much that the results in the downregulated cycles are so superior, but that the results in the non-down-regulated cycles are low for those studies that ultimately contribute to the significance (Table 38.1). Some centers can achieve satisfactory results without agonists, but others do not seem to be able to do so. This may be explained as a reflection of the quality of their follicular monitoring. Agonists may mask inaccuracy in measurements. Lack of experience is not penalized by the occurrence of a premature or mature LH peak the way it would be in gonadotropin-only cycles. The convenience of agonists is beyond doubt. The limits of the size of the dominant follicle at which one can administer the hCG are wide, and range between 16mm and 24mm. Aspirations can be shifted towards convenient days, workload can be equalized, and weekends can be avoided.

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Fig 38.1 Odds ratio and 95% confidence intervals of take home baby rate per started cycle in prospective randomized studies in IVF with or without GnRH as first choice (refs 8– 18). Take home baby rate was published or communicated, or calculated from the pregnancy and abortion rates reported. P values depicted on the right.

Table 38.1. Prospective randomized studies. Year/author No anal. THB/cy % Canc With anal. THB/cy % Canc 1987 Neveu 1/10 10.0% 20% 6/10 60% 0% 1990 Antoine 9/90 10.0% 15% 15/90 16.7% 8% 1990 Benadiva 14/88 15.9% 20% 10/68 14.7% 14% 1990 Ferrier 4/43 9.3% 26% 0/30 0% 23% 1990 Abdalla 3/58 5.1% 19% 7/48 14.6% 9% 1990 vd Berg 45/2 7.7% 17% 16/101 15.8% 14% 1990 Kublik 2/26 7.7% 39% 5/34 14.7% 15% 1991 Maroulis 11/93 11.8% 23% 11/99 11.1% 12% 1991 Ron El 11/151 7.2% 27% 21/151 13.9% 3% 1992 Kingsland 19/158 12.0% 11% 24/150 16.7% 5% 1992 Corson 3/33 9.0% 30% 8/34 23.5% 8% Total 81/802 10.1% 20.3% 123/815 15.1% 10.4%

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SIDE EFFECTS OF DOWN-REGULATED VERSUS “GONADOTROPIN ONLY” CYCLES There is increasing evidence that GnRH agonists have more side effects and risks. It has been published that the incidence of the ovarian hyperstimulation syndrome may be increased, as well as the number of multiple pregnancies. However, as the incidence of OHSS is much lower than the pregnancy rates, only very large studies may shed light in this aspect. Apart from the suffering, treatment cost of the OHSS may be considerable, and these should be added to the cost of the treatment. THE OVARIAN HYPERSTIMULATION SYNDROME The incidence of the OHSS varies per center and seems to be dependent on nomenclature and diagnostics. The literature concerning the risk of OHSS in agonist cycles suggests an increased risk with percentages of 6.6 to 8.4% of cycles.15,20 Half of these were severe. In cycles with HMG alone an incidence of 0.7% was found.20 On the basis of these differences, a prospective study including a very large number of patients would be needed in order to achieve significance, which has not been performed. We have performed a blinded study of patients undergoing IVF stimulated with HMG and recombinant FSH without GnRH agonists.21 In this study, OHSS did not occur in 89 cycles versus about 3.2% hospital admissions due to OHSS in a similar large multicenter study with the use of agonists.22 Recently, some insightful studies have been performed with COH, using either agonists or antagonists, in a prospective, randomized fashion. In one study, the incidence of OHSS for patients in the antagonist arm was 2.4%, in the agonist arm 5.9%. In this study the rate of hospital admission due to OHSS was 0.8 and 2.5% respectively.23 In two other studies, the admission rates due to severe OHSS were 1.1 and 1.7% for the antagonists, versus 6.5 and 5.6% for the agonists.24,25 However, in all studies where GnRH agonists were not used, the admission rate was even below those mentioned for the antagonists. In cycles without agonists hospital admission rates of less then 0.2% can be achieved. Although there is no scientific proof of the protective effect of aspirating all follicles, we certainly believe this to be the case. The most severe forms of OHSS may be life threatening. In one study an intensive care unit (ICU) admission rate of patients with GnRH downregulation of 0.6% was reported.26 One problem with the literature concerning adult mortality is its publication deficit. To date, only one such case study has been published.27 In the Netherlands, however, from all the 10 documented adult mortalities from IVF (1:10,000 stimulation cycles),

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six were due to complications of OHSS. All of these patients were treated with GnRH agonists and none of these cases have been published in the scientific literature. THE MULTIPLE PREGNANCY RATES It is likely that the multiple pregnancy rate is increased in agonist cycles in comparison to gonadotropin-only cycles. In the study by Chetowksi (1989), there were 44% multiples in the agonist arm versus 8% in the HMG-alone group.7 In order to diminish the multiple pregnancy rates, one approach is to reduce the number of embryos replaced. However, that can negate the increased results of agonist cycles. OTHER SIDE EFFECTS? Not very much is known concerning the absence of long-term effects, justifying the firstchoice use of these compounds based purely on convenience. There are reports concerning absence of immediate shortterm effects of agonists inadvertently used during pregnancy, but these are incidental and should be judged with caution. These reports may represent a publication bias typical for such cases. The authors may be inclined to publish only when no side effects are shown. In addition, as will be elaborated upon below, there has been a report on an increase in the attention deficit and hyperactivity disorder (ADHD) in those children, that only becomes manifest 6–8 years after birth. After administration, agonists can be detected in follicular fluid28 and they readily pass the placenta.29 It has been demonstrated in rabbits that there is a direct, adenyl cyclaseindependent effect of these drugs on the oocyte that may lead to premature resumption of the meiosis.30 It is not known, however, whether there are long-term consequences from this feature.

STANDARD TOXICOLOGICAL AND TERATOLOGICAL TESTS There are no signs of an increase in congenital abnormalities after the administration of GnRH agonists. It is not sure, however, whether the standard toxicological or teratological tests are sufficient to exclude a possible influence. Agonists are small molecular decapaptides and are known to be neuropeptides. One might envisage that these compounds may have an effect on brain tissue and influences on brain development and behavioral aspects cannot be ruled out. If such an influence exists, however, it may take years to express itself. Administration of agonists to rats 28 days after ovulation does not lead to demonstrable effects within the first period of development, nor in behaviour and development in

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female rats. In male rats, however, agonist administration leads to clear disturbances that are only expressed after puberty. There is a significant reduction in testicular size, disturbed pubertal development, diminished fertility and aberrant sexual behaviour.31 In rats, 28 days after ovulation or about one week after birth is equivalent to about 22 weeks’ pregnancy in the human with regard to the development of brain centers.32 So if there is an effect on brain development, and extrapolation to the human is considered, agonists present at that moment in time could lead to an influence that will express itself only after puberty. Moreover, if there would be a direct effect on the testes, the fetus would be vulnerable at about 8–12 weeks. GnRH receptors have been demonstrated outside the brain, especially in ovarian tissue, but so far the testis has not been investigated. A direct effect at the testicular level seems feasible, however, although with the short-acting agonists, such an influence is unlikely. Conversely, this can also present a clear warning for the depotpreparations, that may sometimes last as long as 8 weeks or longer in the body, much longer than the minimum duration they are guaranteed to work, namely, about 4 weeks.

FUNCTIONAL NEUROTERATOLOGY Swaab has introduced the concept of “functional neuroteratology.” He has stressed the possibility of influences by substances in pregnancy that may affect behavior that only may come to expression many years afterwards.33,34 Especially with the use of neuropeptides it is clear that caution is warranted. It has recently been published that the inadvertent administration of GnRH agonists after ovulation, in the so called long protocol, may lead to an increase in the incidence of developmental and behavioral problems (especially attention deficit hyperactivity disorder as mentioned earlier) in infancy, many years after birth.35 It is of interest that of the published case reports regarding the erroneous use of agonists in pregnancy even such a basic feature as the sex of the child that is born was often not mentioned. If the child is male, it is imperative that these patients are subjected to a follow up examination until after puberty.

ECONOMIC CONSIDERATIONS Despite some claims that the treatment with agonists might lead to monetary savings there is no doubt that there is an increase in cost in the long run. The advantage in terms of cost lies in the fact that one may reschedule working times to exclude weekends, and that one might decrease the frequency of ultrasound monitoring. However, as the number of days of stimulation is on average at least 4 days longer, there is no decrease in absolute number of examinations.

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The overall costs initially comprise the price of the agonist itself and, secondly, the added cost of the extra ampoules of HMG or FSH. Depending on the type of downregulation (long or short scheme) one may need about 10–20amps more. The cost should be analysed as the cost per healthy child. In addition to the costs directly attributable to the use of agonists are the increased costs due to the higher complication rate, which can involve admission to a hospital or ICU because of OHSS. These costs may add considerably to the total.

SELECTIVE USE OF AGONISTS? Instead of completely down-regulating all patients with agonists and then overdosing them with FSH, we believe hyperstimulation schemes should be individualized for each patient. So far we only reserved agonists for those patients that exhibited a LH peak in the first cycle. These individuals are known to be at increased risk for a repeat immature LH peak, and if they do not become pregnant, they will receive agonists in the second cycle. Unfortunately, the first cycle may be more or less “sacrificed” to determine whether the patients need agonists. As such the patients that use agonists are a negative selection of the total. Using this approach, we have analysed the results in 1990 in relation to the use of agonists (Table 38.2). We now aim to recognize those patients that later will display a premature LH peak. It seems that with the determination of the concentration of inhibin-B on the third day of the cycle one can predict with reasonable accuracy whether or not a LH peak will occur.36 A low inhibin B concentration depicts poor responders, a high inhibin B concentration is seen in patients with polycystic ovarian syndrome (PCO). In our study, when inhibin levels are above 120pg/ml, there is an elevated chance of an LH peak. At this level the sensitivity would be 70%, the specificity 87%, the positive predictive value 87% and the negative predictive value 70% (LR+5.4, LR-: 0.34). With the impending worldwide introduction of GnRH antagonists, that exert their action by competitive binding, it may be possible to select those patients that really need agonists and prevent treatment with agonists for those who do not.

CONCLUSION AND RECOMMENDATIONS FOR CONTROLLED OVARIAN HYPERSTIMULATION IN GONADOTROPIN-ONLY CYCLES The role of agonists in IVF is not disputed, but it is questionable whether they should be used firstly and indiscriminately. The differences between groups of patients treated with or without agonists can, at least in part, be explained by the difference in cancellation rate. Agonists do not notably

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improve pregnancy rates in centers with existing good pregnancy rates, but they may normalize results in centers with low pregnancy rates. They allow unrivalled flexibility, but by the same token, they also can conceal

Table 38.2. Results in deliveries per cycle initiated (incl cancellations) with the birth of at least one healthy child. Analysis of all stimulation cycles in the year 1990 (excluding satellite and transport cycles, unstimulated cycles, donor oocyte cycles and cycles of cryopreserved embryos). Gonadotropin only With agonists year DHV THBR/Cycle % Canc % THBR/Cycle % Canc % 1990 First cycles 55/253 21.7% 5.9% 3/9 33.3% 0 Following cycles 48/240 20.0% 9.2% 23/137 16.8% 3.6% All cycles 103/493 20.9% 7.5% 26/146 17.8% 3.4% incompetence in follicle monitoring, as they do not just prevent “premature” LH peaks, but also “mature” LH peaks. However, application of these simulation schemes for all patients does not take into account the increased complication rate, especially in terms of the incidence of OHSS. In addition, because of the increased multiple pregnancy rate, one has to adopt a more restricted transfer policy, which in itself negates part of the advantages of agonist treatment and, finally, there is a lower cost-benefit ratio. Stimulation schemes should become more individualized and tailored to the specific needs of each patient. More efforts should be directed towards the identification of those that may require agonists from patients that do not. The thought that there is one single “best” universal scheme for all patients should be abandoned. Some patients may benefit from agonists, some from antagonists, and others from gonadotropin-only protocols for COH. When contemplating COH without GnRH agonists there are some important prerequisites. 1) Measurement of follicles should be performed accurately and conscientiously. One should measure in three directions to accurately estimate follicle diameter, especially when there are several follicles that may lead to deviation from the ideal round or oval shape. Inaccurate monitoring will lead to diminished results to a degree not seen in down-regulated cycles. 2) hCG should be given relatively “early”, when the leading follicle is between 15mm and 17mm. Most or all oocytes from follicles above 10mm will be able to start resumption of the second meiotic division, which, together with the cytoplasmatic ripening, is necessary for adequate fertilization. Although the overall number of oocytes will be lower, the aim is to reach an optimal number of mature oocytes (between five and eight) and not just simply maximize the total number of eggs.

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3) Smaller follicle size can challenge the experience of the person performing the aspiration. Aspirations are more difficult to perform, requiring more expertise of the puncture technique. 4) The program should be full time (7 days per week). With non-agonist stimulations, it is not possible to “skip” one or more days since hCG administration must occur when the lead follicle has reached the appropriate diameter. 5) Although this is not necessary, we aim to start stimulation without pre-treating with oral contraceptives. In the so-called long protocol, when agonist administration starts in the midluteal phase oral contraceptives are often used to exclude the formation of ovarian cysts, as well as to prevent erroneous administration of agonists after natural fertilization.35 When no oral contraceptives are used, however, the first day of the cycle cannot be changed. The cycle could thus start on any of the seven days of the week. It is our opinion that future follicle stimulations should become more individualized. Instead of completely and indiscriminately downregulating all patients, clinicians should search for selection criteria to distinguish those patients that really need GnRH agonists; whether some patients may benefit from GnRH antagonists or may not need agonists at all. There is no such thing as one ideal stimulation scheme for all patients, regardless of age, physical characteristics, and cause of infertility.

REFERENCES 1 Hughes EG, Fedorkow DM Daya S, et al. The routine use of gonadotropin releasing hormone agonist prior to in vitro fertilization and gamete intrafallopian transfer: a meta-analysis of randomized controlled trials. Fertil Steril (1992); 58:888. 2 Jansen CAM, Burgers S. Do follicle number, presence of cysts or LH peaks justify cancellation of an IVF cycle? Hum Reprod (1990); 5S:96. 3 Goney N, Dirnfeld M, Goldman S, et al. The use of long acting gonadotropin releasing hormone agonist (GnRHa, Decapeptyl) and gonadotropins versus short acting GnRH-a (Buserelin) and gonadotropins before and during ovarian stimulation for in vitro fertilization. J In Vitro Fertil Embryo Transf (1991); 8:254. 4 Macnamee MC, Tayler PJ, Howles CM, et al. Short term luteinizing hormone-releasing hormone agonist treatment: prospective trial of a novel ovarian stimulation regimen for in vitro fertilization. Fertil Steril (1989); 52:264. 5 Garcia JE, Padilla SL, Bayati J, et al. Follicular phase gonadotropinreleasing hormone agonist and human gonadotropins: a better alternative for ovulation induction in in vitro fertilization. Fertil Steril (1990); 53:302. 6 Thanki KH, Schmidt CL. Follicular development and oocyte maturation after stimulation with gonadotropins versus leuprolide

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acetate/gonadotropins during in vitro fertilization. Fertil Steril (1990); 54:656. 7 Chetowski RJ, Kruse LR, Nass TE. Improved pregnancy outcome with the addition of leuprolide acetate to gonadotropins for in vitro fertilization. Fertil Steril (1989); 52:250. 8 Neveu S, Arnal F, Hedon B, et al. Ovarian stimulation by a combination of gonadotropin-releasing hormone agonist and gonadotropins for in vitro fertilization. Fertil Steril (1987); 47:639. 9 Antoine JM, Salat-Baroux J, Alvarez S, et al. Ovarian stimulation using human menopausal gonadotropins with or without LHRH analogues in a long protocol for in-vitro fertilization: a prospective randomized comparison. Hum Reprod (1990); 5:565. 10 Benadiva CA, Mastroianni L, Blasco L, et al. Comparison of different regimens of a gonadotropin-releasing hormone analog during ovarian stimulation for in vitro fertilisation. Fertil Steril (1990); 53:479. 11 Ferrier A, Prey K, Rasweiler JJ, et al. Evaluation of leuprolide acetate and gonadotropins versus clomiphene citrate and gonadotropins for in vitro fertilization or gamete intrafallopian transfer. Fertil Steril (1990); 54:90. 12 Abdalla HI, Morris NN, Ahuja KK et al. Comparative trial of luteinizing hormone-releasing hormone analog/human menopausal gonadotropin and clomophene citrate/human menopausal gonadotropin in an assisted conception program. Fertil Steril (1990); 53:473. 13 van de Berg-Helder A, Helmerhorst FM; Blankhart A, Brand R, Waegemaekers C, Naaktgeboren N. Comparison of ovarian stimulation regimens for in vitro fertilization (IVF) with and without a gonadotropin releasing hormone (GnRH) agonist: results of a randomized study. J In Vitro Fertil Embryo Transf (1990); 7:358. 14 Kubik CJ, Guzick DS, Berga SL, et al. Randomized, prospective trial of leuprolide acetate and conventional superovulation in first cycles of in vitro fertilization and gamete intrafallopian transfer. Fertil Steril (1990); 54:836. 15 Maroulis GB, Saphier A, Emery M, et al. Prospective randomized study of human menotropin versus a follicular and a luteal phase gonadotropin-releasing hormone analog-human menotropin stimulation protocols for in vitro fertilization. Fertil Steril (1991); 55:1157. 16 Ron El R, Nachum H, Herman A, et al. Gonadotropins and combined gonadotropin-releasing hormone agonistgonadotropins protocols in a randomized prospective study. Fertil Steril (1991); 55:574. 17 Kingsland C, Mason B, Tan SL, et al. The routine use of gonadotropinreleasing hormone agonists for all patients undergoing in vitro fertilization. Is there any medical advantage? A prospective randomized study. Fertil Steril (1992); 57:804. 18 Corson SL, Batzer FR, Gocial B, Eisenberg E, Huppert LC, Nelson JR. Leuprolide acetate-prepared in vitro fertilization-gamete intrafallopian

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transfer cycles: efficacy versus controls and cost analysis. Fertil Steril (1992); 57:601–5. 19 MacLachlan V, Besanko M, O’Shea F, et al. A controlled study of luteinizing hormone-releasing hormone agonist (buserelin) for the induction of folliculogenesis before in vitro fertilization. New Engl J Med (1989); 320:1233. 20 Golan A, Ron-El R, Herman A, et al. Ovarian hyperstimulation syndrome following D-Trp-6 luteinizing hormone-releasing hormone microcapsules and menotropins for in-vitro fertilization. Fertil Steril (1988); 50:912. 21 Jansen CAM, van Os HC, Out HJ, Coelingh Bennink HJT. A prospective randomized clinical trial comparing recombinant follicle stimulating hormone (Puregon) and human menopausal gonadotropins (Humegon) in non-downregulated in-vitro fertilisation patients. Hum Reprod (1988); 13:2995–9. 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); 10:2535–40. 23 The European Orgalutran study Group. Simplification of treatment with the gonadotropin-releasing hormone antagonist ganirelix (Orgalutran®) in women undergoing controlled ovarian hyperstimulation with recombinant follicle stimulating hormone (Puregon®): results of a controlled, randomized, multicenter trial. 2000 (in press). 24 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. 25 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. 26 Smitz J, Camus M, de Vroey P, et al. Incidence of severe hyperstimulation syndrome after GnRH agonist/HMG superovulation for in vitro fertilization. Hum Reprod (1990); 5:933. 27 Cluroe AD, Synek BJ. A fatal case of ovarian hyperstimulation syndrome with cerebral infarction. Pathology (1995); 27:344–6. 28 Loumaye E, Vandrieken L, Coen G, et al. Use of gonadotropinreleasing hormone agonist during ovarian stimulation leads to significant concentrations of peptide in follicular fluids. Fertil Steril (1989); 52:256. 29 Skopelak VM, Hodgen GD. Infusion of gonadotropin-releasing hormone agonist during pregnancy: Maternal and fetal responses in primates. Am J Obstet Gynecol (1987); 156–755.

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30 Joshimura Y, Ubukata Y, Nakamura Y, et al. Gonadotropin-releasing hormone agonists induce meiotic maturation and degenerated oocytes in the in vitro perfused rabbit ovary. Fertil Steril (1991); 55:177. 31 van den Dungen HM. LHRH and gonadotropins in rat pubertal development. Academic thesis, AZVU, 1990. 32 van den Dungen HM. Personal communication. 1992. 33 Swaab DF, GJ Boer, Feenstra MF. Concept of functional neuroteratology and the importance of neurochemistry. Prog Brain Res (1988); 73:3. 34 Boer GJ, Sijdewint GM, Swaab DF. Neuropeptides and functional neuroteratology. Prog Brain Res (1988); 73:245. 35 Lahat E, Raziel A, Friedler S, Schieber-Kazir M, Ron-El R. Long-term follow-up of children born after inadvertent administration of a gonadotropin-releasing hormone agonist in early pregnancy. Hum Reprod (1999); 14:2656–60. 36 Lockwood GM, Muttukrishna S, Ledger WL, Groome NP, Dolfing J, Jansen CAM. Optimisation of non-analogue IVF: a role for inhibin β in predicting patients liable to premature LH surge or inadequate response. Hum Reprod (1997); 12S:157–8.

39 The use of GnRH agonists Roel Schats, Joop Schoemaker

INTRODUCTION Gonadotropin releasing hormone (GnRH) is the primary hypothalamic regulator of reproductive function. With the help of a very small amount (250ug) of GnRH derived from 160000 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 which, 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 synaps and released in a pulsatile fashion into the complex capillary net of the portal system of the pituitary gland.4 The synthesis and release of the pituitary gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), was shown to be dependent on this pulsatile GnRH pattern.5–7 In the human, the critical range of pulsatile release frequencies ranges 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.8– 10 High frequent (>3pulses/hour) and continuous exposure of the pituitary to GnRH failed to produce normal LH and FSH release patterns while its sensitivity appeared to be greatly dependent on gonadal steroids and proteins.11–13 After the discovery of the chemical structure of native GnRH, many analogs were synthetically produced. Most analogs 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 instant

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fall in gonadotropin secretion from the pituitary by competitive receptor binding, and were designated as GnRH antagonists. Nowadays GnRH agonists have gained a wide field of clinical applications.14 Suppression of the pituitary ovarian (or testicular) axis for a limited or even extended period of time is the main goal to be achieved in these treatments. The introduction of the GnRH antagonists into clinical practice has been hampered for a long time by problems concerning solubility, direct allergy like side effects owing to histamine release and cost factors.15,16 GnRH ANALOGS NOWADAYS ON THE MARKET 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.17 Over the past three decades, thousands of analogs of GnRH have been synthesized. Only seven of the agonistic analogs of GnRH have become approved and 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.

Fig 39.1 Hormone levels for FSH, LH and estradiol (E2) in a patient with continuous

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intravenous infusion of 0.5µg/minute LHRH. LH was measured with three and FSH with two different assays (RIA: Radiolmmuno Assay, MLCA: Magic Lite Chemoluminescence Assay and IRMA: ImmunoRadioMetric Assay), bbt: basal body temperature (private collection Prof. J.Schoemaker). Although 90% of the biological activity is lost with splicing of glycine number 10, most of it is restored with the attachment of NH2-ethylamide to the proline at position 9.18 The second major modification was the replacement of the glycine at position 6 by D-amino acids, which decreases enzymatic degradation. The combination of these two modifications was found to have synergistic biological activity and also proved to exhibit a higher receptor binding affinity. The affinity can be increased further by 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 non-ionized fat soluble compounds.18 For details about the structure see Table 39.1. GnRH AGONISTIC ANALOGS: CLINICAL APPLICATIONS The original goal for 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 demonstrated.19 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 non pulsatile 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 hypogonadotrophic hypogonadism, which was also termed “medical gonadectomy” or “medical hypophysectomy” allowed for the relatively rapid extensive introduction of GnRH agonists into clinical practice. For a variety of indications complete abolishment 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,

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Table 39.1. Amino acid sequence and substitution of he GnRH agonists. Compound Position 6 Position 10 Amino acid no 1 2 3 4 5 6 7 8 9 10 Native GnRH Glu His Trp Ser Tyr Gly Leu Arg Pro Gly-NH2 Nonapeptides Leuprolide (Lupron, Leu N-Et-NH2 Lucrin) Buserelin (Suprefact) Ser(O’Bu) N-Et-NH2 Goserelin (Zoladex) Ser(O’Bu) AzaGlyNH2 Histrelin (Supprelin) DAzaGlyHis(Bzl) NH2 Deslorelin (Ovuplant) D-Trp N-Et-NH2 Decapeptides Nafarelin (Synarel) 2Nal Gly-NH2 Triptorelin (Decapeptyl) Trp Gly-NH2 central precocious puberty, endometriosis, uterine fibroids, hirsutism, and other conditions.20 Since the first report on the use of the combination of the GnRH agonist buserelin and gonadotropins for ovarian stimulation for in vitro fertilization back in 1984,21 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 as the scheme of first choice for controlled ovarian hyperstimulation in in vitro fertilization (IVF). The major advantage initially offered by the agonists was the efficient abolishment of the spontaneous LH surge.22 The incidence of premature LH surges and subsequent luteinization in cycles with exogenous gonadotropin stimulation was observed by several investigators to range between 30% and 50%. A deleterious effect on both fertilization and pregnancy rates was noted.22,23 The cycles in which a premature LH surge occurred usually had to be cancelled. 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 recovered. Both led to improved clinical pregnancy rates per embryo transfer and especially per started cycle.24 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) Which route of administration is the best?

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(2) Which agonist(s) should be used in ART? (3) What is the optimal dose? (4) What is the optimal scheme? (1) WHICH ROUTE OF ADMINISTRATION IS THE BEST? In fact there are three routes of administration for the GnRH agonists: (1) intramuscular or subcutaneous depot injection; (2) intranasal administration; and (3) subcutaneous injections. 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.25 Although several authors claim a normal outcome of pregnancy following inadvertent administration of a GnRH agonist during early pregnancy,26–31 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.32 A depot preparation without reduction in the dosage cannot be advocated for the routine use in IVF (see also (2)). With the intranasal route the absorption of the GnRH agonist fluctuates inter- and intra-individually giving an unpredictable desensitization level, but most of the times 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 versus the subcutaneous route of administration. (2) 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,33 while deslorelin never has been applied in human IVF. Also 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 preparations to be used as first choice because of their long duration of action. Broekmans et al showed that rapid induction of a hypogonadotrophic 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.25 This is far longer than is actually needed. Devreker et al found obvious negative effects of depot preparations: longer stimulation phase, consequently more ampoules needed, but more importantly lower

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implantation and delivery rates (32.8% v 21.1%; 48.9% v 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.34 Although depot preparations seem attractive because of their ease of administration for the patient, they can not be advocated at the moment. Dosage reduction might be the key to overcome these unwanted side effects. Thirteen prospective randomized trials were traced in the literature comparing different agonists with each other.35–47 The problem with those studies is that for none of the applied individual analogs the optimal dosage has been determined. Therefore the value of these articles is limited in respect to elucidating the question which one 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 dose finding studies for the use of GnRH agonists in ART are still and urgently needed. In fact it is rather strange that they still have not been done more than 10 years after the introduction of the agonists in IVF. The suppression of the LH surge is the only achievement that should be met. It is obvious that the required dosage for that goal can be completely different from the dosage to treat carcinoma of the prostate (see (3) below). (3) 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.48 The dose finding in pulsatile gonadotropin releasing hormone for hypogonadotropic amenorrhoea was performed by Crowley et al.49 In contrast there is only one prospective, randomized, double blind, placebo controlled dose finding study performed in IVF for the GnRH agonist triptorelin.50 Before this study, the same group had already shown a clear dose dependency of pituitary desensitization during controlled ovarian stimulation in IVF.51 The results show that the dosage necessary for suppressing the spontaneous LH surge is much smaller, namely only 15– 50% of the dosage needed for the treatment of a malignant disease. It is very likely that dose finding studies for the other agonists will give similar results. (4) WHAT IS THE OPTIMAL SCHEME? 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.52 He also reported another

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beneficial effect: a major advantage of the long protocol of GnRH agonist administration is that with its use, precise timing of hCG is not important. It would, therefore, appear that the routine use of GnRH agonists has both medical as well as practical advantages. Daya recently published a metaanalysis concerning the different GnRH agonist protocols in IVF.53 It revealed significant differences in pregnancy rates between GnRH agonist protocols (for details see Table 39.2). 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. The results of those studies are consequently only applicable to the “normal” patients. There is a possibility that especially in the excluded groups other schemes are preferable. Although the metaanalysis of Daya showed the superiority of the long follicular protocol in comparison with the long luteal, short and ultra-short protocols, this does not exclude the possibility that in individual patients for example a short protocol can give better results. The preferable protocol in these patients is in fact not known. Some investigators claim that in this group of patients the short flare protocol,54 in combination with a dose reduction of the agonist and a high dose of FSH is the treatment of choice.55–57 Further studies are needed to evaluate the short55,57 or long56 “mini-dose” protocols. 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 et al observed both a poor stimulation outcome and a reduction in pregnancy rates in a cycle with cyst formation.58 However, Feldberg et al could not confirm this finding.59 Ovarian cyst formation was reduced when pretreatment with an oral contraceptive was applied.60 Damario et al showed the beneficial effect of this strategy in high responder patients with respect to cancellation rates and pregnancy rates.61 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 centres 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 three weeks. Several investigators have tried to shorten this long duration of administration leading to the so called “early cessation” protocols. Although it seems a logical approach no significant beneficial effect with respect to the main outcome measures (LH, estradiol, and progesterone levels, duration of the stimulation phase, number of retrieved oocytes, fertilization rate and pregnancy rates) was found by Pantos et al.62 Faber et al claimed a beneficial effect of early cessation in combination with high dose FSH in poor responders.63 Fujii et al found that early discontinuation (on day of the start of ovarian stimulation (cycle day 7) had adverse effects on follicular development.64 They explained

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this deleterious effect by the phenomenon previously described by Sungurtekin et al of profound paradoxical drop of serum LH (see also Fig 39.1) after early cessation, leading to significantly lower estradiol levels on the day of hCG and a higher cancellation rate.65 However, the pregnancy rates were not different. The duration of the stimulation phase was significantly longer and consequently the number of ampoules was higher. Their conclusion was, although the discontinuation-long protocol can decrease the total GnRH

Table 39.2. GnRH agonist protocols and pregnancy rates. Protocol vs Protocol OR (95% Cl) Long Ultrashort 1.47 (1.20–2.12) Long Short (all) 1.26 (1.10–1.56) Long follicular Short 1.53 (1.06–2.22) Long luteal Short 1.10 (0.82–1.49) Long luteal Long follicular 0.93 (0.41–2.11)

P 0.039 0.036 0.008 0.51 0.78

Table 39.3. Summary of advantages and disadvantages of the different GnRH agonist protocols. GnRH Route of Administration Duration of Advantages Disadvantages agonist administration days of cycle administration protocol (CD) Ultrashort In/sc CD 2,3–4,5 3 days Patient’s Low PR protocol comfort Short In/sc CD 2,3 until 8–12 days Patient’s No protocol day of hCG comfort programming Long In/sc CD 2 until day 28–35 days Programming, Long duration follicular of hCG good PR of administration Long In/sc CD 21 until 21–28 days Programming, Long duration luteal day of hCG good PR of administration Menstrual In/sc CD 21 until 7–12 days Inconclusive Low estradiol early menses levels cessation Follicular In/sc CD 21 until 13–20 days Inconclusive Low estradiol early stim. day 6,7 levels cessation Long Depot CD 2 Once Patient’s (Too) long follicular comfort duration of (depot) action

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Long Depot luteal (depot) Ultralong In/sc/depot

CD 21

Once

CD 2 or 21

800

Patient’s comfort

(Too) long duration of action 8–12 weeks, Only for Side effects depot 2 or 3 special cases due to times estrogen deficiency

agonist dose, this regimen is not cost effective because of the increase in hMG/FSH dose and the adverse effects on follicular development. Hazout et al66 and Yang et al67 also described an early cessation protocol in comparison with a depot preparation of triptorelin. They both found a reduction in hMG requirement as the main advantage for the early cessation protocols. We do not think it is fair to compare a depot preparation with daily subcutaneous administration for one week and therefore the conclusions that can be drawn from their studies are rather limited. The available data are not convincing enough to promote an “early cessation” protocol. In Table 39.3 the most important advantages and disadvantages of the different GnRH agonist protocols are summarized.

CONCLUSIONS The GnRH agonists nowadays are widely used in IVF to control the endogenous LH surge and achieve augmentation of multifollicular development. Disadvantages, such as the necessity of 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 consequently improvement of pregnancy rates. The introduction of the GnRH agonists in IVF is not an example of excellent research, because proper dose finding studies are almost absent. It is a little bit a shame to state, but further research in finding the right dose and protocol can still improve the clinical benefits of the GnRH agonists. The daily administered, short acting preparations deserve preference to the depot formulations. The intranasal administration fits best patient’s comfort considerations, while the subcutaneous route can 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.

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15 Reissmann Th, Diedrich K, Comaru-Schally AV. Introduction of LHRH antagonists into the treatment of gynaecological disorders. Hum Reprod (1994); 9:767–9. 16 Gordon K, Hodgen GD. Will GnRH antagonists be worth the wait? Reprod Med Rev (1992); 1:189–94. 17 Nestor JJ jr. Development of agonistic LHRH analogs. In: Vickery BH, Nestor JJ jr, Hafez ESE, eds LHRH and its analogs. Lancaster: MTP Press, 1984:3–15. 18 Karton MJ, Rivier JE. Gonadotropin-releasing hormone analog design. Structure-function studies toward the development of agonists and antagonists: rationale and perspective. Endocr Rev (1986); 7:44–66. 19 Corbin A, Beattie CW. Post-coital contraceptive and uterothrophic effects of luteinizing hormone releasing hormone. Endocr Res Commun (1975); 2:116–20. 20 Conn PM, Crowley WF jr. Gonadotropin-releasing hormone and its analogues. N Engl J Med (1991); 324:93–103. 21 Porter RN, Smith W, Craft IL, Abdulwahid NA, Jacobs HS. Induction of ovulation for in-vitro fertilisation using buserelin and gonadotropins. Lancet (1984); ii: 1284–5. 22 Flemming R, Coutts JR. Induction of multiple follicular growth in normally menstruating women with endogenous gonadotropin suppression. Fertil Steril (1986); 45:226–30. 23 Loumaye E. The control of endogenous secretion of LH by gonadotropin-releasing hormone agonists during ovarian hyperstimulation for in-vitro fertilisation and embryo transfer. Hum Reprod (1990); 5:357–76. 24 Hughes EG, Fedorkow DM, Daya S, Sagle MA, Van de Koppel P, Collins JA. The routine use of gonadotropinreleasing hormone agonists prior to in vitro fertilization and gamete intrafallopian transfer: a metaanalysis of randomized controlled trials. Fertil Steril (1992); 58:888– 96. 25 Broekmans FJ, Bernardus RE, Berkhout G, Schoemaker J. Pituitary and ovarian suppression after early follicular and mid-luteal administration of a LHRH agonist in a depot formulation: Decapeptyl CR. Gynecol Endocrinol (1992); 6:153–61. 26 Weissman A, Shoham Z. Favourable pregnancy outcome after administration of a long-acting gonadotropinreleasing hormone agonist in the mid-luteal phase. Hum Reprod (1993); 8:496–7. 27 Balasch J, Martinez F, Jove I, Cabre L, Coroleu B, Barri PN, Vanrell JA. Inadvertent gonadotropin-releasing hormone agonist (GnRHa) administration in the luteal phase may improve fecundity in in-vitro fertilization patients. Hum Reprod (1993); 8:148–51. 28 Ron-El R, Lahat E, Golan A, Lerman M, Bukovsky I, Herman A. 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.

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29 Cahill DJ, Fountain SA, Fox R, Fleming CF, Brinsden PR. Outcome of inadvertent administration of a gonadotropin-releasing hormone agonist (buserelin) in early pregnancy. Hum Reprod (1994); 9:1243–6. 30 Gartner B, Moreno C, Marinaro A, Remohi J, Simon C, Pellicer A. Accidental exposure to daily long-acting gonadotropin-releasing hormone analogue administration and pregnancy in an in-vitro fertilization cycle. Hum Reprod (1997); 12:2557–9. 31 Taskin O, Gokdeniz R, Atmaca R, Burak F. Normal pregnancy outcome after inadvertent exposure to long-acting gonadotropinreleasing hormone agonist in early pregnancy. Hum Reprod (1999); 14:1368–71. 32 Lahat E, Raziel A, Friedler S, Schieber-Kazir M, Ron-El R. Long-term follow-up of children born after inadvertent administration of a gonadotropin-releasing hormone agonist in early pregnancy. Hum Reprod (1999); 14:2656–60. 33 de Ziegler D, Cedars MI, Randle D, Lu JK, Judd HL, Meldrum DR. Suppression of the ovary using a gonadotropinreleasing-hormone agonist prior to stimulation for oocyte retrieval. Fertil Steril (1987); 48:807–10. 34 Devreker F, Govaerts I, Bertrand E, Van den Bergh M, Gervy C, Englert Y. The long-acting gonadotropin-releasing hormone analogues impaired the implantation rate. Fertil Steril (1996); 65:122–6. 35 Balasch J, Jové I, Moreno V, Civico S, Puerto B, Vanrell JA. The comparison of two gonadotropin-releasing hormone agonists in an in vitro fertilization program. Fertil Steril (1992); 58:991–4. 36 Parinaud J, Oustry P, Perineau M, Rème JM, Monroziès X, Pontonnier G. Randomized trial of three luteinizing hormone-releasing hormone analogues used for ovarian stimulation in an in vitro fertilization program. Fertil Steril (1992); 57:1265–8. 37 Penzias AS, Shamma FN, Gutmann JN, Jones EE, DeCherney AH, Lavy G. Nafarelin versus leuprolide in ovulation induction for in vitro fertilization: a randomized clinical trial. Obstet Gynecol (1992); 79:739–42. 38 Tapanaienen 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. 39 Dantas ZN, Vicino M, Balmaceda JP, Asch RH, Stone SC. Comparison between nafarelin and leuprolide acetate for in vitro fertilization: preliminary clinical study. Fertil Steril (1994); 61:705–8. 40 Goldman JA, Dicker D, Feldberg D, Ashkenazi J, Voliowich I. A prospective randomized comparison of two gonadotropin-releasing hormone agonists, nafarelin acetate and buserelin acetate, in in-vitro fertilization—embryo transfer. Hum Reprod (1994); 9:226–8. 41 Goldman JA, Dicker D, Feldberg D, Ashkenazi J, Voliowich I. A prospective randomized comparison of two gonadotropin-releasing

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hormone agonists, nafarelin and buserelin acetate, in in-vitro fertilization—embryo transfer. Hum Reprod (1994); 9:226–8. 42 Tarlatzis BC, Grimbizis G, Pournaropoulos F, Bontis J, Lagos S, Pados G, Mantalenakis S. Evaluation of two gonadotropin-releasing hormone (GnRH) analogues (leuprolide and buserelin) in short and long protocols. J Assist Reprod Genet (1994); 11:85–91. 43 Lockwood GM, Pinkerton SM, Barlow DH. A prospective randomized single-blind comparative trial of nafarelin acetate with buserelin in long-protocol gonadotropin-releasing hormone analogue controlled invitro fertilization cycles. Hum Reprod (1995); 11:3079–80. 44 Tanos V, Friedler S, Shushan A, Strauss N, Hetsroni I, Lewin A. Comparison between nafarelin acetate and DTrp6-LHRH for temporary pituitary suppression in in vitro fertilization (IVF) patients: a prospective cross over study. J Assist Reprod Genet (1995); 12:715– 19. 45 Oyensaya OA, Teo SK, Quah E, Abdurazak N, Lee FY, Cheng WC. Pituitary down-regulation 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. 46 Avrech OM, Goldman GA, Pinkas H, Amit S, Neri A, Zukerman Z, Ovadia J, Fisch B. Intranasal nafarelin versus buserelin (short protocol) for controlled ovarian hyperstimulation before in vitro fertilization: a prospective clinical trial. Gynecol Endocrinol (1996); 10:165–70. 47 Corson SL, Gutmann JN, Batzer FR, Gocial B. A double blind comparison of nafarelin and leuprolide acetate for down-regulation in IVF cycles. Int J Fertil Menopausal Stud (1996); 41:446–9. 48 Polson DW, Mason HD, Saldahna MB, Franks S. Ovulation of a single dominant follicle during treatment with low-dose pulsatile follicle stimulating hormone in women with poly cystic ovary syndrome. Clin Endocrin (Oxf) (1987); 26:205–12. 49 Santoro N, Wierman ME, Filicori M, Waldstreicher J, Crowley WFJ. Intravenous administration of pulsatile gonadotropin-releasing hormone in hypothalamic amenorrhea: effects of dosage. J Clin Endocrin Metab (1986); 62:109–16. 50 Janssens RMJ, Lambalk CB, Vermeiden JPW, Schats R, Schoemaker J. Dose-finding study of triptorelin-acetate for prevention of premature LH surge: a prospective, randomised, double blind, placebo controlled study. Hum Reprod (1998); 13 (Suppl 1):49–50, 099. 51 Janssens RM, Vermeiden JP, Lambalk CB, Schats R, Schoemaker J. Gonadotropin-releasing hormone dosedependency of pituitary desensitization during controlled ovarian hyperstimulation in IVF. Hum Reprod (1998); 13:2386–91. 52 Tan SL. Luteinizing hormone-releasing hormone agonists for ovarian stimulation in assisted reproduction. Curr Opin Obstet Gynecol (1994); 6:166–7.

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53 Daya S. Optimal protocol for gonadotropin-releasing hormone agonist use in ovarian stimulation. In: Gomel V, Cheung PCK, eds. In vitro fertilization and assisted reproduction, Monduzzi Editore: Bologna, Italy (1997); 405–15. 54 Tasdemir M, Tasdemir I, Kodana H, Fukuda J, Tanaka T. Short protocol of gonadotropin-releasing hormone agonist administration gave better results in long protocol poor-responders in IVF-ET. J Obstet Gynaecol Res (1996); 22:73–7. 55 Scott RT, Navot D. Enhancement of ovarian responsiveness with microdoses of gonadotropin-releasing hormone agonist during ovulation induction for in vitro fertilization. Fertil Steril (1994); 61:880–5. 56 Feldberg D, Farhi J, Ashkenazi J, Dicker D, Shalev J, Ben Rafael Z. Minidose gonadotropin-releasing hormone agonist is the treatment of choice in poor responders with high follicle-stimulating hormone levels. Fertil Steril (1992); 62:343–6. 57 Surrey ES, Bower J, Hill DM, Ramsey J, Surrey MW. 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. 58 Keltz MD, Jones EE, Duleba AJ, Polcz T, Kennedy K, Olive DL. 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. 59 Feldberg D, Ashkenazi J, Dicker D, Yeshaya A, Goldman GA, Dicker D, Goldman JA. Ovarian cyst formation: a complication of gonadotropin-releasing hormone agonist therapy. Fertil Steril (1989); 51:42–5. 60 Biljan MM, Mahutte NG, Dean N, Hemmings R, Bissonette F, Tan SL. Pretreatment with an oral contraceptive is effective in reducing the incidence of functional ovarian cyst formation during pituitary suppression by gonadotropin-releasing hormone analogues. J Assist Reprod Genet (1998); 15:599–604. 61 Damario MA, Barmat L, Liu HC, Davis OK, Rosenwaks Z. Dual suppression with oral contraceptives and gonadotropin-releasing hormone agonists improves in-vitro fertilization outcome in high responder patients. Hum Reprod (1997); 12:2359–65. 62 Pantos K, Meimeth-Mamianaki T, Vaxevanoglou T, Kapetanakis E. Prospective study of a modified gonadotropin-releasing hormone agonist long protocol in an in vitro fertilization program. Fertil Steril (1994); 61:709–13. 63 Faber BM, Mayer J, Cox B, Jones D, Tonet JP, Oehninger S, Muasher SJ. Cessation of gonadotropin-releasing hormone agonist therapy combined with high-dose gonadotropin stimulation yields favorable pregnancy results in low responders. Fertil Steril (1998); 70:826–30.

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64 Fujii S, Sagara M, Kudo H, Kagiya A, Sato S, Saito Y. A prospective randomized comparison between long and discontinuous-long protocols of gonadotropin-releasing hormone agonist for in vitro fertilization. Fertil Steril (1997); 67:1168–8. 65 Sungurtekin U, Jansen RP. Profound luteinizing hormone suppression after stopping the gonadotropin-releasing hormone-agonist leuprolide acetate. Fertil Steril (1995); 63:663–5. 66 Hazout A, de Ziegler D, Cornel C, Fernandez H, Lelaider C, Frydman R. Comparison of short 7-day and prolonged treatment with gonadotropin-releasing hormone agonist desensitization for controlled ovarian hyperstimulation. Fertil Steril (1993); 59:596–600. 67 Yang TS, Tsan SH, Wang BC, Chang SP, Ng HT. The evaluation of a new 7-day gonadotropin-releasing hormone agonist protocol in the controlled ovarian hyperstimulation for in vitro fertilization. J Obstet Gynaecol Res (1996); 22:133–7.

40 Antagonistic analogs of GnRH: preferable stimulating protocol Basil C Tarlatzis, Helen Bili

OVERVIEW Currently, the most commonly used ovarian stimulation protocols for assisted reproductive technologies (ART) are those utilizing gonadotropin releasing hormone agonists (GnRHa), mainly in the long desensitization protocol. This involves GnRHa administration for at least 14 days until pituitary downregulation, followed by the co-administration of FSH containing gonadotropins (human menopausal gonadotropins—HMG) or recombinant follicle stimulating hormone (-recFSH). The ultimate aim of such protocols is to increase the number of good quality embryos available for transfer, since it has been shown that the transfer of more than one embryo results in higher pregnancy rates, but with an increased incidence of multiple gestations.1 However, multiple pregnancies are associated with a higher risk for obstetric complications, fetal morbidity and mortality, as well as psychosocial problems,2 consequences that are usually underestimated by ART specialists. On the other hand, fetal reduction which offers a solution in these cases, may lead to the loss of pregnancy, notwithstanding the psychological and ethical ramifications. Therefore, the avoidance of high order pregnancies by reducing the number of transferred embryos has become a primary goal and has decreased the need for intense ovarian stimulation.3,4 Several studies have indicated that GnRHa stimulation protocols increase the number of oocytes retrieved, are associated with high pregnancy rates, and allow better programmation of in vitro fertilization (IVF) activities. On the other hand, they are rather complex and expensive, while they prolong the length of treatment, since complete ovarian activity suppression may require 15 days or more of GnRHa administration and a high amount of exogenous gonadotropins to achieve adequate follicular stimulation.3,5 Also, GnRHa protocols bring some danger for the patients, as the ovarian hyperstimulation syndrome (OHSS), which is a serious and potentially life threatening complication of ART, appears to be more frequent with the use of GnRHa.5 There is also some evidence to indicate that the GnRHa may be luteolytic, necessitating luteal support.6

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The introduction of the new generation of GnRH antagonists gives the opportunity to overcome many of the problems associated with GnRHa.6 Furthermore, it allows to reevaluate the current policy of intense ovarian stimulation, and to develop new softer and more flexible stimulation protocols that could be individually adapted. GnRH antagonist action is characterized by an immediate suppression of pituitary gonadotropin release and a short recovery phase, between two and four days after the antagonist is discontinued,7 in contrast with GnRHa, where restoration of gonadal function usually begins within 60– 90 days, when a depot formulation is used. Intermittent secretion of human hypothalamic GnRH induces pulsatile secretion of both luteinizing hormone (LH) and FSH from the anterior pituitary. In addition, it seems that endogenous GnRH is required for estradiol (E2) induced LH surge, even in very small amounts, and it can therefore be prevented or inhibited by the administration of GnRH antagonists.8 GnRH antagonists, unlike GnRHa, do not act by down regulation but by the specific and competitive blockage of the GnRH receptors.9 On the contrary, GnRHa induce desensitization of the gonadotropic cells by reducing the number of GnRH receptors on the cell membrane, so-called down regulation. It is also clear that GnRH antagonists are more potent in suppressing LH and FSH, compared with GnRHa. The third generation GnRH antagonists have modifications in positions 1, 2, 3, 6, and 10 of the sequence of human GnRH and a structure that offers metabolic stability and reduced allergic side effects.6 In fact, the clinical introduction of GnRH antagonists has

Table 40.1. Current GnRH antagonists. GnRH antagonists Stage of development Cetrorelix (Asta Medica, Germany) Completed clinical trial phase III Ganirelix (Organon, The Netherlands) Completed clinical trial phase III Antide (Serono, Switzerland) Early clinical trial phase II Antarelix Preclinical trial Azaline-B Preclinical trial been delayed by the side effects of histamine release of the older compounds. Two of the third generation GnRH antagonists, Cetrorelix (Cetrotide, Serono International, Switzerland), and Ganirelix (Orgalutran, Organon, The Netherlands) are now available for clinical use (Table 40.1). On the other hand, Antide is in early clinical phase II trial, while antarelix and azaline-B are both in the preclinical stage of development (Table 40.1). Hence, the data used in this chapter are derived from the clinical studies with Cetrorelix and Ganirelix.

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ADMINISTRATION PROTOCOLS The main objective of using GnRH antagonists in ovarian stimulation for IVF, is the avoidance of a premature LH rise. Since GnRH antagonists cause an immediate blockage of the GnRH receptors on the pituitary gonadotropes, their administration can be limited to those days when E2 levels are probable to induce the LH surge.7,10,11 Thus, two different protocols with GnRH antagonists have been developed: (a) the single dose protocol,11,12 in which a single high dose of the GnRH antagonist is administered in the late follicular phase (day 8 or 9 of the stimulation cycle), and (b) the multiple dose protocol,7,13 in which daily small doses of the GnRH antagonist are administered, beginning from day 6 or 7 of the stimulation cycle (Fig 40.1). SINGLE DOSE PROTOCOL It appears that in the single dose regimen, one injection of the antagonist on day 8 is usually sufficient, although in slow responders a repeat injection may be needed until HCG administration (Fig 40.1). With the use of Cetrorelix, the lowest effective dose seems to be 3 mg, and each injection allows 3 to 4 days of treatment with gonadotropins without an LH surge.11 This was further confirmed in a subsequent study by Olivennes et al,12 in which they showed that this protocol allowed the prevention of premature LH surges and was associated with a reduction of the gonadotropin dose and stimulation period as compared with the long GnRHa protocol, using a depot formulation. Moreover, they observed a lower

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Fig 40.1 Ovarian stimulation with FSH together with GnRH antagonists in the single dose protocol (upper panel) and in the multiple dose protocol (lower panel).

Table 40.2. IVF results in women treated with cetrorelix (single dose) and triptorelin (depot-long protocol). Treatment Variables Cetrorelix Triptorelin Patients with ovum pickup (N) 113 36 Stimulation length (days) 9.4±1.4* 10.7±1.7* hMG dose (ampules) 24.3±7.4* 35.6±15.1* 1786±808* 2549±1194* E2 on hCG day (pg/ml) Oocytes (N) 9.2±5.1 12.6±7.4 Embryos (N) 5.4±3.5 7.5±4.9 Clinical pregnancies/OPU (%) 22.6 28.2 Miscarriage rate (%) 15.4 27.3 Ongoing pregnancy rate/OPU (%) 18.3 23.1 OHSS (%) 3.5 11.1 *Statistically significant. Adapted from ref. 12. number of follicles and oocytes in the Cetrorelix patients while the percentage of mature oocytes and the fertilization rate were comparable.

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The pregnancy rate, although not statistically significant, was lower in the antagonist group as was also the incidence of OHSS (Table 40.2). On the other hand, the single dose protocol has not been applied in ovarian stimulation using Ganirelix, and, hence, no clinical data are available so far. MULTIPLE DOSE PROTOCOL The multiple dose protocols were proposed by Diedrich et al7 and involve the daily treatment with low doses of GnRH antagonist in combination with exogenous gonadotropins (Fig 40.1).7,14 In order to determine the minimal effective dose, Albano et al15 compared daily doses of 0.5mg, 0.25mg and 0.1mg of cetrorelix in women undergoing IVF. Both doses of 0.5mg and 0.25mg were able to prevent LH surges, while two out of seven patients treated with the 0.1mg had an LH rise. Since the IVF results of the 0.5mg and the 0.25mg doses were comparable, the daily dose of 0.25mg was selected for the multiple dose protocol. Similar results were obtained from a multicenter dose finding study13 comparing six dosages (0.0625mg, 0.125mg, 0.25mg, 0.5mg, 1.0mg and 2.0mg) of the GnRH antagonist Ganirelix, which showed too that 0.25mg of Ganirelix is the minimal dose. This dose was not only effective in preventing a premature LH rise but also yielded a high implantation rate of 22% and an ongoing pregnancy rate of 33% per attempt. Importantly, the six dose groups were similar in terms of the number of oocytes retrieved as well as in the number and quality of embryos obtained. However, very low implantation rates of 8.8% and 1.5% were obtained in the two highest dose groups (1mg and 2mg, respectively), which resulted in an ongoing pregnancy rate of only 14.1% in the 1mg group and no ongoing pregnancies in the 2mg group. On the other hand, LH rises were observed in the lowest doses groups of 0.0625mg and 0.125mg. Thus, based on these findings, a daily dose of 0.25mg has been recommended for both antagonists as the minimal effective dose for clinical use. Subsequently, large randomized multicenter controlled phase III trials were conducted to compare the use of GnRH antagonists with GnRHa for IVF.16,17 Both studies have indicated that in the antagonist group the duration of treatment and the amount of gonadotropins used were significantly lower than in the agonist group (Table 40.3). On the other hand, the number of medium size follicles and the E2 levels were lower in the antagonist than in the agonist group, but the fertilization rate and embryo quality were not different. The pregnancy rates were similar in both groups, although there was a trend for lower pregnancy rates in GnRH antagonist groups (Cetrorelix and Ganirelix). This fact, although statistically not significant, has to be further evaluated. On the other hand, the necessity for the practitioners to familiarize themselves with a new medication and also novel protocols, must not be underestimated.

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Table 40.3. IVF results of multiple dose cetrorelix and ganirelix versus long protocol with Buserelin (phase III studies). Cetrorelix Buserelin Ganirelix Buserelin Cycles (N) 188 85 463 238 LH rise>10lU/L(%) 1.6 0 2.8 1.3 hMG/recFSH dose (IU) 1770±637.5* 1920±570* 1500 1800 (900– (900– 5400) 6450) Stimulation length (days) 10.6±2.3 11.4±1.8* 9 (6–18) 10 (6–19) 1625±836* 2082±1049* 1190 1700 E2 on hCG day (pg/ml) (373– (527– 3105) 4070) Oocytes (N) 8.0±4.9* 10.6±6.6* 9.1±5.4 10.4±5.8 Fertilization rate (%) 53.6 52.9 62.1 62.1 Embryos (N) 3.8 4.5 6.0±4.5 7.1±5.2 Clinical pregnancies/ started 22.3 25.9 21.8 28.2 cycle (%) OHSS (%) 1.1 6.5** 2.4 5.9 *: P<0.01, **: P<0.05. Adapted from refs 16, 17. Concerning the luteal phase, it seems that no serious negative influence exists18 after the GnRH antagonist treatment. Moreover, the incidence of OHSS (Tables 40.2, 40.3) was consistently lower in the antagonist than the agonist groups in all the studies performed so far.12,16,17

“SOFT” OVARIAN STIMULATION FOR IVF During the last few years, the need to attenuate the stimulation protocols for IVF, has been strongly emphasized, aiming to reduce the complications (for example, OHSS, multiples, etc) and the cost of controlled ovarian hyperstimulation, without compromising the success rate.3,19,20 The recent availability of GnRH antagonists encourages the use of older stimulation protocols for IVF, which were abandoned mainly because of the high incidence of premature LH surges. Moreover, with the use of GnRH antagonists during the late follicular phase, the final stages of oocyte meiotic maturation can be induced by the administration of recombinant LH,21 native GnRH,22 or GnRH agonist instead of HCG.23

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(A) “SOFT” STIMULATION PROTOCOLS Clomiphene citrate (CC), alone or in combination with low doses of gonadotropins, together with the GnRH antagonists could be introduced again in clinical IVF practice aiming to reduce the total dose of gonadotropins used and to obtain smaller numbers of better quality oocytes (Fig 40.2). Furthermore, improved laboratory techniques could allow in the future the selection and transfer of better quality embryos compensating for the smaller numbers. The protocol of CC/gonadotropins has been used extensively in IVF/ET with satisfactory pregnancy rates.24,25 The major drawback for the CC/gonadotropin protocol has been the high cancellation rate, mainly related to premature LH surges,24 and the lower number of oocytes and embryos obtained. The association of GnRH antagonists to this combination has gained new interest since it could prevent LH rises and could represent an interesting treatment option in selected patients with good prognosis by

Fig 40.2 Ovarian stimulation with clomiphene citrate (CC) and HMG or recFSH together

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with GnRH antagonists in the single dose protocol (upper panel) and in the multiple dose protocol (lower panel). reducing the side effects, the risks, and the cost. Moreover, a simpler procedure could be repeated to reach high cumulative success rates. Thus, Craft et al studied the use of cetrorelix in combination with CC/gonadotropins in “difficult” patients—poor responders and women with polycystic ovary syndrome (PCOS).26 CC was given daily (100mg) from day 2 of the cycle for five days, and gonadotropin injections were either given daily or on alternate days. Cetrorelix was administered daily, 0.25mg subcutaneously, from the fifth day of gonadotropin stimulation. In the group of poor responders, more oocytes were retrieved (6.4 v 4.7 oocytes/cycle) at a lower dose of FSH (709 v 1163 IU/oocyte; p=0.08) compared with the previous protocol used and two live births were obtained (11.8%). In the PCOS group, fewer oocytes were recovered (10.2 v 14.5 oocytes/cycle), using a lower dose of gonadotropins (170 v 189 IU/oocyte) and resulting in one ongoing pregnancy, while no patients experienced OHSS. These encouraging preliminary results are also explored in other ongoing studies with Cetrorelix, combining the multiple dose or the single dose protocols with the CC/gonadotropin stimulation. (B) MINIMAL OVARIAN STIMULATION FOR IVF BASED ON THE NATURAL MENSTRUAL CYCLE Aiming to simplify the procedure, efforts were made to apply IVF in the natural menstrual cycle.27,28 Despite the encouraging fertilization and implantation rates that have been reported, the cancellation rate of 10– 30% has rendered this approach rather unattractive to most IVF centers. The introduction of GnRH antagonists in clinical practice offers an interesting perspective. Hence, the combination of the natural menstrual cycle and the administration of GnRH antagonists in the late follicular phase to prevent the LH surge, is a promising alternative which is currently being studied. Rongieres-Bertrand et al29 investigated the administration of Cetrorelix in the late follicular phase together with small amounts of supplementary gonadotropins, in the natural cycle of 33 women undergoing 44 cycles of IVF with good prognosis. Cycle monitoring was started on day 8 with ovarian ultrasonography and plasma E2 measurements. A single subcutaneous injection of 1mg or 0.5mg Cetrorelix was administered when E2 plasma levels reached 100pg/ml to 150pg/ml and the leading follicle was between 12mm and 14mm. Moreover, daily injections of 150IU of FSH were given from the time of the first Cetrorelix injection until HCG administration. The mean number of gonadotropin ampules used was 4.7±1.4, and the cycle cancellation rate was very low (5.5%) compared with previous reports on natural IVF cycles, but in 10 cycles

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(25%) no oocytes were retrieved and in six cycles (15%) fertilization was not obtained. Thus, embryo transfer was performed in 22 cycles (55%) leading to seven clinical pregnancies (17.5% per retrieval, 32% per transfer), from which five were ongoing.29 These preliminary results seem quite interesting and require further investigation. Adopting another approach, de Jong et al30 administered recombinant FSH in the early to mid-follicular phase (from cycle day 5 onwards) and Cetrorelix in daily injections (0.25mg subcutaneously/day) from cycle day 8 onwards, when at least one follicle of 13mm was present. The total amount of recombinant FSH administered was substantially less (700– 1200IU) than that usually administered in conventional ovarian stimulation protocols for IVF (around 2200IU). Also, with this minimal ovarian stimulation protocol it was possible to obtain pregnancies, without luteal support. It seems, therefore, that the minimal stimulation protocols based on the spontaneous natural cycle can be a possible alternative for good prognosis patients, but their efficiency needs to be confirmed in larger studies.

POSSIBLE COMPLICATIONS FROM THE APPLICATION OF GnRH ANTAGONISTS IN IVF Until now, the GnRHa long protocol is mostly used in IVF, as it gives good clinical results and programmable IVF procedures. Before the GnRH antagonists protocols can be established in clinical practice, their efficiency and safety has to be examined vis-à-vis those of the GnRHa protocols.20,31 From all the available studies with both GnRH antagonists, no systematic side effects have been detected and the local tolerance was similar with that of GnRHa.16,17 On the other hand, the pregnancy rates, although satisfactory, tend to be lower than with GnRHa.16,17 This finding, if confirmed, could be attributed either to suboptimal use of this new drug in combination with gonadotropins or to an adverse, direct or indirect, effect on the endometrium. Indeed, direct effect of GnRH antagonists on the endometrium or the ovary cannot be excluded, but this issue is also unclear for GnRHa. Data from a recent study32 suggest that high dosages of Ganirelix do not adversely affect the potential of embryos to establish clinical pregnancies in freeze thaw cycles, indicating that there is no direct negative effect of this antagonist on the quality of oocytes and embryos. However, the first part of this study indicates that when high doses of GnRH antagonists are used in conjunction with recFSH, the suppression of endogenous LH might be too intense, leading to LH deprivation and cessation of follicle development as well as poor IVF results.13 Although GnRH antagonists seem to allow more flexible protocols to be applied, their use may disturb the current programmation of the IVF units, since a treatment cycle has to begin with the spontaneous menstrual

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cycle. In addition, the single dose protocol, requires meticulous monitoring of follicular development and E2 production in order to decide whether, if necessary, to administer a second dose to avoid premature LH surges. FUTURE OVULATION STRATEGIES WITH GnRH ANTAGONISTS At present, the application of GnRH antagonists has been adopted to the GnRHa protocols aiming mainly to prevent untimely LH surges. However, GnRHa also decreases high tonic LH levels, which have been associated with poorer oocyte quality and IVF outcome,33,34 and this is not achieved with the current protocols of GnRH antagonists. Thus it needs to be explored whether the immediate suppression of endogenous gonadotropins by the antagonists, without the flare up effect of GnRHa, could offer any

Table 40.4. Advantages and disadvantages of GnRH agonists and antagonists. GnRH agonists GnRH antagonists Initial flare-up effect Immediate suppression of gonadotropin levels Slow recovery of pituitary function Quick recovery of pituitary function Avoidance of endogenous LH Avoidance of endogenous LH surge surge More programmable IVF Less programmable IVF but greater patients’ procedures convenience More oocytes for IVF and Fewer oocytes, but also less OHSS. cryopreservation Expensive protocols with long Less expensive protocols with shorter duration duration additional advantage in the clinical outcome of patients with polycystic ovary syndrome or endometriosis. Furthermore, as experience with these compounds increases, new protocols can be developed aiming to recruit a smaller number of follicles with better synchronization of follicular maturation, since natural factors such as the recruitment process and the inherent size of the follicular cohort, seem to remain undisturbed with the new GnRH antagonist regimens. This strategy would avoid the complications of ovarian stimulation (for example, OHSS and multiples), prevent the ethical problems associated with embryo freezing and reduce the cost of treatment. Moreover, convenience can be an important issue for the patients, particularly with

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regard to the shorter treatment duration with these new simplified protocols (Table 40.4).

CONCLUSIONS The availability of the new GnRH antagonists opens new perspectives in ovulation induction. The different studies presented so far, confirmed the efficiency of GnRH antagonists in preventing the LH surge and leading to viable pregnancies with a low incidence of OHSS. Moreover, the GnRH antagonists give the opportunity to use again some of the old stimulation protocols (CC, HMG/recFSH or CC/HMG/recFSH) or develop new “mild” ones, even involving the natural cycle, and to trigger ovulation by other means than hCG. However, these protocols need to be evaluated further. Similarly, the two GnRH antagonists, Cetrorelix and Ganirelix, as well as the two regimens, single and multiple dose, need to be further explored and compared properly. In conclusion, the new generation GnRH antagonists represent a significant progress in ovarian stimulation. They are potent suppressors of endogenous gonadotropins, they can be used in shorter, more flexible and convenient protocols, and they are associated with satisfactory pregnancy rates with less complications. Nevertheless, further studies are needed to determine the most efficient protocol of ovarian stimulation for IVF, using these new compounds.

REFERENCES 1 Society for Assisted Reproductive Technology. Assisted reproductive technology in the United States and Canada: 1994 results generated from the American Society of Reproductive Medicine, Society for Assisted Reproductive Technology registry. Fertil Steril (1996); 66:697–705. 2 Garel M, Salobir C, Blondel B. Psychological consequence of having triplets: a 4-year follow-up study. Fertil Steril (1997); 67:1162–5. 3 Fauser BCJM, Devroey P, Yen SSC, et al. Minimal ovarian stimulation for IVF: Appraisal of potential benefits and drawbacks. Hum Reprod (1999); 14:2681–6. 4 Olivennes F, Frydman R. Friendly IVF: the way of the future? Hum Reprod (1998); 13:1121–4. 5 Smitz J, Ron-El R, Tarlatzis BC. The use of gonadotrophin releasing hormone agonists for in vitro fertilization and other assisted procreation techniques: Experience from three centers. Hum Reprod (1992); 7:49–66. 6 Karten MJ, Hoeger CA, Hooh WA, Lindberg MC, Nagri R. The development of safer antagonists: strategy and status. In: Bouchard P,

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Haour F, Franchimont P, Schatz B, eds. Recent progress on GnRH and gonadal peptides. Paris: Elsevier, Session II, (1990): 147–58. 7 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. 8 Leroy I, Frydman R, d’Acremont MF, deMouzon J, Brailly-Tabard S, Bouchard P. A single injection of a GnRH antagonist (Cetrorelix); postpones the luteinizing hormone (LH) surge: further evidence for the role of GnRH during the LH surge. Fertil Steril (1994); 62:461–7. 9 Rivier JE, Porter J, Rivier CL. New effective GnRH antagonists with minimal potency for histamine release in vitro. J Med Chem (1986); 29:1846–51. 10 Itzkovitz-Eldor J, Kol S, Mannaerts B, Coelingh-Bennink H. Case report: first established pregnancy after controlled ovarian hyperstimulation with recombinant follicle stimulating hormone and gonadotropin releasing hormone antagonist ganirelix (Org 37462). Hum Reprod (1998); 13:294–5. 11 Olivennes F, Fanchin R, Bouchard P, Taieb J, Selva J, Frydman R. Scheduled administration of GnRH antagonist (Cetrorelix) on day 8 of in vitro fertilization cycles: a pilot study. Hum Reprod (1995); 10:1382–6. 12 Olivennes F, Belaisch-Allart J, Emperaire J-C, et al. Prospective randomized controlled study of in vitro fertilization-embryo transfer with a single dose of a luteinizing hormone-releasing hormone (LHRH); antagonist (cetrorelix) or a depot formula of an LH-RH agonist (triptorelin). Fertil Steril (2000); 73:314–20. 13 Ganirelix Dose-Finding Study Group. A double-blind, randomized, dose-finding study to assess the efficacy of the GnRH antagonist Ganirelix (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. 14 Diedrich K, Felberbaum R. New approaches to ovarian stimulation. Hum Reprod (1998); 13:1–13. 15 Albano C, Smitz J, Camus M, Riethmuller-Winzen H, Van Steirteghem A, Devroey P. Comparison of different doses of gonadotropin releasing hormone antagonist Cetrorelix during controlled ovarian hyperstimulation. Fertil Steril (1997); 67:915–22. 16 Albano C, Felberbaum R, Smitz J, et al. On behalf of the European Cetrorelix Study Group. Ovarian stimulation with HMG: results of a prospective randomized phase III European study comparing the luteinizing hormonereleasing hormone (LHRH)-antagonist cetrorelix and the LHRH-agonist buserelin. Hum Reprod (2000); 3:526–31. 17 The European Orgalutran Study Group. Treatment with the gonadotrophin-releasing hormone antagonist ganirelix in women

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undergoing ovarian stimulation with recombinant follicle stimulating hormone is effective, safe and convenient: results of a controlled, randomized multicenter trial. Hum Reprod, in press. 18 de Jong D, Macklon NS, Fauser BC. A pilot study involving minimal ovarian stimulation for in vitro fertilization: extending the “folliclestimulating hormone window” combined with the gonadotropinreleasing hormone antagonist cetrorelix. Fertil Steril (2000); 73:1051– 4. 19 Edwards RG, Lobo R, Bouchard P. Time to revolutionize ovarian stimulation. Hum Reprod (1996); 11:917–9. 20 Bouchard P, Fauser BCJM. Gonadotropin-releasing hormone antagonist: new tools vs. old habits. Fertil Steril (2000); 73:18–20. 21 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- and FSH-deficient anovulatory women: a dose-finding study. J Clin Endocrinol Metab (1998); 83:1507–14. 22 Gordon K, Williams RF, Danforth DR, Hodgen GD. A novel regimen of gonadotropin-releasing hormone (GnRH) antagonist plus pulsatile GnRH: controlled restoration of gonadotropin secretion and ovulation induction. Fertil Steril (1990); 54:1140–5. 23 Gonen Y, Balakier H, Powell W, Casper RF. Use of gonadotropinreleasing hormone agonist to trigger follicular maturation for in vitro fertilization. J Clin Endocrinol Metab (1990); 71:918–22. 24 Tummon IS, Daniel SAJ, Kaplan BR, Nisker JA, Yuzpe AA. Randomized, prospective comparison of luteal leuprolide acetate and gonadotropins versus clomiphene citrate and gonadotropins in 408 first cycles of in vitro fertilization . Fertil Steril (1992); 58:563–8. 25 Dhont M, Onghena A, Coetsier T, De Sutter P. Prospective randomized study of clomiphene citrate and gonadotrophins versus goserelin and gonadotropins for follicular stimulation in assisted reproduction. Hum Reprod (1995); 10:791–6. 26 Craft I, Gorgy A, Hill J, Menon D, Podsiadly B. Will GnRH antagonists provide new hope for patients considered “difficult responders” to GnRH agonist protocols? Hum Reprod (1999); 14:2959–62. 27 Lenton EA, Cooke ID, Hoope M. In vitro fertilization in the natural cycle. Bailliere’s Clin Obstet Gynecol (1992); 6:629–45. 28 Seibel MM. The natural cycle for in vitro fertilization. In: Seibel MM, Blackwell RE, eds. Ovulation Induction (1994); New York: Raven Press: 223–8. 29 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.

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30 de Jong D, Macklon NS, Fauser BCJM. Minimal ovarian stimulation for IVF: extending the FSH window. In: Jansen R, Mortimer D, eds. Towards reproductive certainty: fertility and genetics beyond 1999. London: Parthenon Publishing (1999): 195–9. 31 Devroey P. GnRH antagonists. Fertil Steril (2000); 73:15–7. 32 Kol S, Lightman A, Hillensjo T, et al. High doses of gonadotrophinreleasing hormone antagonist in in vitro fertilization cycles do not adversely affect the outcome of subsequent freeze-thaw cycles. Hum Reprod (1999); 14:2242–4. 33 Homburg R, Levy T, Berkovitz D, et al. Gonadotropin-releasing hormone agonist reduces the miscarriage rate for pregnancies achieved in women with polycystic ovarian syndrome. Fertil Steril (1993); 59:527–31. 34 Tarlatzis BC, Grimbizis G, Pournaropoulos F, et al. The prognostic value of basal luteinizing hormone: folliclestimulating hormone ratio in the treatment of patients with polycystic ovarian syndrome by assisted reproduction techniques. Hum Reprod (1995); 10:2545–9.

41 Monitoring IVF cycles Matts Wikland, Torbjörn Hillensjö

INTRODUCTION Historically, monitoring of ovarian response, by means of measuring estrogen, came into the use for ovulation induction owing to the complications of gonadotrophin treatment. In ovulation induction cycles with gonadotrophins Klopper and co-workers showed that success rates and complication rates were not dependent on monitoring as such, but on the treatment protocol used. If more gonadotrophins are given, successes increase, as do complications. Monitoring merely gives us the possibility to descide how far we want to go.1 This is probably true for ovulation induction cycles but not for ovarian stimulation with gonadotrophins in ART cycles where the mature oocytes should not be ovulated. In these cycles the method for monitoring should be a clinically useful method to identify the optimal time for retrieving the mature oocytes. For this particular purpose ultrasound for monitoring follicular growth seemed to be a promising method. The method was first evaluated in the natural cycles by Hackeloer et al.2 The technique was soon evaluated for use in stimulated cycles.3 One major problem that soon was realized 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 estrogen and ultrasound monitoring of follicular maturation in stimulated cycles.6–9 This combination of ultrasound and hormonal monitoring seemed to be important in cycles stimulated with clomiphene citrate or gonadotrophins or both in combination in order to identify the optimal time for administration of human chorionic gonadotrophin (hCG) without a negative interference of a premature peak of luteinizing hormone (LH). With the introduction of gonadotrophin releasing hormone (GnRH) analogues combined with ovarian superovulation in cycles of assisted reproductive technologies (ART) the risk for high tonic levels of LH or premature LH peaks has disappeared.10 Thus, with the introduction of GnRH analogues or lately GnRH antagonists there seems to be no need for extensive hormonal monitoring of ART cycles.11 Ultrasound alone or in certain cases combined with one or two estrogen measurements seemed to be sufficient in most women entering an ART cycle.12 Thus, nowadays

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most groups performing ART use either ultrasound alone or a combination of ultrasound and estradiol measurements. In this chapter we will describe the use of a clinically simple method for monitoring ART cycles that has been used in our ART program for almost 10 years.

REASONS FOR MONITORING THE CYCLE Ovarian stimulation with gonadotrophins in ART cycles is performed merely for one reason. To achieve as many mature oocytes as possible in one stimulated cycle. The more mature oocytes can be retrieved in one ART cycle the better is the chance of having several good embryos to choose between for embryo transfer. Up to date available data from analysis of a large database clearly indicate that the more of high quality embryos in one stimulated cycle the better chance for a pregnancy.13 Thus when performing ovarian stimulation in ART cycles there are three obvious reasons why the cycle has to be monitored: (1) to evaluate if the dose of gonadotrophins is optimal; (2) to avoid ovarian hyperstimulation syndrome (OHSS); and (3) to find the optimal time for hCG administration. The monitoring of the ovarian response to hormonal stimulation in ART cycles will identify those who have not responded adequately or perhaps are poor responders as well as women at risk for ovarian hyperstimulation syndrome.14,15 Furthermore, at the end of the ovarian stimulation it is important to find out the optimal time to give hCG in order to achieve mature oocytes as well as endometrial maturity.

METHODS OF MONITORING FOLLICULAR MATURATION There are three principal ways of monitoring the follicular maturation in ART cycles: (1) estrogens alone; (2) ultrasound measurements of follicular diameter and endometrial thickness; and (3) ultrasound and estrogens combined. There is an extensive literature regarding the use of all the above methods for monitoring the ovarian response in ART cycles. Even though no study has shown that estrogens or ultrasound alone are superior to the other for monitoring follicular maturation in ART cycles, data in the literature show the advantage of using both ultrasound and estrogens as the method of choice for monitoring. It has been said that one method should be used for timing and the other to avoid complications.16 Which of the two methods

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the clinician relies most on for his or her decision to increase, decrease, or stop the gonadotrophin seems to be very much dependent on the clinician’s own experience and/or routines used at the clinic. Furthermore, there is no consensus about how often the monitoring has to be done during the ovarian stimulation. The number of times for monitoring seems to be arbitrarily chosen and thus varies considerably between different clinics. The literature include studies employing very simple as well as very complicated methods how to use estradiol and/or ultrasound for monitoring ART cycles with comparable pregnancy rates and complication rates. Thus, it seems preferable to choose a simple method such as ultrasound and combine it with estrogen measurements only when it seems clinically relevant such as in cases with poor response and in patients at risk for OHSS. ESTROGENS ALONE Estradiol measurements as the only method for monitoring ART cycles stimulated with gonadotrophins was mainly used in the early days of ART. The method for monitoring was based on the experience from monitoring ovulation induction cycles. Some groups have tried to identify a certain estrogen level that should be reached before hCG was given.17 Others have claimed that the number of days estradiol increased was important and thus gave hCG on the basis of that.18 Even though some groups are still using estradiol measurements as the sole monitoring system for ART cycles, most groups either combine estradiol measurements with ultrasound monitoring of follicular diameter or use the later alone. ULTRASOUND FOR MONITORING ART CYCLES Ultrasound monitoring of follicular diameter and endometrial thickness is a non-invasive 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 since it responds to increasing estradiol production. In our experience ultrasound is a very simple and reliable method for monitoring ovarian stimulation in ART cycles. Since the end of 1991, our group has used a monitoring system where ultrasound alone was used in most ART cycles. In women not at risk for OHSS or poor responders (evaluated 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 18mm (mean of two diameters) less than 15 follicles and an endometrial thickness of 7mm or more, hCG was given and oocyte pickup performed 36–38 hours later. If the follicles did not fulfill these criteria on the day of the scan, follicular growth rate of

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2mm/24 hours was calculated and hCG given accordingly. The 18mm diameter as well as the figure of 15 follicles were arbitrarily chosen. Retrospectively comparing 361 ART cycles performed during 1989, in which several ultrasound and serum estradiol measurements were used for monitoring, with 500 cycles performed during 1991 with the above simplified method, the take home baby rates were 17% and 26% respectively.19 In another retrospective analysis of our own data, using the same simplified method of a single ultrasound scan for monitoring follicular maturation, take home baby rates per started cycle of 31% and 1.8% of OHSS were achieved.11 Between 1991 and 1998 this simplified monitoring system has been used in 4826 ART cycles. During that period the take home baby rate per started cycle was 26% and the rate of mild OHSS 2.8%, indicating that it is possible to use a rather simple monitoring system while still achieving a good pregnancy rate and a low complication rate of OHSS. Our data as well as data from other studies indicate that it is possible to use a very simple monitoring method with ultrasound only in women not at risk for OHSS or previously known to be poor responders.14 The disadvantage of using a simple monitoring system as described above is that there is no possibility of increasing the dose of gonadotrophins early in the cycle. However, whether such an early increase of the dose means anything for the outcome (number of mature oocytes) or not has not yet been shown. If one believes that there is a need for early monitoring of the cycle it should probably be performed on stimulation day 5 or 6 since a response in follicular size or estradiol could not be expected much earlier in protocols with long pituitary downregulation with GnRH agonists. The major factor making it possible to apply a simplified monitoring system by ultrasound only is the use of a stimulation protocol including a GnRH agonist. MONITORING CYCLES WITH GnRH ANTAGONISTS GnRH antagonists have been clinically available for the use in ovarian stimulation cycles for a year now. However, the experience of how to best monitor such cycles is limited. Most studies so far published have adopted a monitoring system combining ultrasound, estrogen, and LH measurements.20 Ultrasound monitoring of GnRH antagonist cycles often starts on stimulation day 6 since that normally is the day for starting the GnRH antagonist (0.25mg) in a multiple dose protocol.21 If the largest follicles on stimulation day 6 is <12mm the day for starting to give the GnRH antagonist should be preferably delayed by one day. The reason for this is that starting too early with the antagonist may influence the further development of the follicles and the estrogen production. The risk of an LH peak to occur before the follicles are <12mm seems very small. Normally the next ultrasound is performed on stimulation day 9. If there

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are at least two follicles of >=18mm HCG is given on that day. If the follicles do not fulfill this criterion, a growth rate for the largest follicles of 2mm/24 hours is calculated. The day for giving HCG is based on such a calculation. When using the single dose protocol of GnRH antagonist (3mg) we also start monitoring with ultrasound on stimulation day 6, and if none of the follicles are greater than 12mm the next ultrasound is performed on stimulation day 8. On that day we give the antagonist if the largest follicle is >=14mm. Next ultrasound is performed on stimulation day 10. The criteria for giving HCG are the same as for the multiple dose protocol. In most cycles where GnRH antagonists are used we perform ultrasound monitoring only twice. Using ultrasound monitoring only once or occasionally twice is beneficial for the couple since that means less travelling, time off work, and time in hospital as well as less work for the clinic with the patient. All these advantages make the treatment less costly. COMBINING ULTRASOUND AND ESTROGENS Combining ultrasound and estrogens seems to be important in those women who are at risk for OHSS.22 In those women ultrasound alone can be difficult to relay on whether the simulation should be stopped or if it possible to coast the cycle.23 Furthermore, in women who on a particular day have smaller follicles than expected, measurement of their serum estradiol concentrations could be helpful in judging whether the gonadotrophin dose should be increased or not. Another situation where an early estradiol measurement can be valuable is to check that the chosen starting dose of gonadotrophin resulted in an adequate response. In such cases we analyse serum estradiol on stimulation day 5. If this serum estradiol is <700 pmol/l we increase the dose of gonadotrophin with one ampoule (75IU) and then perform the scan on stimulation day 9 or 10. This is a simple way of early finding out whether the starting dose has been sufficient.

CONCLUSION In our experience it is possible to use ultrasound as the main method and only in certain cases combine this with serum estradiol when monitoring ovarian stimulation with gonadotrophins in ART cycles. More trials are needed before a valid conclusion concerning the optimal monitoring regimen during antagonist cycles can be made.

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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 Frauenheilk (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 gonadotrophins. 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, Tureck RW, Mastroianni L Jr, Lessey BA. 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. Monitoring 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 gonadotrophin 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 sequentional use of clomiphene citrate and pulsatile human menopausal gonadotrophin. J Clin Endocrinol 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. Hum Reprod (1997); 12:2133–9.

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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, Ross C, Gosden C, Barlow D. Follicular monitoring and outcome of in vitro fertilization in gonadotrophin-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 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. The Parthenon Publishing Group (1993):23–7. 17 Wramsby H, Sundstrom P, Liedholm P. Pregnancy rate in relation to number of cleaved eggs replaced after invitro fertilization in stimulated cycles monitored by serum levels of oestradiol and progesterone as sole index. Hum Reprod (1987); 2:325–8. 18 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. 19 Wikland M. Vaginal ultrasound in assisted reproduction. In: Hamberger H, Wikland M, eds. Assisted reproduction (Bailliere’s Clinical obstetrics and Gynaecology); (1992); 2:283–96. 20 Borm G, Mannaerts B. The European Orgalutran study group: 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, multicentre trial. Hum Reprod (2000); 15:1490–8. 21 Ganirelix 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. 22 Forman R, Frydman R, Egan D. Severe ovarian hyperstimulation syndrome using agonists of gonadotropin-releasing hormone for in vitro fertilization: a European series and a proposal for prevention. Fertil Steril (1990); 55:502. 23 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.

42 Follicle aspiration Carl Wood

The initial studies on the maturation of human oocytes in vitro were carried out on oocytes that were obtained when ovaries or pieces of ovaries were acquired by laparotomy.1 By 1970, Steptoe and Edwards had developed a laparoscopic method for aspirating oocytes from their Graafian follicles, with a method that yielded oocytes from about one third of follicles.2 Initially, they used a needle and syringe to provide the suction, but later they developed an aspiration device, which provided continuous suction, with control being exerted by the assistant’s finger on the bypass valve. A similar technique, using a Venturi system activated by a foot operated “on-off” valve, was utilized by Lopata et al.3 Following the first few births from in vitro fertilization (IVF) and embryo transfer, attention was focused on the instruments used for oocyte recovery. Equipment, including a variety of needles and regulated aspiration pumps, became commercially available in the early 1980s: for example, a Teflon coated needle was developed that resulted in oocyte collection rates of >90%.4 The next major development was the change from laparoscopic to transvaginal ultrasound guided aspiration.5

EXPERIMENTAL AND PHYSICAL ASPECTS OF OOCYTE RETRIEVAL Apart from the comparison of manual and mechanical suction on the effect of zonal damage,6 surprisingly little has been published on the theory of oocyte collection until the studies performed by Cook Medical Technology, Brisbane.7 A number of factors may affect oocyte collection and/or damage the ova. These include variables such as pump vacuum flow, velocity, needle lumen size and length, follicular pressure and size, and collection techniques. In order to study factors influencing the success of oocyte collection and the cause of trauma to oocytes, Cook Medical Technology, Brisbane, developed appropriate equipment.7 Velocity and flow rate through the needle and attached lines were calculated from the pressure difference between the collection tube and the needle tip. The velocity and flow rate were slightly underestimated,

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especially at the moment the needle punctured the wall of the follicle. The studies were done on bovine follicles.

APPLICATION OF VACUUM TO A FOLLICLE VACUUM APPLIED AFTER NEEDLE ENTRY INTO THE FOLLICLE Upon application of vacuum, the vacuums throughout the system equilibrate to steady flow conditions, the period of time depending on the follicle volume, the vacuum used and the capacity of the needle. During this time the follicle wall collapses as the fluid volume decreases until the follicle totally collapses and blocks the needle tip. Maximum flow is achieved during the steady state then slows dramatically as the follicle collapses, blocking the needle tip. In some cases the fluid continues to flow very slowly up the needle, possibly owing to air being sucked into the follicle around the point of entry of the needle (the system was not fully closed). VACUUM DEACTIVATED BEFORE EXIT FROM THE FOLLICLE Changes in vacuum and flow occur if the regulated vacuum is discontinued whilst the needle tip is still in the follicle, providing the system remains closed (there are no air leaks). After the pump is deactivated and the pressure in the collection tube returns to atmospheric pressure, there is a back flow of fluid towards the follicle. The magnitude of the back flow depends on (i) how much air enters the system, and (ii) the height of the collection tube above the needle tip. VACUUM ACTIVATED AND DEACTIVATED OUTSIDE THE FOLLICLE A comparison of flow rates, during aspiration of a 15mm follicle, where both activation and deactivation of the vacuum occur outside the follicle shows that flow decreases as the follicle collapses but there is a sudden rapid flow, towards the collection tube, as the needle is withdrawn from the follicle.This may assist emptying the follicle. This may be the best technique.

DAMAGE TO OOCYTES The effects of flow rates and maximum velocities achieved in the aspiration system using a 17 gauge needle at various vacuums on the

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morphology of the oocyte cumulus mass shows that all oocytes had lost their cumulus mass after aspiration at 20kPa (150mm Hg).

VACUUM PROFILES IN ASPIRATION SYSTEM Usually, for oocyte collection, the vacuum in the collection system is <20kPa (150mm Hg). It is estimated that, using 20kPa (150mm Hg), it will take up to five seconds for the system described above to stabilize to the selected vacuum. Large follicles have a small positive pressure of 3.75–75mm Hg. Follicular pressure is dependent on the size (and hence the maturity), shape and position of the follicle, with the pressure increasing with increasing follicular size. The pressure of the fluid in the follicle at the moment of penetration of the needle may be much higher than the normal follicular pressure. As the needle tip is being forced into the wall, the deformation of the surface of the follicle will cause the pressure to rise. The more blunt the needle, the higher the pressure will become (up to 60mm Hg) and consequently, the larger the amount of fluid which spurts out of the follicle when punctured. Some of this fluid will flow up the needle, while the remainder escapes between the outer needle wall and the follicular wall. If a vacuum has already been applied before the needle punctures the follicle, little follicular fluid is lost.

FOLLICULAR AND NEEDLE VOLUMES With an increased interest in oocyte collection from immature follicles, it is important to note that the volume of such follicles is small (Fig 42.1). For example, an immature follicle with a diameter of 5mm has a volume of ~0.065ml. It would take the contents of over 17 such follicles to fill the lumen of a standard 16 gauge needle and line (total length 100cm).

APPLICATION OF VACUUM Once a needle punctures the follicle, the pressure within the follicle and needle equilibrates. The follicular wall will generally make a tentative seal around the needle and, in the absence of an applied vacuum, resistance within the needle will bring the fluid to rest. As the regulated vacuum is applied, the vacuums throughout the system tend to equilibrate to steady flow conditions. In the above situation, where a good seal exists around the needle tip, if the regulated vacuum is discontinued while the needle tip is still in the follicle, there is a back flow of

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Fig 42.1 Relation between diameter of follicle and volume of follicle. Arrows indicate the internal volume of standard 16-gauge and 17-gauge needles and lines (100cm length). fluid toward the follicle. In this closed system the magnitude of the reverse flow is similar to the maximum flow toward the collection tube but only lasts for a fraction of a second and slows rapidly. If the needle is withdrawn from the follicle while the vacuum is still applied, there is a dramatic surge of fluid toward the collection tube. The needle tip goes from the high vacuum of the follicle to atmospheric pressure. If the oocyte is contained in the last fraction of the collected follicular fluid, or comes from an immature follicle where the volume is small, it can be subjected to speeds well above those expected. It can also be subjected to increased turbulence in both the needle and collection tube.

DAMAGE WITHIN THE NEEDLE/VACUUM LINES There is a vacuum gradient down the collection system, with the vacuum at the needle tip being only 5% of the vacuum selected at the vacuum pump. The ovum is therefore exposed to an ever increasing vacuum during its travel along the collection system. This increased vacuum may cause the ovum to swell and the zona to crack. High velocities may strip the cumulus of the oocyte. Even in laminar flow, there will be a significant difference between the velocity of the fluid in the centre of the needle and that towards the periphery. Thus the outer layers of the cumulus may be subjected to “drag,” which may strip them. The longer the needle, or the smaller its internal diameter, the greater the vacuum required to maintain the same velocity and the greater the risk

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of damaging the oocyte. If turbulent flow is present the ovum may be tossed about, which could result in either stripping off of the cumulus or cracking of the zona.

DAMAGE WITHIN THE FOLLICLE The ovum has to be accelerated from a resting state within the follicle to the velocity of the fluid within the needle. Moreover, it has to accelerate to this velocity as it enters the needle tip. This rapid acceleration may strip off the cumulus. In theory, this damaging effect should be greatest in smaller follicles, especially immature follicles, where there may be some adherence of oocytes, necessitating the use of higher suction vacuums. Additionally, the oocyte will be drawn closer to the needle tip as the follicle collapses. This means that it could be subjected to an increasing accelerative force once it detaches from the wall. This may cause the cumulus to tear off from the oocyte. In addition, there is a rapid increase in vacuum at the needle tip, which may also affect the oocyte.

DAMAGE TO THE CUMULUS The above results indicate that an intact cumulus may be an important factor in the resistance of oocytes to damage. The morphology of cumulus is not changed after in-vitro aspiration at vacuums and velocities above those normally used in vivo, providing the cumulus is regular, compact and refractive. The cumulus is less resistant if it is damaged or degenerate.

CONCLUSION The above findings highlight two important issues relating to oocyte collection. Firstly, maintenance of suction: follicular fluid (and oocytes) may be lost if entry into and exit from the follicle are made in the absence of suction. This gain, however, may be offset by possible damage due to the dramatic forward flow of fluid toward the collection tube. Secondly, movement of the needle tip within the follicle: damage to the oocyte, particularly the cumulus, may occur because of collection technique. It is a common practice during oocyte collection to “spin” the needle within the follicle. It is possible that significant damage may occur as the oocyte is “scraped” from the follicular wall by the edge of the needle, particularly in small follicles or in the collapsed follicle, where the needle size becomes large compared to the follicular volume. There is a need to undertake further studies on the effect of needle movement in follicles on oocyte quality and subsequent blastocyst

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development. One possible solution, however, may be to combine flushing of follicles with lower suction vacuums.

CLINICAL ASPECTS OF OOCYTE RETRIEVAL TIMING OF OOCYTE RETRIEVAL The development of the ovarian follicles is monitored by vaginal ultrasound scanning of the ovaries and measurement of serum estradiol. The clinical criteria for administration of human chorionic gonadotrophin (hCG) will vary with the stimulation protocol. For instance, in gonadotrophin releasing hormone/follicle stimulating hormone (GnRH/FSH) stimulated cycles, a cohort of at least three follicles with a diameter of more than 17mm is required. In addition, serum estradiol concentrations should approximate 800–1000pmol/l per follicle. For natural cycles, on the other hand, one mature follicle is all that can be expected. When the criteria are met, the final maturation of the oocytes is initiated by an intramuscular injection of hCG (5000–10000IU) to mimic the endogenous luteinizing hormone (LH) surge. After the administration of hCG, the oocytes are expected to ovulate about 37 hours later. The oocyte retrieval is scheduled to precede ovulation, about 34–36 hours after the hCG injection. During this period cytoplasmic changes take place in the oocyte and meiosis is resumed. The intercellular cytoplasmic connections between the granulosa cells and the oocyte are interrupted. The cortical granules, vesicles synthesized by the Golgi-apparatus, migrate towards the oolemma. The prophase of the first meiotic division is resumed and the oocyte progresses to the metaphase of the second meiotic division at which stage it becomes arrested for the second time. During this process the nuclear envelope has broken down and the first polar body has been expelled.

EGG PICKUP TECHNIQUE ANAESTHESIA The use of analgesia and anaesthesia varies in different countries and different patients. Light anaesthesia is most acceptable as the patient is asleep, has no memory of the procedure, wakes within five minutes of completion of the oocyte collection, and is able to return home within one to two hours. Preoperative counselling and physical examination is necessary. Because it is a low risk surgical procedure routine checks have been occasionally omitted. The procedure should be cancelled or performed

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under local anaesthesia if an upper respiratory infection is present or a fever of unknown cause is evident.

Table 42.1. Anaesthetic protocol for IVF procedures. Fentanyl 1–2g/kg iv (average dose 100g) Midazolam 0.05–0.1mg/kg iv (average dose 2–5mg) If required, add Propofol 1–2mg/kg Monitor oxygen saturation and administer oxygen as indicated PREPARATION The success of oocyte retrieval is dependent on good visualization, accessibility of both ovaries, and on the materials and methods used for the oocyte collection. Pretreatment diagnostic laparoscopy will reveal difficult vaginal access to one or both ovaries and should be corrected prior to controlled ovarian hyperstimulation. When confronted with inoperable pelvic adhesions the feasibility of transvaginal retrieval is assessed by transvaginal ultrasound. Occasionally it may be necessary to perform laparoscopic oocyte pickup because the ovaries are adherent high on the lateral pelvic sidewall. Depending on the procedure (transvaginal or laparoscopic) and specific requests from patients the degree of anaesthesia may vary. For transvaginal procedures pain relief may be obtained with a paracervical block (for example, xylocaine or mepivacaine) or mild sedation (diazepam given intramuscularly or intravenously) in combination with opioid analgesics (pethidine hydrochloride). Alternatively, spinal or general anaesthesia may be used. One anaesthetic protocol is outlined in Table 42.1. An excellent degree of relaxation is obtained allowing for a quick and safe procedure, and immediate recovery at conclusion of the operation. On average, the oocyte retrieval takes no longer than 10 min minimizing the exposure of the oocytes to the anaesthetic agents. These pharmaceutical agents accumulate rapidly in follicular fluid during the procedure.8 There is little evidence that sedative and anaesthetic agents have an adverse effect on the postconceptional development of the exposed human oocyte.9 Even though the concentrations of these drugs in the follicular fluid are much lower than their serum concentrations, it is advisable to reduce the procedure time to a minimum.8

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MATERIALS CHECKLIST FOR TRANSVAGINAL OOCYTE RETRIEVAL (FIGS 42.2–6) Dry block heater with thermostat (Thermoline Scientific Equipment, Wetherill Park, Australia) Warm blocks (U-Lab, Melbourne, Australia) Falcon test tubes 2047 (Becton Dickinson, Sydney, Australia) Glass syringe with blunt needle (Lab Supply, Melbourne, Australia) Glass beaker (Lab Supply, Melbourne, Australia) Thermometers (Lab Supply, Melbourne, Australia) Automated pumps for flushing (William A Cook Australia, Brisbane, Australia) Aspirating needle, 17g, single/double lumen (William A Cook Australia, Brisbane, Australia) Suction pump with vacuum regulator (William A Cook Australia, Brisbane, Australia) Ultrasound scanner with a 7.5MHz transvaginal probe and needle guide (Acuson 128 and EV519 transducer; Acuson, Mountain View, CA, USA) K-Y Jelly (Johnson & Johnson, Arlington, TX, USA) Latex probe cover (G.E. Medical Systems, Melbourne, Australia) Heating plate covering the microscope stage (Thermoline Scientific Equipment, Wetherill Park, Australia) Microscope WILD MS (Leica, Heerburgg, Switzerland) 92×21mm petri dishes (Medos Company, Melbourne, Australia) Glass Pasteur pipettes (Crown Scientific, Melbourne, Australia) Small injection needles 25g (Terumo, Melbourne, Australia) Most materials in this list except the dry heater block are preheated at 37°C in a warming box. Immediately prior to the procedure, the heating stage with thermostat control is positioned on a trolley covered with a sterile drape. The heating stage, set at 37°C, is covered with a transparent plastic drape to minimize contact with textile fibres. The “warm blocks” with the test tubes, glass syringe, and glass beaker are placed in the heating stage. The thermometer is placed in one of the test tubes filled with handling medium and the temperature is checked and adjusted. The glass syringe and beaker are filled with handling medium and kept ready for flushing, remaining in the warm blocks at all times. Glass syringes are still used to avoid possible toxicity associated with rubber plungers and silicone lubricants. Automated pumps facilitate the flushing procedure. These pumps also provide preset flushing volumes and injection rates. The aspirating needle can be either a single or double lumen needle. The double lumen needle is useful when multiple follicle flushes are needed (for example, in natural cycle IVF). It must be remembered that the dead space volume of the single lumen needle and its tubing are approximately 1ml, and the oocyte may thus move backward and forward

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within this dead space during aspiration and flushing. The design of the double lumen needle eliminates this problem since the aspirating channel and the flushing channel are separated, ensuring a unidirectional flow in the aspirating channel. Prior to use, the aspiration needle and its Teflon tubing are flushed thoroughly with heparinized handling medium. The length of the Teflon tubing between the needle and the collecting test tube should be minimized to avoid unnecessary cooling of the oocytes. A pedal operated suction pump with vacuum regulator is used. A wide range of different models are now available. The maximum aspiration pressure is set at approximately −15kPa or −125mmHg.

RETRIEVAL TECHNIQUES LAPAROSCOPIC OOCYTE RETRIEVAL This technique may still be indicated when the ovaries are out of reach with the transvaginal approach either because of their elevated position in the pelvis or behind an enlarged uterus or when the ovaries are too mobile, rendering the transvaginal technique impossible or unsafe. This procedure requires general anesthesia and endotracheal intubation, which is a major disadvantage.

Fig 42.2 K-MAR-5100 aspiration unit (Cook IVF).

Fig 42.3 K-FTH 1012 Falcon tube heater and connecting tubing (Cook IVF).

Fig 42.4 Needle showing echo tipping to obtain a bright image on ultrasound (Cook IVF).

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Fig 42.5 Needle with different bevels (Cook IVF).

Fig 42.6 Handle on ovum pickup needle—handpiece allows rotation of needle tip to maximize fluid and oocyte collection (Cook IVF). The ovary is immobilized by holding it at the utero-ovarian ligament with a grasping forceps. Laparoscopic oocyte retrieval is only possible when the ovarian cortex is partly exposed and follicles are visible on the surface. After the follicles are identified, they are punctured with the aspiration needle, which has been flushed previously with heparinized flushing medium. To prevent the follicle wall from tearing and breaking the seal around the needle, the needle should be inserted, where the follicular wall is slightly thicker. While the needle remains in the follicle, the aspirate in the collecting tube is handed to the embryologist and checked for the presence of an oocyte. When no oocyte is identified the

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follicle may be gently flushed until the oocyte is retrieved. The procedure is repeated until all accessible oocytes are aspirated. ULTRASOUND GUIDED RETRIEVAL In our centre an Accuson 128 with a 7.5MHz transvaginal probe is used for all transvaginal oocyte retrievals. After applying the conducting jelly to the tip of the transducer, the transvaginal probe is covered with a latex probe cover. Care is taken to position the needle guide correctly onto the probe. Regular checks to make sure that the indicator line on the imaging screen actually coincides with the path of the aspirating needle are essential to the safety of the procedure. The needle tip is specially treated to enhance echogenicity and shows up as a bright spot on the monitor. During the procedure it should follow the indicator line. When a lot of lateral tension is exerted on the needle, it may bend and the needle tip may leave the path of the indicator line. In such circumstances, the needle may be withdrawn and the curvature corrected manually, or better still, the needle may be replaced. The ovaries are localized and lined up with the indicator line on the imaging screen. The follicle closest to the probe is entered with a short, controlled stabbing motion. A more progressive drilling motion may be indicated when follicles are localized at the posterior side of the ovary in the vicinity of major pelvic blood vessels or bowel loops. The needle tip is kept at the centre of the follicle while the follicle wall collapses around it. The operator should make sure that the follicle is completely emptied. Rotating the needle around the longitudinal axis may help drain small pockets of follicular fluid. When the follicle is aspirated, the follicular fluid is immediately sent to the embryologist. To reduce adverse effects of temperature fluctuations on the oocytes, the distance between the patient and the embryology lab should be minimal. Alternatively, transportation of the oocytes should be done with the collecting tubes in warm blocks. When follicles are flushed, the assistant injects the prewarmed heparinized handling medium through the Teflon tubing. The injected volume depends on the size of the follicle; to prevent rupture of the follicle it should not exceed the volume of the aspirated follicular fluid. While the medium is being injected, the operator can observe the follicle filling. In many cases only one puncture through the ovarian capsule is needed to aspirate all or most follicles: this greatly reduces the risk of postoperative haemoperitoneum. Many follicles may be aligned in the path of the needle to minimize the number of puncture wounds in the ovary. When all follicles in one ovary are aspirated, the needle is withdrawn and flushed by holding the needle tip in a test tube filled with heparinized handling medium. Subsequently, the vaginal vault is punctured a second time to reach the contralateral ovary and the procedure

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is repeated. When all follicles have been aspirated in both ovaries the pouch of Douglas is inspected for any fluid collection. Fluid is aspirated transvaginally and checked by the embryologist for the presence of spontaneously ovulated oocytes or oocytes lost at the time of puncture. When the pelvic aspirate contains mainly blood, this routine also helps to reduce postoperative pain caused by peritoneal irritation. After the needle is withdrawn it is flushed once again. All needle flushes need to be checked for oocytes. The vaginal vault is swabbed and checked for any bleeding. After each retrieval the transvaginal probe is cleaned with warm soapy water, rinsed, and dried. It is then soaked in 0.5% aqueous chlorhexidine for 10min. Ovarian endometriomas as usually visible on ultrasound when follicles are more than 15mm in diameter but may only be detected in smaller follicles when the follicular aspirate reveals chocolate coloured fluid. Similar fluid may sometimes be found in a haemorrhagic corpus luteum. The fluid aspirate may be embryotoxic and thorough washing of the needle and aspirating system should be carried out before another follicle is aspirated. If cleansing is difficult a new needle and aspirating set are used. If large endometriomas are aspirated at the time of egg pickup, an intravenous antibiotic is given to prevent the risk of pelvic infection which may occur secondary to chemical peritonitis resulting from peritoneal spill of endometriotic fluid. When the needle is in or close to the ovary, blood may be aspirated with follicular fluid. This may result from prior bleeding inside the follicle, which can be recognized by a speckled appearance on ultrasound, or by bleeding commencing after the needle punctures the wall of the follicle. Bleeding resulting from needle puncture is less likely using a sharp (new) needle and entering the follicle at right angles to the circumference of the follicle. Tearing of the wall of the follicle is less likely, as is loss of fluid from the follicle before effective aspiration. The needle should be kept in the centre of the follicle as contact between the needle and the inside of the wall of the follicle is avoided until the follicle is empty, reducing trauma, possible bleeding and blood in the aspirate. Sometimes pure venous or arterial blood is seen in the aspiration needle, indicating an ovarian vessel has been entered. The needle should be withdrawn and the needle and aspirating system flushed clean before reuse. Re-entry to the same follicle may be worthwhile when ultrasound indicates bleeding has stopped, and few follicles are available for oocyte retrieval. FOLLICULAR FLUSHING The value of follicular flushing is debatable.10,11 Its value is evident where low number of follicles are present such as in patients on a natural or a minimal stimulation (Clomid only) IVF cycle or in poor responders to

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controlled ovarian hyperstimulation. However, when more than 10 follicles have been recruited during controlled ovarian hyperstimulation, the benefits of flushing are less clear. Flushing all follicles prolongs the procedure considerably, increasing the patient’s discomfort and raising the overall cost of the procedure.12 Flushing all follicles up to six times may increase the yield by 20%. The time factor can be minimized by flushing all follicles only once. Interestingly, equal proportions of oocytes were found in the first aspirate and in the dead space of the needle and its tubing, indicating that the cumulus-oocyte complex is frequently aspirated when the follicle is almost completely collapsed.12 This underlines the importance of aspirating the complete content of the follicle. Both heparinized culture medium and heparinized normal saline can be used for follicular flushing. A randomized study13 has shown that heparinized normal saline is an equally good but cheaper and more convenient medium than standard heparinized culture medium and could replace it for flushing follicles during oocyte recovery for IVFET procedures.

EGG PICKUP TECHNIQUE-IMPORTANT POINTS • Clean vagina of particulate matter before needle entry, as reduces contamination of needle and vaginal bacterial count. • Vaginal ultrasound focused to maximize size of each follicle so needle can enter centre of the follicle. • Enter the follicle at its maximum diameter. • Aspiration commenced before enter follicle to prevent leakage. • Avoid excess aspiration pressure as cumulus may be torn from oocyte. • Flush follicles at low pressure. • Flush aspirating system after the first follicle is emptied to remove vaginal mucous or tissue. • An empty follicle is determined by (i) several ultrasound views, and (ii) observation of aspiration of the tube. • Aspiration is easier if the ovary is fixed by firm manual pressure with one hand—it reduces rotation of the ovary. • Operator observes both ultrasound picture and tubal aspirate to co-ordinate movement of ultrasound probe inside follicle.

EGG PICKUP-DIFFICULTIES • Transuterine needle puncture—minimize distance by manipulation of uterus or pressure on ovary—needle may bend or break. • Endometriosis fluid may be embryotoxic—leave endometriomas alone or aspirate endometriomas and flush cyst and needle repeatedly to clean. • Bleeding—ovarian vessels, remove needle, bleeding stops.

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— iliac vein remove needle gently, if rapid bleeding perform laparotomy. — vaginal bleeding apply pressure for 2 minutes; if bleeding continue suture. • Infection—Intravenous antibiotics if vaginal or cervical infection, pelvic infection in past history, bowel or pelvic adhesions.

COMPLICATIONS The ultrasound guided transvaginal technique is a very efficient and simple procedure. However, this should not distract from the fact that a number of potentially dangerous complications exist, consisting mainly of hemorrhage, trauma to pelvic anatomical structures, and infection.14 For a detailed review on the complications inherent with egg retrieval the reader is referred to Chapter 54.

REFERENCES 1 Edwards RG. Maturation in vitro of human ovarian oocytes. Lancet (1965); ii:926–9. 2 Steptoe PC, Edwards RG. Laparoscopic recovering of preovulatory human oocytes after priming of ovaries with gonadotrophins. Lancet (1970); ii:683–9. 3 Lopata A, Johnstone IWH, Leeton JF, Muchnicki D, Talbot TM, Wood C. Collection of human oocytes at laparoscopy and laparotomy. Fertil Steril (1974); 25:1030. 4 Renou P, Trounson AO, Wood C, Leeton JF. The collection of human oocytes for in vitro fertilization. I. An instrument for maximising oocyte recovering rate. Fertil Steril (1981); 35:409–12. 5 Feichtinger W, Kemeter P. Transvaginal sector scan sonography for needle guided transvaginal follicle aspiration and other applications in gynecologic routine and research. Fertil Steril (1986); 45:722–5. 6 Cohen J, Avery S, Campbell S, Mason BA, Riddle A, Sharma V. Follicular aspiration using a syringe suction system may damage zona pellucida. J In Vitro Fertil Embryo Transfer (1986); 4:224–6. 7 Horne R, Bishop CJ, Reeves G, Wood C, Kovaks GT. Aspiration of oocytes for in-vitro fertilization. Hum Reprod Update (1996); 2(1):77– 85. 8 Soussis I, Boyd O, Paraschos T, Duffy L, et al. Follicular fluid levels of midazolam, fentanyl and alfentanyl during transvaginal oocyte retrieval. Fertil Steril (1995); 64:1003. 9 Coetsier T, Dhont M, DeSuter PM, et al. Propofol anaesthesia for ultrasound guided oocyte retrieval: accumulation of the anaesthetic agent in follicular fluid. Hum Reprod (1992); 7:1422.

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10 Kingsland CR, Taylor CT, Aziz N, Bickerton N. Is follicular flushing necessary for oocyte retrieval? A randomized trial. Hum Reprod (1991); 6:382. 11 Tan SL, Waterstone J, Wren M, Parsons J. A prospective randomized study comparing aspiration only with aspiration and flushing for transvaginal ultrasound-directed oocyte recovery. Fertil Steril (1992); 58:356–60. 12 El Hussein E, Balen AH, Tan SL. A prospective study comparing the outcome of oocytes retrieved in the aspirate with those retrieved in the flush during transvaginal ultrasound directed oocyte recovery for invitro fertilization. Br J Obstet Gynaecol (1992); 99:841. 13 Biljan MM, Dean N, Hemmings R, Bissonnette F, Tan SL. Prospective randomized trial of the effect of two flushing media on oocyte collection and fertilization rates after in vitro fertilization. Fertil Steril (1997); 68:1132–4. 14 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.

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. After a review of the differences between natural cycles and those seen in assisted reproductive technologies (ART) (stimulated and programmed), the elements (E and P), timing, and route of replacement are reviewed. Lastly, standard protocols for replacement are provided. Progesterone (P) and estradiol (E) 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 peak about four days after ovulation and continue 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 one to four hours, with measured levels ranging between 4 and 20ng/ml during peak production. This P production is enormous: it is 40-fold maximal E production, some 25mg daily versus 0.6mg for E. In normal cycles, P and E production wanes about 10 days after ovulation. Menses follows that event about four 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 surgically removing the corpus luteum in early pregnancy. Classic work by Csapo showed that luteectomy led to miscarriage in almost every case if performed before seven weeks of gestational age, and almost never if performed after that time.5

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Fig 43.1 E and P levels in normal cycles. 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 (adapted from reference 3).

THE SPECIAL PROBLEM OF THE LUTEAL PHASE AFTER OVARIAN STIMULATION In stimulated cycles typical of in vitro fertilization treatment, the luteal phase is different from the natural 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. Second, and perhaps more importantly, the duration of ovarian steroid production in stimulated cycles is usually shorter than normal by one to three days. This truncated luteal phase has been noted since the earliest days of IVF (Fig 43.2),6 and created concern that an early menses might prevent a successful implantation, since menses were on occasion observed to occur as early as 10 days after egg retrieval. Moreover, the decline of serum E and P concentrations is also more abrupt compared with the rate of fall in natural cycles (Figs 43.1 and 43.2). This early and rapid fall was the reason luteal support was adopted in the early days of IVF. With the advent of gonadotropin releasing hormone (GnRH) agonist use in the late

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1980s, the problem of the short luteal phase became even more common. Multiple studies show the importance of some form of luteal support in such cycles.7–10 The supraphysiological E and P concentrations 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.11–13 Increased uterine contractions in high responding women at the time of embryo transfer have been associated with lower pregnancy rate.14,15

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

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supplementation. (With permission from reference 6.) THE SPECIAL PROBLEM OF PROGRAMMED CYCLES While the success of donor egg therapy has clearly shown 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” on histological appearance. Since the normal cycle has a small amount of pre-ovulatory P production, it is possible that the dyssynchrony is due to the omission of this P in the typical programmed cycle regimens. In fact, deZiegler suggests that providing a small amount of P in programmed cycles eliminates this disparity.16 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.16 Whether correction of this disparity increases pregnancy rates is untested.

Table 43.1. Relation between the duration of P treatment before embryo transfer and the subsequent pregnancy rate17. # days after P started “Cycle day” No Implantation rate (%) Pregnancy rate (%) 2 16 18 0 0 3 17 25 3.5 12 4 18 40 14.1 40 5 19 60 15.8 48 6 20 49 5.6 20 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 week wide window. In programmed cycles, however, the timing is critical, since the only source of sufficient P is exogenous. Navot et al showed that two day old embryos transferred on the second through fourth day of P therapy produced pregnancies, while transfers out of that window did not.1 A larger study confirmed the importance of the timing of P therapy.17 In this study, embryos from donor eggs were transferred into recipients 44 to 48 hours after egg retrieval. Recipients had a standard form of E and P replacement, but the day on which P was begun was variable. The highest pregnancy rates occurred when two day old embryos were transferred on

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the fourth or fifth day of P therapy, a bit longer than observed by Navot.1 This corresponds to beginning P on the day the donor got hCG or the day afterwards, but before egg retrieval. It suggests that a longer exposure than is natural might improve the odds for pregnancy. But even if one is not willing to start P that early in clinical practice, it is nonetheless very clear that there is a strong effect of timing of P replacement on pregnancy potential in programmed cycles. 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 likely 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) is a superior form of luteal support as compared to P alone, while P alone is in turn better than no such luteal support.8 However, given the increased risk of ovarian hyperstimulation syndrome that hCG produces, use of hCG to provide combined E and P secretion has not been widely adopted. If the superiority of hCG over P is in fact due to its ability to stimulate E production, then simple replacement of E in addition to P might be helpful. A recent study supports this hypothesis.18 High responding patients (>2,500pg/mL E at time of hCG) pretreated with a long GnRH agonist protocol were randomized to receive either P alone (50mg p.v. bid and 50mg i.m. qd) or both E (2mg p.o. 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. ROUTE OF SUPPORT Possible routes of P delivery include transdermal, oral, intramuscular, transvaginal, sublingual, nasal, and rectal. Of these, only three—oral,

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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 administration (Fig 43.3)19 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.20 Any effort to increase the oral dose sufficiently to achieve the requisite serum P concentrations produces a degree of somnolence unacceptable to most patients. 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. 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 P delivery in the US for ART cycles. The IM route delivers P at relatively high efficiency and without the

Table 43.2. The effect of oral versus non-oral P supplementation on clinical outcomes in IVF cycles. Note the poorer outcomes after oral use in both studies. Study Regimens Clinical pregnancy Implantation rate Miscarriage rate rate (%) (%) (%) Licciardi et IM 50mg qd 58 41 n/a al., Oral 200mg 46 18 n/a 19999 TID Freidler et Vag 100mg 47 31 13 al., BID Oral 200mg 33 11 40 199910 QID

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Fig 43.3 Serum levels of P after oral (100mg) versus vaginal (90mg) administration, as measured by standard radioimmunoassay (RIA) or liquid chromatography-mass spectrometry (true). Note that the oral use has a short half-life, and is over-reported by RIA. Alternatively, vaginal use yields a longer half-life and a more accurate representation of the true amount of P in the circulation. (Adapted from reference 19.) loss encountered with the oral route as a result of hepatic “first pass” metabolism. However, the IM 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. The usual IM dosing is from 25mg to 100mg daily, sometimes in divided doses. This regimen produces peak serum P levels that can be well above the physiologic range. Endometrial architecture has generally

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shown appropriate “in phase” development, and pregnancy and miscarriage rates have seemed to be “normal”.21,22 It is useful to note that IM P at the usual doses is able to delay menses in most women. VAGINAL ROUTE The vaginal route offers several important advantages over IM 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 micronized P tablets, pharmacist-formulated 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.

Fig 43.4 Comparison of serum P levels and endometrial development during 3 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.

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Initial trials comparing various routes of P administration in ART cycles supported the advantages of vaginal therapy. Devroey et al in a series of studies demonstrated that vaginal therapy was at least as good (if not better than) IM replacement, and clearly better than oral (Fig 43.4).21– 23 This was borne out in endometrial histology, but more importantly, pregnancy and miscarriage rates, and in both IVF and Donor Egg cycles. And paradoxically, this superiority in clinical outcomes was observed even though serum progesterone levels were abnormally low.24 This led to the demonstration of a “targeted drug delivery” from vagina to uterus.25–27 Based on these findings, the group in Belgium adopted vaginal therapy and has continued to use it ever since (as Utrogestan (Prometrium in the US) at a dose of 200mg three times daily). In the US, the first and only FDA approved system for pregnancy support is Crinone 8%. It is a bioadhesive vaginal gel containing 90mg 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 90mg is about four times more than the dose at which endometrial development is not satisfactory. Much higher doses of micronized P capsules (Prometrium or Utrogestan, 200mg thrice daily) given vaginally are required to produce the same endometrial effects.

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

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fall after oral use, and the prolonged levels after Crinone use.

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.

Table 43.3. Comparison of clinical outcomes at the Jones Institute Donor Egg program using different means of P replacement in the years 1996 through 1998. No differences were apparent, whether using IM P, twice daily Crinone, or once daily Crinone.29,30 Crinone 8% twice Crinone 8% once IM P 50mg once daily daily daily N 54 46 249 Clinical 48% 46% 41% pregnancy Miscarriage 33% 14% 25% Ongoing 33% 40% 31% pregnancy Implantation rate 20% 22% 21% 31 A large, multicenter experience is also reassuring (Table 43.4).

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Thus the advantages of Crinone over other vaginal therapies are a longer half-life (Fig 43.5) and lower patient-to-patient variability in absorption (Fig 43.6).14 Clinical trials of Crinone have been encouraging to date. While an occasional report has been unfavorable,28 other published experiences have been favourable: in donor egg cycles at the Jones Institute, reassuring pregnancy and miscarriage rates were seen at both the twice and once daily dosing levels as compared to IM therapy (Table 43.3).29,30 PECULIARITIES OF VAGINAL PROGESTERONE THERAPY BLEEDING While most US practitioners with experience in IM P therapy have come to expect that menses will be delayed as long as P therapy continues, 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. And the timing of the bleeding is “physiological”; it comes when it normally does in natural cycles that are not conception cycles. Roman et al33 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 three 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 pruritis 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. 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.

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INTERCOURSE There is no prohibition from coital activity during the use of vaginal P products. Though there may be some P absorption by the male through penile exposure, it carries no known or suspected risks.

Table 43.4. Interim analysis of multicenter trial of Crinone 8% in IVF.31 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.32 Also note the low rate of miscarriage once a sac was documented (<10%). Age N Clinical pregnancy Ongoing pregnancy <35 605 39.7% 35.0% 35–39 437 34.6% 30.7% 40+ 142 16.9% 14.8% Total 1184 35.1% 31.0% SART 199732 4801 33.6% –

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Fig 43.7 Histogram of the frequency of first bleeding after various luteal days in IVF cycles supplemented with vaginal P (Utrogestan 200mg 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. PROGESTERONE OPTIONS Based on these considerations, I believe the following regimens to be equally effective in endometrial preparation and hence pregnancy support. 1. Progesterone in oil 50mg intramuscularly, once daily.

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2. Crinone 8% vaginally, once or twice daily. 3. Prometrium 200mg vaginally, three or four times daily. I believe practitioners could 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, although support is probably not needed until five to seven days after retrieval. The evidence that hCG (or estradiol replacement) is superior to P supplementation alone is not conclusive but is sensible, so 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 five days after) egg retrieval and continuing until pregnancy testing some 14 days after egg retrieval, or, • hCG every three to five days throughout luteal phase (in cycles at low risk of OHSS)

Fig 43.8 In stimulated cycles, progesterone can be started from as soon as the day of egg retrieval to as late as 1 week later. The optimal timing has not been defined.

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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 be varied from as short as seven days to as long as 35 days without ill effects.34,35 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 gets her hCG until the day the donor has her egg retrieval; similar pregnancy rates were observed across this range of days. In our program, we have traditionally begun P the day of egg retrieval (conventionally designated “day 14” (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. We have conventionally called the day progesterone replacement is begun in such cycles “day 15,” in recognition of the fact that significant P secretion does not begin in natural cycles until the day after ovulation (thus day 15; Fig 43.10). Based on this naming, we expect to achieve synchrony between embryos and endometrium when: • we thaw embryos frozen one day after retrieval (prezygotes) on day 16, and transfer them on day 17 (or later);

Fig 43.9

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In programmed cycles of donor egg recipients, progesterone is most often begun about the time of the donor’s egg retrieval. Good results have been reported with progesterone started as early as the day of the donor’s hCG injection.

Fig 43.10 In programmed cycles for transfer of cryopreserved embryos, progesterone is most often begun according to the day the embryos were frozen. For example, when embryos were frozen as zygotes, progesterone has conventionally been started the day before the thaw. There is some evidence that earlier starts (by a day or even 2) may be equivalent or perhaps even superior. • we thaw embryos frozen two days after retrieval (two to four cells) on day 17, and transfer them on that day (or later); • we thaw embryos frozen three days after retrieval (four to eight cells) on day 18, and transfer them on that day (or later); • we thaw embryos frozen five days after retrieval (blastocysts) on day 20, and transfer them on that day.

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SUMMARY P and E play central parts in preparation for and maintenance of human pregnancy. Until the luteoplacental shift occurs at about seven weeks of gestational age, the ovary’s production of these hormones is critical to pregnancy maintenance. Beyond seven 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 IM delivery 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. Aside from its high patient acceptability, it also seems to offer more “targeted” delivery of progesterone to the uterus.

REFERENCES 1 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(13):806–11. 2 De Ziegler D, Cornel C, Bergeron C, Hazout A, Bouchard P, Frydman R. Controlled preparation of the endometrium with exogenous estradiol and progesterone in women having functioning ovaries. Fertil Steril (1991); 56:851–5. 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 Endo 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 Al, Pulkkinen MO, Rutter B, Sauvage JP, Wiest WG. 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 Hutchinson-Williams K, DeCherney AH, Lavy G, Diamond MP, Naftolin F, Lunenfeld B. Luteal rescue in in vitro fertilization-embryo transfer. Fertil Steril (1990); 53:495–9. 8 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. 9 Licciardi RL, Kwiatkowski A, Noyes NL, Berkeley AS, Krey LL, Grifo JA. Oral vs. intramuscular progesterone for in vitro fertilization: a prospective randomized study. Fertil Steril (1999); 71:614–8.

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10 Friedler S, Raziel A, Schachter M, Strassburger D, Bukovsky I, Ron-El R. Luteal support with micronized progesterone following in-vitro fertilization using a down-regulation protocol with gonadotropinreleasing hormone agonist: a comparative study between vaginal and oral administration. Hum Reprod (1999); 14:1944–8. 11 Toner JP, Hassiakos DK, Muasher SJ, Hsiu JG, Jones HW Jr. Endometrial receptivities after leuprolide suppression and gonadotropin stimulation: histology, steroid receptor concentrations, and implantation rates. NY Acad Sci (1991); 622:220–9. 12 Toner JP, Singer GA, Jones HW Jr. Uterine receptivity after ovarian stimulation for assisted reproduction. In: Implantation in Mammals. Giarardi L, Compara A, Trounson AO, eds. New York; Serono Symposia Publications/Raven Press (1993); 9:231–8. 13 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. 14 Abramowicz JS, Archer DF. Uterine endometrial peristalsis—a transvaginal ultrasound study. Fertil Steril (1999); 54:451–4. 15 Fanchin R, Righini C, Olivennes F, Taylor F, de Ziegler D, Fryman R. Uterine contractions as visualized by ultrasound after pregnancy rates in IVF and embryo transfer. Hum Reprod (1998); 13:1968–74. 16 de Ziegler D, Fanchin R, Massonneau M, Bergeron C, Frydman R, Bouchard P. Hormonal control of endometrial receptivity; the egg donation model and controlled ovarian hyperstimulation. Ann NY Acad Sci (1994); 734:209–220. 17 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. 18 Farhi J, Weissman A, Steinfeld Z, Shorer M, Nahum H, Levran D. 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. 19 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. 20 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. 21 Devroey P, Palermo G, Bourgain C, Van Waesberghe L, Smitz J, Van Steirteghem AC. Progesterone administration in patients with absent ovaries. Int J Fertil (1989); 34:188–93. 22 Smitz J, Devroey P, Faguer B, Bourgain C, Camus M, Van Steirteghem A. A prospective randomized comparison of intramuscular

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or intravaginal natural progesterone as a luteal phase and early pregnancy supplement. Hum Reprod (1992); 7:168–75. 23 Bourgain C, Devroey P, Van Waesberghe L, Smitz J, Van Steirteghem AC. Effects of natural progesterone on the morphology of the endometrium in patients with primary ovarian failure. Hum Reprod (1990); 5:537–43. 24 Miles R, Paulson R, Lobo R, Press M, Dahmoush L, Sauer M. Pharmacokinetics and endometrial tissue levels of progesterone after administration by intramuscular and vaginal routes: a comparative study. Fertil Steril (1994); 62:485–90. 25 Bulletti C, de Ziegler D, Flamigni C, Giacomucci E, Polli V, Bolelli G, Franceschetti F. Targeted drug delivery in gynaecology: the first uterine pass effect. Hum Reprod (1992); 12:1073–9. 26 Cicinelli E, DeZiegler D, Bulletti C, Matteo MG, Schonauer LM, Galantino P. Direct transport of progesterone from vagina to uterus. Obstet Gynecol (2000); 95:403–6. 27 Fanchin R, de Ziegler D, Bergeron C, Righini C, Torrisi C, Frydman R. Transvaginal administration of progesterone: dose-response data support a first uterine pass effect. Obstet Gynecol (1997); 90:396–401. 28 Damario MA, Goudas VT, Session DR, Hammitt DG, Dumesic DA. Crinone 8% vaginal progesterone gel results in lower embryonic implantation efficiency after in vitro fertilization-embryo transfer. Fertil Steril (1999); 72:830–6. 29 Gibbons WE, Toner JP, Hamacher P. Experience with a novel vaginal progesterone preparation in a donor oocyte program. Fertil Steril (1998); 69:96–101. 30 Jobanputra K, Toner JP, Denoncourt R, Gibbons WE. Crinone 8% (90mg) given once daily for progesterone replacement therapy in donor egg cycles. Fertil Steril (1999); 72:980–4. 31 Levine H. Luteal support from the vaginal progesterone (P) gel Crinone 8%: preliminary results of multicenter trial show higher pregnancy rates than historical controls. Poster #571, presented at the 47th annual meeting of the Society for Gynecological Investigation, March 22–25, 2000, Chicago, IL. 32 Assisted reproductive technology in the United States: 1997 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril (2000); 74:641–53. 33 Roman E, Aytoz A, Smitz JE, Faguer B, Camus M, Van Steirteghem A, Devroey P. Analysis of the bleeding pattern in assisted reproductive cycles with luteal phase supplementation using vaginal micronized progesterone. Hum Reprod (2000); 15:1435–9. 34 Yaron Y, Amit A, Mani A, Yovel I, Kogosowski A, Peyser M, David M, Lessing J. Uterine preparation with estrogen for oocyte donation: assessing the effect of treatment duration on pregnancy rates. Fertil Steril (1995); 63:1284–6.

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35 Younis J, Simon A, Laufer N. Endometrial preparation: lessons from oocyte donation. Fertil Steril (1996); 66:873–84.

44 Evaluation and treatment of the low responder patient Richard T Scott Jr

One of the greatest challenges in clinical reproductive endocrinology is evaluation and management of patients who respond poorly to exogenous gonadotropins and who are categorized as “low responders”. Identifying potential low responders is of critical clinical importance. These patients require specialized management to optimize the number and quality of oocytes that may be available for assisted reproductive technologies (ART) procedures. Even with optimal management, their clinical pregnancy and delivery rates are diminished compared with age matched controls, and patients should be counseled accordingly. This chapter will review how low responders are defined, what screening tests are available to predict diminished gonadotropin responsiveness and oocyte quality, and basic treatment protocols that may be used to optimize outcomes for this difficult group of patients.

PHYSIOLOGIC CHANGES IN OVARIAN FUNCTION WITH AGE Virtually all clinicians are aware of the age related diminution in reproductive potential. As women become older, their chances for becoming pregnant decline. A detectable decrease in reproductive efficiency is present by the time women are in their late twenties and very few women are able to conceive beyond their mid-forties. This age related decline in reproductive function has several important characteristics. First, it is related principally to changes at the oocyte level. Navot et al, using the donor egg model, have showed that there is little if any diminution in implantation rates with increasing “uterine” age.1 In a study where donor oocytes from the same cohort were divided equally between a young and an older recipient, there were no differences in implantation or delivery rates. One of the most critical aspects to understanding the impact of the age related diminution in ovarian reproductive function is the difference between a decline in quantitative ovarian responsiveness (a low responder) and a decline in ovarian reserve (low quality oocytes with very poor potential to produce a viable pregnancy). These two factors are closely related but not identical, and the screening tests available to assess ovarian reserve (quality) and ovarian responsiveness (quantity) will be presented separately.

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DEFINING LOW RESPONDERS There is no consensus in the literature on what defines a low responder.2–4 This probably reflects the fact that differing levels of responsiveness may be considered normal at different ages. In spite of that, the production of less than four mature follicles at the time of administration of human chorionic gonadotropin (hCG) or a peak estradiol (E2) of less than 500pg/ml has been used by many authors and is a good general definition. This definition, even if accepted, may be difficult to apply. It is not uncommon to have a patient with more than four mature follicles produce less than 500pg/ml of E2, especially in the era of pure recombinant follicle stimulating hormone (FSH) stimulations. Similarly, some patients will have peak E2 concentrations over 500pg/ml while producing less than four mature follicles.

TESTS THAT PREDICT OVARIAN RESERVE (PREDICTIVE VALUE IS FOR IMPLANTATION AND DELIVERY RATES) BASAL FSH CONCENTRATIONS AGE RELATED CHANGES IN FSH LEVELS A series of studies in the 1970s and 1980s characterized the endocrinologic aspects of the transition through the climacteric. Sherman and Korenman documented that women with normal ovulatory cycles commonly begin having subtle elevations in their FSH levels beginning in their mid-30s.5,6 Other authors confirmed these findings, consistently showing that the first elevations occur in the early follicular phase.7,8 Although these studies did not evaluate the relation between FSH levels and ovarian reserve (pregnancy potential), they documented that FSH concentrations increase around the same general time that the incidence of diminished ovarian reserve increases. These data provided the initial background data when evaluating various forms of FSH screening. The elevations in FSH concentrations are not accompanied by changes in circulating E2 or progesterone (P) levels throughout the menstrual cycle.9,10 Thus 19 and 49 year old women with regular menstrual cycles have comparable E2 and P levels through the various phases of their cycles. Thus there is no lack of hormonal support of the endometrium to explain the age related decline in fertility. Batista et al evaluated LH, FSH, E2, inhibin, P, PP-14, and endometrial biopsies (EMB) in younger (age 20–30) and older (age 40–50) volunteers.11 FSH concentrations were increased and inhibin levels (Monash assay) were decreased in the older group. None of the other variables were different. Further studies could not find any decrease in E2,

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P, LH, or inhibin (Monash assay) levels with reproductive ageing. The same investigators also measured the 24 hour mean FSH and LH levels in the early follicular and midluteal phases of the cycle in both the younger and the older groups. The 24-hour mean FSH concentrations were significantly higher in the older group in both the phases of the cycle compared with the younger group, whereas no differences were noted for LH. BASAL DAY 3 FSH CONCENTRATIONS AND PREGNANCY RATES Scott et al found in a large retrospective study of 758 IVF cycles that pregnancy rates decreased markedly as basal day 3 FSH concentrations rose.12 Ongoing pregnancy rates were less than 2% in those whose basal FSH levels exceeded 25IU/I (Fig 44.1). This reduction in pregnancy rates was attributed to changes in ovarian function since these patients developed fewer follicles, produced fewer oocytes, and had fewer embryos transferred. Although the relation with quantitative aspects of ovarian function was clear, the dramatic decline in implantation rates (approximately 10% v <0.1%) indicated that the most important differences were qualitative in nature. Importantly, age would not have predicted the differences in clinical response since the ages of the women in the different groups were equivalent (mean age of 35 years).

Fig 44.1 Elevated basal day 3 FSH levels prognosticate very low pregnancy rates. (Data from reference 12.) A further study from the same center evaluated the relative predictive values of basal FSH concentrations and age in 1478 consecutive IVF

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cycles (Fig 44.2).13 Although there was a decline in pregnancy rates as age increased, basal FSH levels provided much better predictive values for pregnancy and cancellation rates. INTERCYCLE VARIABILITY IN BASAL FSH LEVELS It has been established for over three decades that basal FSH concentrations vary dramatically from cycle to cycle. The original research on basal FSH concentrations evaluated pregnancy rates in stimulated cycles that immediately followed measurement of the FSH concentrations. It seemed possible that patients might have better outcomes in cycles where their basal FSH concentrations were normal— even if they had had an elevated concentration in the past. The question arose whether women should be followed serially and stimulated in the cycle where their basal FSH concentrations were normal. Scott et al evaluated this question in women undergoing multiple in vitro fertilization (IVF) cycles who had FSH concentrations that varied in and out of the normal range (they had at least one normal and at least one elevated FSH concentration).14 Significant variability in basal FSH concentrations did occur with a range that extended from <1IU/I to 42IU/I. A paired analysis of the high and low FSH cycles in these patients found no differences in stimulation quality, the number of oocytes retrieved, or fertilization rates. Interestingly, the patients all behaved as low responders in both cycles. These data indicate that by the time patients develop higher variability in their basal FSH concentrations, they have already had a significant diminution in their ovarian reserve. Furthermore, it is strongly suggested that serial screening of FSH concentrations to select the optimal cycle for stimulation is of limited clinical value.

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Fig 44.2 Basal day 3 FSH levels (A) are more predictive of pregnancy rates and outcome than age (B). (Data from reference 13.) Martin et al have reported a long term study that evaluated the value of repetitive basal FSH screening.15 They noted that the pregnancy rate with a single abnormal and with all others being normal was 5%. Two or more abnormal results had a 0% pregnancy rate. These data initially seem in contrast to these reported above. However, the authors note that if the FSH threshold value was slightly higher (to a level generally in keeping with that used by most programs), that there would be no ongoing pregnancies in the group with even a single elevated basal FSH value (Fig 44.3).

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BASAL FSH SCREENING IN WOMEN WITH ONE OVARY Khalifa et al compared the basal FSH concentrations in women with one or two ovaries and evaluated the predictive values of the test in each group.16 The 162 women with one ovary had higher mean basal FSH concentrations and correspondingly had a poorer response to gonadotropin stimulation than the 1066 patients with two ovaries. However, after controlling for basal FSH level, there were no differences in gonadotropin responsiveness or pregnancy and delivery rates. These data indicate that basal FSH screening retains its predictive value in women with one ovary, even when using the same thresholds for defining an abnormal test. ESTRADIOL CONCENTRATIONS AND BASAL DAY 3 FSH CONCENTRATIONS The validity of FSH screening is dependent on the time in the cycle the sample is collected. Timing is considered optimal when circulating E2 concentrations are at their nadir, typically around cycle day 3. Some patients will have inappropriately high E2 concentrations on day 3 suggesting that they may be further into their follicular phase than is clinically apparent. In these circumstances, it is possible that the higher E2 concentration might suppress FSH concentrations back into the normal range, thus masking an abnormal result in a patient who has diminished ovarian reserve.12,17 The original studies evaluated the relation between basal concentrations of E2 and FSH in an effort to determine if there was a threshold value above the predictive value of a normal FSH concentration was lost. No such threshold value could be identified. Several investigators have revisited this question. Licciardi et al determined that progressive increases in basal day 3 E2 concentrations were associated with declining ovarian responsiveness and pregnancy rates.18 However, after controlling for FSH concentrations there was no difference in pregnancy rates in women with normal or “elevated” basal E2 levels. Thus, the authors did not clearly show that the E2 concentrations added information beyond that seen with FSH concentrations alone. In contrast, Smotrich et al were able to demonstrate a significant decline in pregnancy rates with elevated E2 levels, even after controlling for FSH concentrations.19 These authors chose a higher threshold E2 value for defining a significant elevation (80pg/ml compared with either 45 or 75pg/ml in Licciardi’s paper) although no data regarding the comparability of the assays are available.

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Fig 44.3 Elevated day 3 FSH levels on a single occasion prognosticate poor pregnancy rates and outcome. (Data from reference 15.)

Fig 44.4 Basal day 3 E2 levels do not predict pregnancy rates or outcomes in women with normal day 3 FSH levels. (Data from reference 20.) In the largest study to date, Frattarelli et al have recently evaluated the relation between basal E2 concentrations and IVF outcome in over 800 IVF cycles (Fig 44.4).20 As long as the basal FSH concentrations were normal, basal E2 concentrations did not predict pregnancy rates or

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outcome. This topic remains controversial and awaits data from other centers. BASAL FSH SCREENING VERSUS OVARIAN RESPONSIVENESS It is tempting to equate elevations in basal FSH concentrations with the diminished ovarian responsiveness to gonadotropin stimulation. We have recently evaluated this question in 141 patients who have cycled in our program. Although most patients with elevated levels were low responders, some had normal or even high responsiveness to gonadotropin to stimulation. Unfortunately, there was a very poor pregnancy rate in all patients independent of their ovarian responsiveness (Scott, unpublished data) (Fig 44.5). CURRENT STATUS OF BASAL FSH SCREENING Elevated basal day 3 FSH concentrations are highly predictive of diminished ovarian reserve as defined by poor gonadotropin responsiveness and pregnancy rates in patients undergoing complex ovulation induction or one of the assisted reproductive technologies. The test is simple, inexpensive, and routinely available. The studies performed to date are limited to clinical circumstances requiring complex ovulation induction.

THE CLOMIPHENE CITRATE CHALLENGE TEST The clomiphene citrate (CC) challenge test was described by Navot et al as a means of assessing ovarian reserve in women 35 years of age and older.21 This simple test consisted of measuring serum FSH concentrations on cycle day 3 (basal), and then again on cycle day 10 after the administration of 100 mg of CC on cycle days 5 through 9. In the original

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Fig 44.5 Ovarian responsiveness does not prognosticate increased pregnancy rates in women with elevated basal FSH levels (Scott, unpublished data). study, which was published prior to any of the studies addressing the value of basal FSH concentrations, an abnormal test was defined as an elevated level on cycle day 10. Obviously, an abnormal day 3 value also results in the test being considered abnormal. Similar to the information regarding basal FSH concentrations, the precise physiology of the CC challenge test has not been clearly defined. The premise of the test is that in women with normal ovarian reserve, the overall metabolic activity of the developing follicles should be able to overcome the impact of the CC on the hypothalamic-pituitary axis and suppress FSH concentrations back into the normal range by cycle day 10. The addition of the CC creates a “provocative” test that unmasks patients who might not be detected by basal FSH screening alone. THE CC CHALLENGE TEST AND PREGNANCY RATES In its original description, the CC challenge test was used to evaluate 51 infertile women over age 35.21 All 51 of these women had normal basal FSH concentrations, but 18 had elevated values after CC administration and were categorized as having diminished ovarian reserve. Demographically, the patients with diminished reserve were similar to those with adequate reserve with equivalent ages, durations of infertility, and requirements for augmentation of ovulation. However, only one of the 18 (6%) patients with diminished reserve conceived while 14 of 33 (42%) of the adequate reserve group became pregnant.

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Following this initial report, several groups evaluated the predictive value of CC challenge test screening in patients participating in ART programs. Tanbo et al studied 91 women over age 35 and found abnormal CC challenge tests in 37. Twenty of the 37 patients also had an elevated basal FSH concentration on cycle day 3.22,23 Only one patient had an abnormal value on day 3 with a normal value on day 10. The predictive value of an abnormal test was 85% for cycle cancellation due to poor ovarian responsiveness, and 100% for failing to conceive. Loumaye et al also evaluated the CC challenge test, but defined an abnormal test by adding the day 3 and day 10 FSH values together.24 In their series of 114 patients, the predictive value of an abnormal test for failing to conceive was 100%. CC CHALLENGE TEST SCREENING IN THE GENERAL INFERTILITY POPULATION The data generated during the initial evaluation of the CC challenge test were similar in nature to that evaluating basal FSH concentrations alone. The CC challenge test evaluates the predictive value of the test in assisted reproduction programs or in patients undergoing complex ovulation induction. There were legitimate concerns that since the CC challenge test reflected the inability of the developing cohort of follicles to suppress FSH concentrations into the normal range, that the test would be predictive only of the quality of the cohort as a whole. If a single follicle within a cohort possesses good reproductive potential (even if the others did not), the natural processes of recruitment and selection could lead to ovulation of the highest quality follicle, and the predictive value of the CC challenge test would be diminished. Scott et al completed a long term prospective evaluation of CC challenge test screening in women from the general infertility population.25 Approximately 10% of the 236 patients who were evaluated and followed for a minimum of one year had an abnormal CC challenge test. The incidence of abnormal tests rose with age and was 3% when <30 years, 7% at 30–34 years, 10% at 35–39 years, and 26% for women over age 40. Most importantly, the pregnancy rates in the patients with diminished ovarian reserve were markedly lower (9%) than those with adequate reserve (43%). The pregnancy rates were still significantly decreased after controlling for age. Of note, only 7 of the 23 patients with abnormal tests had an elevated FSH concentration on day 3, again suggesting that the CC challenge test may be substantially more sensitive than screening with day 3 samples alone. An examination of the estradiol response between days 3 and 10 failed to show any correlation differentiating those women with normal or abnormal FSH responses. Evaluation of the relation between the eventual clinical diagnoses and CC challenge test results in these 236 couples showed a very high incidence of abnormal tests in the patients with unexplained infertility. In

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fact, the incidence of abnormal CC challenge tests was highest among patients with unexplained infertility (38%) and was unaffected by age. This supports that diminished ovarian reserve is an etiology of infertility, and that couples with abnormal tests should not be considered to have unexplained infertility. PREDICTIVE VALUE OF AGE AND THE CC CHALLENGE TEST The data from the studies described above clearly define that the CC challenge test has better predictive values for pregnancy rates than does age alone. However, in clinical practice both age and CC challenge test results are now available. Scott et al performed life table analyses of pregnancy rates of 589 couples from the general infertility population who were followed for up to 45 months.26 Analysis of the patients with abnormal CC challenge tests showed that the pregnancy rates were uniformly poor independent of the patients age. This finding provides further support to the contention that diminished ovarian reserve is a specific etiology of infertility (Fig 44.6). In contrast, evaluation of the patients with adequate ovarian reserve (normal tests) still showed a significant diminution in pregnancy rates with increasing age (Fig 44.6). This underscores the importance of considering a patients age when counseling them regarding their long term chances for conception, even when their CC challenge test results are normal. Pearlstone et al noted similar findings when evaluating the combined predictive value of age and basal FSH concentrations in women over the age of 40 undergoing complex ovulation induction.27 DAY 3 VERSUS DAY 10 ELEVATIONS DURING THE CC CHALLENGE TEST Hofmann et al evaluated the predictive value of day 3 v day 10 elevations. It seemed possible that day 10 values may have a different prognosis than those on day 3 since they require a provocative test to be unmasked. In fact, pregnancy rates were extremely poor even if only the day 10 sample was abnormal. No difference in counseling may be justified if only the day 10 level is elevated.28

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Fig 44.6 Clomiphene citrate challenge test results prognosticate pregnancy rates (A) in an unselected general infertility population and (B) relative to age. (Data from reference 26, reproduced by permission of ESHRE and Oxford University Press/Human Reproduction.) CURRENT STATUS OF CC CHALLENGE TEST SCREENING An abnormal CC challenge test has excellent predictive values for diminished ovarian reserve and poor long-term pregnancy rates in natural cycles, during ovulation induction, and in IVF. Although the test is quite specific, it has limited sensitivity with a significant age related diminution in reproductive potential occurring even among women with normal test results.

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The test may be superior to basal FSH screening because it is two to three times more sensitive than basal FSH screening alone. Although abnormal day 3 FSH values appear to be accompanied by abnormal day 10 values in most cases, the current literature does not contain enough data to recommend omission of the day 3 sample.

INHIBIN B The lack of a clearly definable relation between circulating E2 and FSH day 3 during ovarian reserve testing caused several authors to speculate that other ovarian endocrine products may be involved in regulating basal FSH levels. Principal among these was inhibin A and inhibin B, with its known secretory patterns from the granulosa cells of antral follicles and its direct suppressive effect on FSH secretion. Early efforts to characterize a relation between basal FSH day 3 and circulating inhibin B day 3 were disappointing. Hughes et al found that while there was an age related decline in peak inhibin B day 3 during complex ovulation induction, those day 3 results were not different earlier in the cycle (ie basal).29 Other authors described differences in inhibin B day 3 between low and high responders during ovulation induction, but again there were no differences in basal values. However, it should be noted that these studies were done using the Monash assay for total immunoreactive inhibin. This heterologous double antibody RIA is based on purified 31 kD bovine follicular fluid inhibin, which also binds to the free alpha subunit and its precursors. Using a newly developed and characterized dimeric inhibin B enzyme linked immunosorbent assay (ELISA) that is more specific, the rise in early follicular FSH concentrations has been correlated with a fall in circulating inhibin B concentrations as women progress through their reproductive life. Seifer et al examined outcomes of ART in women characterized as having low or high day 3 serum inhibin-B concentrations.30 Women with low day 3 serum Inhibin-B concentrations showed a lower E2 response, a reduction in the number of oocytes retrieved, a higher cancellation rate, and a decreased clinical pregnancy rate. It should be noted that the study did not control for basal FSH concentrations and that the pregnancy rate in the low inhibin B group were not as low as those seen in women with abnormal basal FSH screening. We recently evaluated the predictive value of basal inhibin B levels in 292 ART patients who had normal basal FSH levels (Scott, unpublished data). Inhibin B concentrations had a positive correlation with various parameters of ovarian response including number of follicles, oocytes, and embryos. However, there was no relation to pregnancy or delivery rates (Fig 44.7). These data indicate that inhibin B concentrations probably do not add

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Fig 44.7 Basal day 3 inhibin-B levels do not prognosticate pregnancy rates or outcomes in women with normal basal day 3 FSH levels (Scott, unpublished data). significant clinical predictive value beyond that obtained with FSH screening. Given the limited availability and high cost of the test physicians may want to wait until other more convincing clinical data are available before applying this test clinically.

THRESHOLD VALUES FOR OVARIAN RESERVE SCREENING When applying these tests to a given patient population, the practicing clinician is critically dependent on the validity of the assay results and the threshold values used for counseling. The importance of validating any given assay system is described below. The broader issue of selecting a threshold value for normal and abnormal is also very important. In some of the early reports, authors used the distribution of results among healthy and apparently normal women to determine the 95% confidence interval of anticipated results. Values above this range were considered abnormal. While this approach is intuitively logical, it is not appropriate for the validation of this type of test result. For example, if the women screened were all in their early twenties, it would be illogical and probably incorrect to assume that 5% of them had a degree of diminished ovarian reserve adequate to compromise their fertility. Similarly, if a group of women in their early 40s were evaluated, the number with diminished ovarian reserve would greatly exceed the 5% that would be defined as

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abnormal. Clearly, defining threshold values by creation of a general population confidence interval is inappropriate. The threshold values for a normal and abnormal test should be based on clinically defined endpoints. Since the specific changes that account for the loss of reproductive potential remain undefined, all the studies published to date are observational in nature. The only way to determine where a threshold value is located is to perform the screening test in a large group of women and then follow them clinically to see who is able to conceive. Evaluation of the distribution of these data may then be used to define normal and abnormal test results. For those centers which do not have a large clinical volume, or who would like to apply these screening tests without waiting the required time to accumulate all the follow up data, comparison of their assay system with those from one of the centers where the original research was done is indicated.

PREDICTORS OF OVARIAN RESPONSIVENESS (CLINICAL ENDPOINTS OF OOCYTES PRODUCED OR PEAK E2 CONCENTRATIONS) TESTS OF INITIAL OVARIAN RESPONSIVENESS Expanding on earlier work by Padilla et al,31 Winslow et al evaluated the change in estradiol concentrations 24 hours after the administration of 1mg of leuprolide acetate on cycle day 2 (the gonadotropin releasing hormone (GnRH)-agonist stimulation test or GAST).32 The magnitude of the increase in E2 correlated strongly with IVF success. They note that the correlation with ovarian responsiveness was better than that obtained with basal FSH concentrations. While undoubtedly true, this most probably reflects the fact that basal FSH concentrations have no correlation with ovarian responsiveness (nor should they). A recent study evaluating a similar test, used basal and stimulated (two hours after the use of 0.3mg buserelin acetate) FSH concentrations on day 1 of gonadotropin stimulation and found no improvement over basal FSH in predicting IVF success. The exogenous FSH ovarian reserve test (EFORT) was proposed by Fanchin et al (Fig 44.8).33 The EFORT is similar to the GAST which gauges ovarian responsiveness to exogenous FSH. The test is predictive of ovarian response and to a lesser degree pregnancy rates. Specific thresholds that may be used to direct treatment await more rigorous definition in further studies. These tests may have value in prognosticating ovarian response but have not been validated as providing clinical thresholds in or outside of ART adequate to direct patient care. Furthermore, their applicability to the general infertility population is precluded by their expense.

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Fig 44.8 (A) EFORT: exogenous follicle stimulating hormone ovarian reserve test. (B) The ovarian response to the administration of exogenous FSH may be used to prognosticate pregnancy rates. (Data from reference 33, reproduced by permission of ESHRE and Oxford University Press/Human Reproduction.) OVARIAN VOLUME Physical measurements of the ovary have also been proposed as predictors of ovarian response. Ovarian volume has been evaluated by Syrop et al and found to have an excellent correlate with ovarian responsiveness to exogenous gonadotropin stimulation (Fig 44.9).34 The predictive value for ovarian response was superior to basal FSH measurements and other routine clinical parameters. This information is useful in predicting ovarian response and has been used in some oocyte donation programs when selecting potential oocyte donors. Importantly, there were still a substantial number of pregnancies among the women with smallest ovarian volumes. Thus, the clinical utility of the test in counseling patients regarding other treatment options such as oocyte donation or adoption may be limited.

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BASAL ANTRAL FOLLICLE COUNTS Several authors have suggested the possibility of predicting ovarian responsiveness and pregnancy rates by evaluation of the number of small antral follicles in the early follicular phase (Fig 44.10).35 The magnitude of the gonadotropin response and the number of oocytes that may be retrieved correlates quite nicely with these studies. Our group’s initial experience has been that basal follicle counts may be used to direct stimulation protocols and for counseling purposes about cancellation risk. However, there is no threshold value that

Fig 44.9 Clinical pregnancy and delivery rates decline with decreasing basal ovarian volume. (Data from reference 34.)

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Fig 44.10 The umber of basal antral follicles present in the ovary predicts stimulation quality v (A) and pregnancy rates (B) in women undergoing IVF. (Data from reference 35.) (C) Pregnancy rates and outcome in IVF patients with abnormal ovarian reserve screening (Scott, unpublished data). essentially predicts extremely low pregnancy rates. Patients with less than five follicles visible in the basal state (usually examined at the same time that the day 3 FSH concentrations were obtained before starting any

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medications) needed more medication and had produced fewer oocytes but still had ongoing pregnancy rates of up to 35%. Although significantly lower than their age related counterparts, these rates are still high enough to justify an IVF cycle.

TREATMENT OF LOW RESPONDERS The problem of maximizing follicular responsiveness has been extensively studied since investigators began describing dramatically lower success rates in IVF patients who produced fewer follicles during stimulation. Strategies have ranged from simply increasing the dose of exogenous gonadotropins, the use of adjunctive agents such as GnRH analogues and growth hormone, and the use of micromanipulation. With virtually every protocol, improvements in overall responsiveness have been shown for some patients. In spite of this fact the incremental improvement in pregnancy rates has generally been quite small. These data continue to emphasize the importance of the qualitative changes in these patients’ oocytes since many will have sufficient improvements in the quantity of oocytes recovered to have routine numbers of embryos transferred. For these reasons, it may not be sufficient to simply evaluate various treatment regimens by comparing peak E2 concentrations, the number of follicles that develop, the number of oocytes recovered, or the number of embryos that are available for transfer. While pilot studies may legitimately compare ovarian responsiveness, any meaningful and definitive evaluation must also include an assessment of implantation and pregnancy rates. INCREASING EXOGENOUS GONADOTROPIN DOSAGE The first and perhaps simplest approach to increase the magnitude of ovarian response is to increase circulating gonadotropin concentrations during stimulation.36–39 Higher circulating concentrations may reliably be achieved by increasing the quantity of gonadotropins being administered. Patients who responded poorly to lower doses (150IU of FSH; 2 ampoules per day) may commonly produce more follicles when given 300IU or 450IU per day. These enhanced responses lead to increases in the number of oocytes obtained and the number of embryos transferred, and a significant number of pregnancies have been attained. In spite of improvements in some patients, there are clear limits on the effectiveness of this strategy. At some point saturation kinetics are attained, and the ovarian response is determined more by the number of follicles available for recruitment than circulating gonadotropin concentrations. This is particularly important since low responders

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generally have markedly diminished numbers of follicles available for recruitment. Hoffman et al showed that doses above 450IU per day rarely produced a meaningful improvement in ovarian response or the ensuing pregnancy rates.38 Manzi et al were able to significantly increase peak E2 (from 384±26pg/ml to 900±83pg/ml) and number of preovulatory follicles (1.4±0.1 v. 2.7±0.2) by increasing the number of daily hMG ampoules from three to between five and eight, but this did not translate into an increase in cycle fecundity (3.1% v. 4.3%).40 More recently, Land et al found no improvement in pregnancy rates with doses above 225IU per day.41 While the dose necessary to optimize ovarian responsiveness may vary from patient to patient and should certainly be optimized, it is likely that clinically meaningful improvements are only rarely obtained with doses over 450IU per day (6 amps). It remains to be seen whether the introduction of recombinant FSH will alter the pregnancy rates in poor responders. GnRH AGONIST DOWN-REGULATION The introduction of stimulation protocols containing GnRH agonists in the late 1980s provided new opportunities to stimulate patients who previously had limited responses to gonadotropins. Initial reports indicated that some low responders stimulated better after administration of a GnRH agonist in the luteal phase.42 Subsequent clinical experience has provided disappointing results. In fact, many patients who are low responders may be completely refractory to stimulation after being down regulated with a GnRH agonist. While the concurrent use of a GnRH agonist now approaches being the standard for follicular stimulation in most assisted reproductive technology programs, the enhancements in peak E2 concentrations, oocytes obtained, and pregnancy rates generally reflect the near elimination of premature luteinizing hormone (LH) surges and the longer and more aggressive stimulation protocols that are possible. The fact that these are usually not the limiting factors in low responders may explain the generally unfavorable clinical results obtained with these protocols. Recently there have been some questions with regard to direct impact of GnRH agonists on ovarian responsiveness. GnRH receptors have been found in the ovary although their role in follicular development is not understood. Feldberg et al recently showed increased ovarian responsiveness in low responders who were maintained on lower doses of GnRH agonists after pituitary suppression (minidose GnRH agonists) (Fig 44.11).43 While these preliminary data are provocative, prospective randomized doseresponse studies are clearly needed to adequately address the issue of GnRH agonist dose and ovarian performance. Recent experience in our center continues to show that a number of patients are refractory to stimulation after downregulation with GnRH agonist at any dose. It is obvious that low responders represent a heterogeneous

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population of patients and responses to various protocols are likely to vary widely. GnRH AGONIST FLARE Since one goal in optimizing the stimulation of low responders was to increase the quantity of circulating gonadotropins, several investigators administered GnRH agonists to their patients beginning in the early follicular phase. The endogenous gonadotropins flare that occurs in response to the GnRH agonist was used to augment the exogenous gonadotropins. The duration of this endogenous gonadotropin flare has not been completely characterized, but pituitary desensitization is generally achieved within five days of initiating treatment.44 Therefore, the patients are still protected from premature LH surges. While many patients showed improvements in ovarian responsiveness using flare up protocols, these protocols had some drawbacks. Some patients produced degenerate or very low quality oocytes. Others rescued the corpus luteum from their prior cycle and produced very large concentrations of progesterone in the early follicular phase. The impact of these elevations on folliculogenesis, endometrial development, and subsequent implantation rates has not been adequately studied. Finally, the overall impact on pregnancy rates has been mixed. While flare protocols are certainly not as successful for the average ART patient as the luteal phase suppression protocols, they do offer an opportunity to obtain controlled ovarian hyperstimulation in some patients who cannot be stimulated with other protocols. MICRODOSE GnRH AGONIST FLARE There have been no published dose-response studies of the pharmacodynamics of GnRH agonists during flare up ovulation induction cycles. The doses have generally been taken from treatment protocols for men with prostate cancer where minimizing the duration and the effect of the endogenous gonadotropin flare would be desirable. Navot et al reported in 1990 that the rate of pituitary desensitization and ovarian down-regulation was significantly prolonged by using 1% of the normal dose of histerelin.45 They subsequently extended their findings in the primate model by demonstrating that the pituitary could respond with supraphysiologic gonadotropin release in response to low doses of GnRH agonists for very prolonged intervals without inducing desensitization. These data demonstrated that the rate of pituitary desensitization to GnRH agonists stimulation may be dose-dependent. These investigators did not evaluate the potential clinical impact of

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Fig 44.11 Lowering the dose of GnRH-a used to achieve down regulation may enhance ovarian responsiveness. (Data from reference 43.) those findings to determine if they could be used to alter or enhance ovarian responsiveness during controlled ovarian hyperstimulation cycles. Scott et al studied the impact of microdose GnRH agonist administration by giving patients who were low responders 20µg of leuprolide acetate (1/50 the normal dose) every 12 hours beginning on cycle day 2 and continuing until the administration of hCG.46 These patients also received exogenous gonadotropins beginning on cycle day 4. Most patients showed a marked improvement in ovarian responsiveness as indicated by higher peak E2 concentrations, an increase in the number of developing follicles, and the recovery of more oocytes at the time of retrieval. Serial testing of pituitary sensitivity was not done, but it is likely that desensitization was attained by completion of stimulation since none of the patients had detectable premature LH surges. Of more importance, several pregnancies were attained in this previously refractory group. Schoolcraft et al, using a similar protocol, had excellent clinical results with pregnancy rates that increased to 50% in a group of patients previously considered to refractory to stimulation.47 We have recently extended this study. The protocol has been modified and now uses 50µg bid of leuprolide acetate with the onset of exogenous gonadotropins on day 3. These patients almost uniformly achieve greater peak E2 concentrations and require fewer ampoules of gonadotropins. Additionally, 85% will have a greater number of follicles recruited. Pregnancy results have been mixed. Patients who are low responders but

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who have normal ovarian reserve screening have clinical pregnancy rates of 40%. In sharp contrast, patients with abnormal FSH concentrations generally have higher peak E2 concentrations and may produce additional follicles, but pregnancy rates are still very poor (<5%). Thus it seems that a microdose agonist flare up protocol may increase the quantitative follicular response in many patients, but it may not appreciably enhance the quality of the developing cohort of oocytes. ADJUNCTIVE GROWTH HORMONE A detailed discussion of the growth hormone (GH), Insulin-like Growth Factor (IGF) and the Insulin-like Growth Factor Binding Protein (IGFBP) axis is beyond the scope of this chapter; however, there are now extensive data that show 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.48 This led several investigators to evaluate the effect of GH administration as an adjunct during follicular stimulation. GH most probably acts directly on GH receptors noted in granulosa cells (GC) rather than through augmentation of follicular IGF-I, as IGF-I mRNA, and receptors are not expressed in GC of the dominant follicles (IGF-II mRNA and receptors are, however, expressed abundantly in the dominant follicle, the significance of which needs further investigation since IGF-II is not GH-dependent). Early trials were promising, with Homburg et al reporting substantial improvements in follicular responsiveness and pregnancy rates.49 Other investigators also suggested benefit.50 Unfortunately, recent studies have been less encouraging, and some controlled studies have been unable to show clinical benefit.51,52 Considering the large expense and the discouraging results in controlled trials, it must be concluded that there is no well established clinical role for GH in the treatment of low responders at the current time. Further studies directed at defining the dose of GH, and determining if select populations will benefit from treatment (for example, hypogonadotropic and polycystic ovary patients) are currently ongoing. ASSISTED HATCHING Some of the treatments designed to improve pregnancy rates in low responders have not been directed toward improving ovarian responsiveness. Cohen et al reported in 1992 that the use of selective assisted hatching in women with borderline FSH levels improved their implantation and ongoing pregnancy rates.53 This work was extended by Schoolcraft et al who specifically studied patients previously identified as low responders.54 They found substantially higher pregnancy rates in the women whose embryos were hatched.

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These data indicate that the embryos from women who are low responders may have an impaired ability to produce a hatching enzyme (the putative factor responsible for dissolving an opening in the zona pellucida at the time of natural hatching), or that their zona pellucida may be hardened or thickened. In either event the data available at this time indicates some benefit may be obtained through the application of this technique. LOW DOSE ASPIRIN Low dose aspirin treatment has been shown to enhance blood in multiple different organ systems. This may be accomplished by proportionally greater inhibition of vascoconstricting prostaglandins (thromboxane A2) than the vasodilating prostaglandins (prostacyclin). A recent trial from Argentina details a prospective randomized and controlled evaluation of the impact of 100 mg of aspirin on multiple variables including ovarian responsiveness, oocyte number, implantation rates, and pregnancy rates.55 The authors report dramatic improvements in gonadotropin responsiveness, pregnancy rates, and implantation rates. The patient population was not limited to low responders and thus apply to the general population. This treatment will require evaluation in other centers before definitive conclusions are possible, but it certainly deserves further study.

REFERENCES 1 Navot D, Drews MR, Bergh PA, Guzman I, Karstaedt A, Scott RT Jr, et al. Age-related decline in female fertility is not due to diminished capacity of the uterus to sustain embryo implantation. Fertil Steril (1994); 97:97–101. 2 Ben Rafael Z, Feldberg D. The poor-responder patient in an in vitro fertilization-embryo transfer program. J Assist Reprod Genet (1993); 10:118–20. 3 Jacobs SL, Metzger DA, Dodson WC, Haney AF. Effect of age on response to human menopausal gonadotropin stimulation. J Clin Endocrinol Metab (1990); 71:1525–30. 4 Olivennes F, Fanchin R, De Ziegler D, Frydman R. “Poor responders”: screening and treatment possibilities. J Assist Reprod Genetics (1993); 10:115–7. 5 Sherman BM, Korenman SG. Hormonal characteristics of the human menstrual cycle throughout reproductive life. J Clin Invest (1975); 55:699–706. 6 Sherman BM, West JH, Korenman SG. The menopausal transition: Analysis of LH, FSH, estradiol, and progesterone concentrations during menstrual cycles of older women. J Clin Endocrinol Metab (1976); 42:629–36.

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7 Klein NA, Battaglia DE, Fujimoto VY, et al. Reproductive aging: accelerated ovarian follicular development associated with a monotropic follicle-stimulating hormone rise in normal older women. J Clin Endocrinol Metab (1996); 81:1038–45. 8 Klein NA, Battaglia DE, Clifton DK, Bremner WJ, Soules MR. The gonadotropin secretion pattern in normal women of advanced reproductive age in relation to the monotropic FSH rise. J Soc Gynecol Invest (1996); 3:27–32. 9 Lenton EA, Sexton L, Lee S, Cooke ID. Progressive changes in LH and FSH and LH:FSH ratio in women throughout reproductive life. Maturitas (1988); 10:35–43. 10 Lee SJ, Lenton EA, Sexton L, Cooke ID. The effect of age on the cyclical patterns of plasma LH, FSH, oestradiol and progesterone in women with regular menstrual cycles. Hum Reprod (1988); 3:851–5. 11 Batista MC, Cartledge TP, Zellmer AW, et al. Effects of aging on menstrual cycle hormones and endometrial maturation. Fertil Steril (1995); 64:492–9. 12 Scott RT, Toner JF, Muasher SJ, Oehninger SC, Robinson S, Rosenwaks Z. Follicle stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome. Fertil Steril (1989); 51:651–4. 13 Toner JP, Philput CB, Jones GS, Muasher SJ. Basal follicle stimulating hormone level is a better predictor of in vitro fertilization performance than age. Fertil Steril (1991); 55:784–91. 14 Scott RT, Hofmann GE, Oehninger S, Muasher SJ. Intercycle variability of day 3 follicle-stimulating hormone levels and its effect on stimulation quality in in vitro fertilization. Fertil Steril (1990); 53:297– 302. 15 Martin JSB, Nisker JA, Tummon IS, Daniel SAJ, Auckland JL, Feyles V. Future in vitro fertilization pregnancy potential of women with variably elevated day 3 follicle-stimulating hormone levels. Fertil Steril (1996); 65:1238–40. 16 Khalifa E, Toner JP, Muasher SJ, Acosta AA. Significance of basal follicle-stimulating hormone levels in women with one ovary in a program of in vitro fertilization. Fertil Steril (1992); 57:835–9. 17 Scott RT, Hofmann GE. Prognostic assessment of ovarian reserve. Fertil Steril (1995); 63:1–11. 18 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. 19 Smotrich DB, Widra EA, Gindoff PR, Levy MJ, Hall JL, Stillman RJ. Prognostic value of day 3 estradiol on in vitro fertilization outcome. Fertil Steril (1995); 64:1136–40.

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20 Fratterelli JL, Bergh PA, Drews MR, Sharan FI, Scott RT. Evaluation of basal estradiol levels in assisted reproductive technology cycles (2). Fertil Steril (2000); 74:518–24. 21 Navot D, Rosenwaks Z, Margalioth EJ. Prognostic assessment of female fecundity. Lancet (1987); ii:645–7. 22 Tanbo T, Dale PO, Abyhom T, Stokke KT. Follicle-stimulating hormone as a prognostic indicator in clomiphene citrate/human menopausal gonadotrophin-stimulated cycles for in vitro fertilization. Hum Reprod (1989); 6:647–50. 23 Tanbo T, Dale PO, Ludne O, Norman N, Abyholm T. Prediction of response to controlled ovarian hyperstimulation: a comparison of basal and clomiphene citrate-stimulated follicle stimulating hormone levels. Fertil Steril (1990); 53:295–301. 24 Loumaye E, Billion JM, Mine JM, Psalit I, Pensis M, Thomas K. Prediction of individual response to controlled ovarian hyperstimulation by means of a clomiphene citrate challenge test. Fertil Steril (1990); 53:295–301. 25 Scott RT, Leonardi MR, Hofmann GE, Illions EH, Neal GS, Navot D. A prospective evaluation of clomiphene citrate challenge test screening in the general infertility population. Obstet Gynecol (1993); 82:539–45. 26 Scott RT, Opsahl MS, Leonardi MR, Neal GS, Illions EH, Navot D. Life table analysis of pregnancy rates in a general infertility population relative to ovarian reserve and patient age. Hum Reprod (1995); 10:1706–10. 27 Pearlstone AC, Fournet N, Gambone JC, Pang SC, Buyalos RP. Ovulation induction in women age 40 and older: the importance of basal follicle-stimulating hormone level and chronological age. Fertil Steril (1992); 58:674–9. 28 Hofmann GE, Scott RT Jr, Horowitz GM, Thie J, Navot D. Evaluation of the reproductive performance of women with elevated day 10 progesterone levels during ovarian reserve screening. Fertil Steril (1995); 63:979–83. 29 Hughes EG, Robertson DM, Handlesman DJ, Hayward S, Healy DL, de Kretser DM. Inhibin and estradiol responses to ovarian hyperstimulation: Effects of age and predictive value for in vitro fertilization outcome. J Clin Endocrinol Metab (1990); 70:358–64. 30 Seifer DB, Lambert T, Messerlian G, Hogan JW, Gardiner AC, Blazar AS, Berk CA. Day 3 serum inhibin-B is predictive of assisted reproductive technologies outcome. Fertil Steril (1997); 67:110–14. 31 Padilla SL, Bayati J, Garcia JE. Prognostic value of the early serum estradiol response to leuprolide acetate in in vitro fertilization. Fertil Steril (1990); 53:288–94. 32 Winslow KL, Toner JP, Brzyski RG, Oehninger SC, Acosta AA, Muasher SJ. The gonadotropin-releasing hormone agonist-stimulation test—a sensitive predictor of performance in the flare-up in vitro fertilization cycle. Fertil Steril (1991); 56:711–17.

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33 Fanchin R, de Ziegler D, Olivennes F, Taieb J, Dzik A, Frydman R. Exogenous follicle stimulating hormone ovarian reserve test (EFORT): a simple and reliable screening test for detecting ‘poor responders’ in in vitro fertilization. Hum Reprod (1994); 9:1607–11. 34 Syrop CH, Willhoite A, Van Voorhis BJ. Ovarian volume: a novel outcome predictor for assisted reproduction. Fertil Steril (1995); 64:1167–71. 35 Chang MY, Chiang CH, Hsieh TT, Soong YK, Hsu KH. Use of the atrial follicle count to predict the outcome of assisted reproductive technologies. Fertil Steril (1998); 69:505–10. 36 Ben Rafael Z, Strauss JF, Mastroianni L, Flickinger GL. Differences in ovarian stimulation in human menopausal gonadotropin treated women may be related to FSH accumulation. Fertil Steril (1986); 46:586–9. 37 Ben Rafael Z, Benadiva CA, Ausmanas M, et al. Dose of human menopausal gonadotropin influences the outcome of an in vitro fertilization program. Fertil Steril (1987); 48:964–8. 38 Hofmann GE, Toner JP, Muasher SJ, Jones GS. High-dose follicle stimulating hormone (FSH) ovarian stimulation in low responder patients for in vitro fertilization, J In Vitro Fert Embryo Transf (1993); 6:285–9. 39 Karande VC, Jones GS, Veeck L, Muasher SJ. High-dose FSH stimulation at the onset of the stimulation at the onset of the menstrual cycle does not suppress the IVF outcome of low-responder patients. Fertil Steril (1990); 53:486–90. 40 Manzi DL, Thornton KL, Scott LB, Nulsen JC. The value of increasing the dose of human menopausal gonadotropins in women who initially demonstrate a poor response. Fertil Steril (1994); 62:251–6. 41 Land JA, Yarmolinskaya MI, Dumoulin JCM, Evers JLH. High-dose human menopausal gonadotropin stimulation in poor responders does not improve in vitro fertilization outcome. Fertil Steril (1996); 65:961– 5. 42 Serafini P, Stone B, Kerin J, Banrofin J, Quinn P, Marrs R. An alternate approach to controlled ovarian hyperstimulation in “poor responders”. Pretreatment with a gonadotropin-releasing hormone analog. Fertil Steril (1988); 49:90–4. 43 Feldberg D, Farhi J, Ashkenazi J, Dicker D, Shalev J, Ben Rafael Z. Minidose gonadotropin-releasing hormone agonist is the treatment of choice in poor responders with high follicle-stimulation hormone levels. Fertil Steril (1994); 62:343–6. 44 Bider D, Ben-Rafael Z, Shalev J, Goldenberg M, Mashiach S, Blankstein J. Pituitary and ovarian suppression rate after high dosage of gonadotropin-releasing hormone agonist. Fertil Steril (1989); 51:578–81. 45 Navot D, Rosenwaks Z, Anderson F, Hodges GD. Gonadotrophinreleasing hormone agonist-induced ovarian hyperstimulation: low-dose side-effects in women and monkeys. Fertil Steril (1991); 55:1069–75.

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46 Scott RT, Navot D. Enhancement of ovarian responsiveness with micro-doses of GnRH-agonist during ovulation induction for in vitro fertilization. Fertil Steril (1994); 61:880–5. 47 Schoolcraft W, Schenker T, Gee M, Stevey J, Wagley L. Improved controlled ovarian hyperstimulation in poor responder in vitro fertilization patients with a microdose follicle-stimulating hormone flare. Fertil Steril (1997); 67 (1):93–7. 48 Adashi EY, Resnick CE, Hernandez ER, Hurwitz A, Roberts CT, LeRoith D, Rosenfeld R. Insulin like growth factor I as an intraovarian regulator: basic and clinical implications. Ann NY Acad Sci (1991); 626:161–7. 49 Homburg R, Eshel A, Abdalla HI, Jacobs HS. Growth hormone facilitates ovulation induction by gonadotropins. Clin Endocrinol (1988); 29:113–5. 50 Ibrahim ZHZ, Lieberman BA, Matson PL, Buck P. The use of biosynthetic growth hormone to augment ovulation induction with buserelin acetate/human menopausal gonadotropin during controlled ovarian hyperstimulation for in vitro fertilization in women with a poor ovarian response. Fertil Steril (1991); 55:202–5. 51 Younis JS, Simon A, Koren R, Dorembus D, Shenker JG, Laufer N. The effect of growth hormone supplementation on in vitro fertilization outcome: A prospective randomized, placebo controlled double blind study. Fertil Steril (1992); 58:575–80. 52 Shoham Z. European and Australian Multicenter study. Cotreatment with growth hormone and gonadotropin for ovulation induction in hypogonadotropic patients: a prospective, randomized, placebocontrolled, doseresponse study. Fertil Steril (1995); 64:917–23. 53 Cohen J, Alikani M, Trowbridge J, Rosenwaks Z. Implantation enhancement by selective assisted hatching using zona drilling of embryos with poor prognosis. Hum Reprod (1992); 7:685–91. 54 Schoolcraft WB, Schlenker T, Gee M, Jones GS, Jones HW. Efficacy of assisted hatching in poor prognosis IVF candidates. Fertil Steril (1994); 62:551–4. 55 Rubinstein M, Marazzi A, Polak de Fried E. Low dose 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 placebocontrolled study. Fertil Steril (1999); 71:825–9.

45 Repeated implantation failure: the preferred therapeutic approach Mark A Damario, Zev Rosenwaks

OVERVIEW The treatment of human infertility through the assisted reproductive technologies continues to be comparatively inefficient. Despite the common practice of multiple embryo transfers, all in vitro fertilizationembryo transfer (IVF-ET) procedures performed in the United States in 1996 resulted in a mean 26.0% delivery rate per oocyte retrieval.1 Although this IVF-ET delivery rate is actually a slight 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 loss does occur, the largest percentage of failed IVFET 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 age on clinical IVF-ET is manifested not only in the pattern of ovarian response to gonadotrophin stimulation, but also in reduced implantation efficiency and an increased spontaneous abortion rate.2 Determinations 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% total sensitivity in the detection of women with reduced IVF-ET treatment potential. Embryonic loss, which occurs repeatedly after assisted reproduction in women 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

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This chapter summarizes several of the contemporary strategies used to enhance IVFET 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 obvious that patients with severe tubal damage have a poor prognosis with IVF-ET. In many retrospective reports, patients with hydrosalpinges have been identified to have 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 outcomes. Reflux of hydrosalpinx fluid into the uterine cavity may simply result in mechanical factors diminishing embryonicendometrial 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 control patients with hydrosalpinges.17,18 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 (partial zona dissection) seemed to have higher rates of implantation.21

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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 Zona thinning has also been accomplished by 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 New York Presbyterian Hospital.27 The initial trials included patients with normal basal FSH concentrations. In these trials, assisted hatching appeared to benefit patients with thick zonae (>15µm). Further trials employed zona biometric criteria as the indication for zona drilling. These latter selective assisted hatching trials indicated that women aged >38 years 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 fluorescence in situ hybridization for the preimplantation diagnosis of X, Y, 18, 13 and 21 aneuploidy, Munne 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, respectively.30 A further relationship between maternal age and aneuploidy for chromosome 16 was identified.31 Therefore, considering only these data, rates of aneuploidy in women aged ≥40 years would be expected to exceed 40%. Indeed, there remains a possibility that the actual rate of embryonic aneuploidy may be even higher in these women by including the assessment of additional chromosomes. 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 a 8–10 cell embryo. Early work suggested that removing single blastomeres from 8 cell embryos did not effect their viability nor ability to progress to the blastocyst stage.32 While analyses of two blastomeres may be preferable in order to reduce misdiagnoses, there

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is presently less clinical experience with two blastomere biopsies. Following blastomere biopsy, the cells are fixed on a slide and analyzed by fluorescent in situ hydridization (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 3–4).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 rounds of hydridization are employed.34 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 non-disjunction.35,36 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 multi-probe FISH has also been reported.37 Polar body analyses, however, are hampered by the inability to diagnose paternal derived chromosome abnormalities as well as those resulting from postfertilization 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 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 and improved chance for pregnancy. Early attempts to culture human embryos to the blastocyst stage, however, were discouraging as it was clear that the culture media 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.38 The availability of new culture media has recently furthered interest in the culture of human embryos to the blastocyst stage.39 New sequential culture media have been designed specifically for the first 2 days after fertilization (early cleavage) and the third to fourth days of embryo growth (morula and blastocyst). These new sequential culture media systems clearly have resulted in improved blastocyst culture and transfer results over that seen with conventional culture media.40

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A few investigators have suggested that blastocyst culture and transfer may potentially improve clinical outcome in women with repeated implantation failure.41,42 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.43 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.44–46 Embryonic developmental blocks are frequently encountered during the transition from maternal to embryonic genomic activation. Various attempts at improving in vitro culture conditions by modifying the medium electrolyte and energy sources have met with limited success.47 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 human or animals), autologous cumulus or granulosa cells, or an established cell line (monkey kidney epithelial cells (Vero cells)).48–52 Use of coculture methods has produced somewhat variable results, although most investigators have noted at least improvements in embryonic developmental rates.50,51 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 factors53 as well as serve to detoxify the culture medium.54 There is a fair consensus that coculture

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improves embryo morphology, blastocyst development, and hatching.55 In addition, a better synchrony between embryo development and the uterine environment may occur. At the Center for Reproductive Medicine and Infertility at the 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.56,57 In this system, patients undergo an endometrial biopsy performed in the mid- to lateluteal 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 are 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 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.58 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.

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. Two prospective, randomized trials involving prophylactic salpingectomy in patients with severe tubal factor infertility and

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hydrosalpinges have explored whether implantation rates and clinical outcomes can be improved in these patients.59,60 In the first limited monocentric study, Dechaud et al59 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 IVFET 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. Recently, a prospective, randomized multicenter trial of salpingectomy prior to IVF was conducted in Scandinavia.60 Inclusion criteria included the presence of unilateral or bilateral hydrosalpinges as determined by either hysterosalpingography or laparoscopy and age <39 years. A total of 204 patients were available for an intention to treat analysis and 192

Fig 45.3 Day 3 embryos developed on autologous endometrial coculture. actually started IVF. Clinical pregnancy rates per included patient were 36.6% in the salpingectomy group and 23.9% in the non-intervention 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% versus 12.3%, P=0.038) and in patients with ultrasound visible hydrosalpinges (clinical pregnancy rates of 45.7% versus 22.5%, P=0.029; delivery rates of 40.0% versus

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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). One can therefore conclude that patients with severe tubal factor infertility may have improved clinical outcomes following prophylactic salpingectomy, particularly if they have either bilateral hydrosalpinges or hydrosalpinges large enough to be visualized by ultrasound. It remains to be proven, however, if prophylactic salpingectomy improves clinical outcomes in patients who have hydrosalpinges and repeated implantation failures. In addition, 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 (<13µm), while facilitating the implantation of embryos in the setting of a thick zona pellucida (>15µm), 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 (16% v 3%, P<0.05). Women with elevated basal FSH concentrations greater than 15mIU/ml also seemed to particularly benefit, although this has not been subsequently corroborated. In non-randomized studies using historical controls, several investigators reported improved implantation efficiency following assisted hatching in poor prognosis patients (>40 years or several IVF-ET failures).61–63 Most of these centers attempted to use assisted hatching globally in these patients 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 (18.5µm) as opposed to non-pregnant (18.7µm) patients.64 Other investigators also failed to demonstrate clinical benefits from assisted hatching in patients selected for advanced age, zona thickness or previous failed attempts.65 There have been relatively few prospective, randomized controlled trials examining the efficacy of assisted hatching in poor prognosis patients. Magli et al66 reported the clinical efficacy of assisted hatching in 135 cycles with a poor prognosis for pregnancy: (i) maternal age ≥38 years (45 cycles), (ii) three or more previous failed IVF-ET attempts (70

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cycles), and (iii) 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 cycle was significantly higher for the first (31% v 10%, P<0.05) and second groups (36% v 17%, P<0.05). No significant difference in pregnancy rates were noted in the third group, although the numbers were limited. Similarly, higher rates of implantation were obtained compared to the respective controls for the first two groups. Chao et al67 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,68 however, were unable to ascertain any statistically significant benefits of assisted hatching in a prospective, randomized study utilized in unselected patients ≥36 years. Meldrum et al64 have suggested that results of assisted hatching are highly technique dependent. They noted a time dependent improvement in clinical results associated with this technique which they attributed to increasing laboratory 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 the inconsistent clinical results seen with assisted hatching. 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.69 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). 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 aneuploidyfree 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).72–75 In

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an initial non-randomized trial, Gianaroli et al70 reported on 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: (i) maternal age ≥38 years (n=11), (ii) repeated IVF failure (n=22), and (iii) altered karyotype (46XX/45XO mosaics) (n=3). The percentage of abnormal embryos was comparable in the three groups of patients: maternal age (63%), repeated IVF failure (57%) and mosaic karyotype (62%). In particular, 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 or ≥3 previous IVF failures.71 Assisted zona 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 4 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 or greater.72 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 average maternal age, number of previous IVF cycles, duration of stimulation, estradiol concentrations on day +1, and average 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 alphasatellite 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% v/9.6%, P<0.05) and ongoing fetuses or delivered babies per embryo transferred increased after PGD (15.9% v 10.6%, P<0.05). From this trial, the authors concluded that

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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.33,73 In these trials, testing included two rounds of FISH (initial analysis of chromosomes X, Y, 13, 16, 18, and 21 followed by reanalysis for chromosomes 14, 15, and 22) on patients of maternal age ≥36 years, ≥3 previous IVF failures or abnormal karyotypes. In many of these cases, embryo transfer was carried 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% v 10.2%, P<0.001) was achieved in the PGD patients compared with controls. The clinical benefits of PGD was most notable in women ≥38 years and in carriers of an altered karyotype. 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.74 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.43 Since the proportion of aneuploidic embryos appears to increase directly with the number of failed previous IVF-ET cycles,70 blastocyst culture and transfer may be of clinical benefit in patients with a history of repeated implantation failure. Cruz et al75 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 non-randomized 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. 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 not only are likely to have embryos available for transfer, the ability to select among available blastocysts in many instances probably also enhances the implantation

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rate.76 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 frequently will not be good responders to gonadotropin therapy. 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.77 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.78,79 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 v 28% control).80 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 ampulla portion of the fallopian tube have been used.81,82 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.81 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.82 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 can be time consuming as each coculture is individualized. Further, since the granulosa or cumulus cells are plated after retrieval, any coculturing benefits provided to either the gametes or early embryo are probably limited. Nevertheless, Plachot et al83 noted convincing evidence of the benefits of granulosa cell coculture. Using each patient as their own control, one half of the zygotes were cultured using either standard methods or autologous granulosa cell coculture. Eighty-three per cent of granulosa cell coculture embryos were available for transfer compared with only 3% of controls. Other investigators have also noted beneficial

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morphological effects with cumulus cells utilized in the coculture of supernumerary embryos.84 At The Center for Reproductive Medicine and Infertility at the New York Presbyterian 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 of 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.85,86 Coculture with human endometrial epithelial cells has been noted to be beneficial to blastocyst development presumably owing to the induction of embryonic paracrine secretion.87 Further, endometrial cells may be cryopreserved such 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.88 With this approach, these investigators reported a pregnancy rate of 21% versus 8% in patients’ previous cycles. These investigators used a mixture of stromal and epithelial cells following one month of subculture and multiple tissue flask passages. Nieto et al89 reported the use of cryopreserved autologous endometrial (predominantly epithelial) cells and reported a positive effect on the proportion of embryos with minimal or no fragmentation. Simon et al90 further developed a coculture system using 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 use of 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 greater than 90% purity. Cells are then cryopreserved and subsequently thawed in synchrony with the patient’s IVF cycle such 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, at which time selective assisted hatching (if necessary) is performed and transfer 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 preembryo quality (defined as <6 cells or < grade 2 morphology on day 3).56 In this trial, about half of available embryos were allocated to coculture and one half allocated to

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conventional medium. The morphologically best embryos were transferred back 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 a least one previously failed IVF-ET attempt with poor pre-embryo quality.57 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 non-coculture 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).91 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. Recent application of autologous endometrial cell coculture in 19 patients <36 years of age with a history of a single failed IVF-ET cycle associated with very poor embryo quality (grades 3 or below) is encouraging. Clinical pregnancies have been achieved in greater than 80% of treatment cycles to date.92

Table 45.1. Characteristics of human embryos developed on autologous endometrial coculture and conventional medium. Embryo characteristics Coculture Conventional Wilcoxon’s matchedmedium pairs test No. of embryos 203 186 Mean (±SD) no. of 5.9±1.5 5.5±1.4 0.19 blastomeres (day 3) Mean (±SD) % of 21±13 24±11 0.045 fragmentation No. of embryos transferred 90 83 Mean (±SD) no. of 7.4±1.3 6.7±1.9 0.032 blastomeres (transfer)

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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. # blastomeres AECC # blastomeres CM P value All patients (n=79) 6.2±1.4 5.5±1.3 0.0015 Early luteal (n=33) 6.0±1.6 5.6±1.4 0.19 Mid/late luteal (n=46) 6.3±1.2 5.5±1.2 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. % fragmentation AECC % fragmentation CM P value All patients (n=79) 17.7±12.3 21.6±11 0.04 Early luteal (n=33) 19.4±13 20±11 0.87 Mid/late luteal (n=46) 16.5±12 22.8±11 0.012 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 responsiveness 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 al93 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.94 For women with already suspected diminished ovarian reserve, however, the potential detrimental effect of unilateral or bilateral salpingectomy on ovarian responsiveness must be considered. In these cases, either interruption of tubal-uterine patency or ultrasound-guided drainage of hydrosalpinges might also be considered.95 Moreover, salpingectomy is not a procedure without the recognized complications of operative laparoscopy and/or laparotomy in addition to the rare complications of subsequent interstitial96 or abdominal pregnancy.97

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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 that received immunosuppressive treatment, whereas implantation rates were only 7% in patients who did not (2 out of 31).98 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.99,100 Another concern regarding zona manipulation procedures is a possible increased rate of monozygotic twins.101,102 This risk has been attributed to the use of small openings in the zona, which may be prone to pinching the embryo during the hatching process. More recent reports have not found an increased risk of monozygotic twins associated with assisted hatching when acidic Tyrode’s solution was used in experienced laboratories.63 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.61–63,66–68 PREIMPLANTATION GENETIC DIAGNOSIS (ANEUPLOIDY SCREENING) Potential adverse effects of preimplantation genetic diagnosis (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.103,104 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 biological 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.105 Therefore, detecting and discarding mosaic embryos, which is the current approach, might lead to the loss of potentially normal embryos. Although FISH is relatively efficient, FISH failure or misinterpretation can also occur. Harper et al106 reported that a clear FISH signal is obtained

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in 97% of fixed blastomere nuclei. Interpretation of FISH signals can also be complicated by overlapping probe signals. In an early trial, Munne 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 abnormalies as well as abnormalities derived from post-fertilization events. BLASTOCYST CULTURE AND TRANSFER One of the main questions regarding blastocyst culture and transfer is whether all embryos which arrest in culture have no capacity to implant. 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 to develop to the blastocyst stage in culture are chromosomally abnormal, it is possible that some may still not progress because of suboptimal environmental 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 the 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 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, must also be considered. Use of autologous cells averts these infectious risks. Since various cellular preparations and protocols exists, however, laboratories

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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 judgement is required in these instances to ensure that we are providing the best environment for human embryos.

FUTURE DIRECTIONS AND CONTROVERSIES Technologies for preimplantation genetic screening for aneuploidies in women with diminished IVF-ET prognoses are evolving and their clinical utility has not been fully defined. In addition, methods to culture embryos to the blastocyst stage using sequential culture media are also relatively new. In particular, little is known whether this latter technique may aid women with repeated implantation failure. Results with assisted hatching and coculture methodologies seem variable, although they seem to generally result in improved clinical outcomes in women with repeated implantation failure. 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, placentation, and early embryonic vascular compromise. Intravenous immunoglobulin and antithrombogenic therapy including aspirin and heparin have been proposed as treatments.107,108 Although an association between antiphospholipid abnormalities and IVF failure has been shown in some retrospective studies,109,110 recent prospective studies have failed to reveal an association.111 Certain micromanipulation techniques have recently been described that attempt to “rescue” the poor quality embryo. These include microsurgical embryonic fragment removal and cytoplasmic transfer.112,113 Results from 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 prior to generalized clinical use.

CONCLUSION Although treatment of patients with a history of repeated implantation failure has been historically discouraging, new techniques and methodologies are being developed that may provide this difficult group of patients a better prognosis. If severe tubal factor and bilateral

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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. Preimplantation genetic diagnostic techniques for the detection of aneuploidic embryos has considerable promise. Its precise clinical role, however, needs to be further defined. Blastocyst culture and transfer may offer some theoretic advantages in patients with previous IVF failures, particularly in patients who are adequate responders to gonadotropin therapy. Studies incorporating its use in patients with repeated implantation failure will need to be done. Lastly, coculture methods have also been very encouraging in improving both embryo quality as well as clinical outcomes in patients with previous IVF failures. In particular, we have found that the use of autologous cryopreserved endometrial cells offers significant advantages.

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). 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 (1–2mm2) and washed with Hank’s balanced salt solution (HBSS) (Gibco BRL, Grand Island, NY) supplemented with 5000µg per 100ml of penicillin-streptomycin (Gibco BRL, Grand Island, NY) 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.114 Initially, we incubate the tissue pieces for 5 minutes at 37°C in a shaking water bath in 10ml of HBSS containing 0.2% collagenase type 2 (Sigma, St. Louis, MO) and 5,000µg per 100ml of penicillin-streptomycin. Cells 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 15ml polyethylene test tube and centrifuged at 400g for 5 minutes. The pellet is then resuspended in RPMI medium 1640 (Gibco BRL) supplemented with 10% patient’s serum (RPMI-10% serum) and 5,000µg per 100 ml of penicillin-streptomycin.

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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) and cell yield and viability determined quantitatively on a hemacytometer. Tissue culture flasks (25cm2) 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 10ml of HBSS. After approximately 30 seconds, the largest fragments (stromal clumps and undigested tissue) settle on the bottom of the 15ml test tube while the top 8ml contains glands and single stromal cells. The top 8ml (which has a typical “snowflake” appearance) is then transferred to another 15ml 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 to three 25cm2 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 one 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 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. In general, approximately 75% confluence is achieved at the time embryos are placed into the coculture system. Following indentification of fertilization, zygotes are removed from the insemination droplet and allocated to growth in conventional medium (human tubal fluid plus 15% maternal serum) or autologous endometrial

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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. Selective assisted hatching is also performed prior to transfer. 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.

REFERENCES 1 Society for Assisted Reproductive Technology and the American Society for Reproductive Medicine. Assisted reproductive technology in the United States: 1996 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril (1999); 71:798–807. 2 Rosenwaks Z, Davis OK, Damario MA. The role of maternal age in assisted reproduction. Hum Reprod (1995); 10 (Suppl 1):165–73. 3 Scott RT, Toner JP, Muasher SJ, Oehninger S, Robinson S, Rosenwaks Z. Follicle-stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome. Fertil Steril (1989); 51:651–4. 4 Licciardi FL, Liu H-C, Rosenwaks Z. Day 3 estradiol serum concentrations as prognosticators of stimulation response and pregnancy outcome in patients undergoing in vitro fertilization. Fertil Steril (1995); 64:991–4. 5 Plachot M. Chromosome analysis of oocytes and embryos. In: Verlinsky Y, Kuriev A, eds. Preimplantation genetics. Plenum Press: New York, (1991); 103–12. 6 Edwards RG, Fishel S, Cohen J, et al. Factors influencing the success of IVF for alleviation of human infertility. J in Vitro Fertil Embryo Transfer (1984); 1:3–23. 7 Strandell A, Waldenstrom U, Nilsson L, Hamberger L. Hydrosalpinx reduces in vitro fertilization/embryo transfer pregnancy rates. Hum Reprod (1994); 9:861–3. 8 Vandromme J, Chasse E, Jejeune B, Van Rysselberge M, Delvigne A, Leroy F. Hydrosalpinges in in vitro fertilization: an unfavorable prognostic feature. Hum Reprod (1995); 10:576–9. 9 Fleming C, Hull MG. Impaired implantation after in vitro fertilization treatment associated with hydrosalpinx. Br J Obstet Gynecol (1996); 103:268–72. 10 Zeyneloglu HB, Arici A, Olive DL. Adverse effects of hydrosalpinx on pregnancy rates after in vitro fertilizationembryo transfer. Fertil Steril (1998); 70:492–9.

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11 Camus E, Poncelet C, Goffinet F, et al. Pregnancy rates after in-vitro fertilization in cases of tubal infertility with and without hydrosalpinx: a meta-analysis of published comparative studies. Hum Reprod (1999); 14:1243–9. 12 Sharara FI. The role of hydrosalpinx in IVF: simply mechanical? Hum Reprod (1999); 14:557–78. 13 David A, Garcia CR, Czernobilsky B. Human hydrosalpinx: histologic study and chemical composition of fluid. Am J Obstet Gynecol (1969); 105:400–11. 14 Mukherjee T, Copperman AB, McCaffrey C, Cook CA, Bustillo M, Obasaju MF. Hydrosalpinx fluid has embryotoxic effects on murine embryogenesis: a case for prophylactic salpingectomy. Fertil Steril (1996); 66:851–3. 15 Ben-Rafael Z, Orvieto R. Cytokines—involvement in reproduction. Fertil Steril (1992); 58:1093–9. 16 Meyer WR, Castelbaum AJ, Somkuti S, et al. Hydrosalpinges adversely affect markers of endometrial receptivity. Hum Reprod (1997); 12:1393–8. 17 Murray DL, Sagostin 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. 18 Bredkjaer H, Ziebe S, Hamid B, Zhou Y, Loft A, Lindhard A, Nyboe Andersen A. Delivery rates after in-vitro fertilization following bilateral salpingectomy due to hydrosalpinges: a case control study. Hum Reprod (1999); 14:101–5. 19 Fehilly CB, Cohen J, Simons RF, Fishel SB, Edwards RG. Cryopreservation of cleaving embryos and expanded blastocysts in the human: a comparative study. Fertil Steril (1985); 44:638–44. 20 Cohen J, Inge KL, Suzman M, Wiker S, Wright G. Videocinematography of fresh and cryopreserved embryos: a retrospective analysis of embryonic morphology and implantation. Fertil Steril (1989); 51:820–7. 21 Cohen J. Assisted hatching of human embryos, J In Vitro Fertil Embryo Transfer (1991); 8:179–80. 22 Cohen J, Elsner C, Kort H, Malter H, Massey J, Mayer MP, Wiemer K. Impairment of the hatching process following in-vitro fertilization in the human and improvement of implantation by assisting hatching using micromanipulation. Hum Reprod (1990); 5:7–13. 23 Malter HE, Cohen J. Blastocyst formation and hatching in vitro following zona drilling of mouse and human embryos. Gamete Res (1989); 24:67–80. 24 Gordon JW, Talansky BE. Assisted fertilization by zona drilling: a mouse model for correction of oligospermia. J Exp Zool (1986); 239:347–54.

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25 Nakayama T, Fujiwara H, Tastumi K, Fujita K, Higuchi T, Mori T. A new assisted hatching technique using a piezo-micromanipulator. Fertil Steril (1998); 69:784–8. 26 Antinori S, Panci C, Selman HA, Caffa B, Dani G, Versaci C. Zona thinning with the use of laser: a new approach to assisted hatching in humans. Hum Reprod (1996); 11:590–4. 27 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. 28 Hassold T, Chiu D. Maternal age-specific rates of numerical chromosome abnormalities with special reference to trisomy. Hum Genet (1985); 70:11–7. 29 Dailey T, Dale B, Cohen J, Munne S. Association between nondisjunction and maternal age in meiosis-II human oocytes detected by FISH analysis. Am J Hum Genet (1996); 59:176–84. 30 Munne S, Alikani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmental rates and maternal age are correlated with chromosome abnormalities. Fertil Steril (1995); 64:382–91. 31 Benadiva CA, Kligman I, Munne S. Aneuploidy 16 in human embryos increases significantly with maternal age. Fertil Steril (1996); 66:248– 55. 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); 5:708–714. 33 Gianaroli L, Magli MC, Munne 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, Munne S. Preimplantation genetic diagnosis of aneuploidy: were we looking at the wrong chromosomes? J Assist Reprod Genet (1999); 16:176–81. 35 Munne S, Dailey T, Sultan KM, Grifo J, Cohen J. The use of first polar bodies for preimplantation diagnosis of aneuploidy. Hum Reprod (Mol Hum Reprod vol. 1) (1995); 10:1014–20. 36 Verlinsky Y, Cieslak J, Friedine M, et al. Pregnancies following preconception diagnosis of common aneuploidies by fluorescent in-situ hybridization. Hum Reprod (Mol Hum Reprod vol. 1) (1995); 10:1923– 7. 37 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. 38 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. 39 Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Schoolcraft WB. Culture and transfer of human blastocysts increases implantation

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rates and reduces the need for multiple embryo transfers. Fertil Steril (1998); 69:84–8. 40 Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod (1998); 13:3434–40. 41 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. 42 Meldrum DR. Blastocyst transfer-a natural evolution. Fertil Steril (1999); 72:216–7. 43 Gras LR, Gianaroli L, Magli MC, Jones GM, Konnan I, Trounson AO. 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, May 9–14, 1999 . 44 Harlow GM, Quinn P. Development of mouse preimplantation embryos in vivo and in vitro. Aust J Biol Sci (1982); 35:187–93. 45 Jung T, Fischer B. Correlation between diameter and DNA or protein synthetic activity in rabbit blastocysts. Biol Reprod (1988); 39:1111– 16. 46 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. 47 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. 48 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. 49 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. 50 Plachot M, Antoine JM, Alvarez S, et al. Granulosa cells improve human embryo development in vitro. Hum Reprod (1993); 8:2133–40. 51 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. 52 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. 53 Barmat LI, Worrilow KC, Payton BV. Growth factor expression by human oviduct and buffalo rat liver coculture cells. Fertil Steril (1997); 67:775–9.

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54 Fukui Y, McGowan LT, James RW, Pugh PA, Tervit HR. Factors affecting the in vitro development of blastocysts of bovine oocytes matured and fertilized in vitro. J Reprod Fertil (1991); 92:125–31. 55 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. 56 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. 57 Barmat LI, Liu H-C, Spandorfer SD, et al. Autologous endometrial coculture in patients with repeated failures of implantation after in vitro fertilization-embryo transfer. J Assist Reprod Genet (1999); 16:121–7. 58 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-vitro fertilization. Hum Reprod (1998); 13:1696–701. 59 Dechaud H, Daures JP, Arnal F, Humeau C, Hedon B. 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. 60 Strandell A, Lindhard A, Waldenstrom U, Thorburn J, Janson PO, Hamberger L. Hydrosalpinx and IVF outcome: a prospective, randomized multicentre trial in Scandinavia on salpingectomy prior to IVF. Hum Reprod (1999); 14:2762–9. 61 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. 62 Stein A, Rufas O, Amit S, Avrech O, Pinkas H, Ovadia H, Fisch B. 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. 63 Meldrum DR, Wisot A, Yee B, Barzo G, Yeo L, Hamilton F. 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. 64 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. 65 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. 66 Magli MC, Gianaroli L, Ferraretti AP, Fortini D, Aicardi G, Montanaro N. Rescue of implantation potential in embryos with poor prognosis by assisted zona hatching. Hum Reprod (1998); 13:1331–5.

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67 Chao K-H, Chen S-U, Chen H-F, Wu M-Y, Yang Y-S, Ho H-N. 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. 68 Lanzendorf SE, Nehchiri F, Mayer JF, Oehninger S, Muasher SJ. A prospective, randomized, double-blind evaluation of assisted hatching in patients of advanced maternal age. Hum Reprod (1998); 13:409–13. 69 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. 70 Gianaroli L, Magli MC, Munne S, Fiorentino A, Montanaro N, Ferraretti AP. Will preimplantation genetic diagnosis assist patients with a poor prognosis to achieve pregnancy? Hum Reprod (1997); 12:1762–7. 71 Gianaroli L, Magli MC, Ferraretti AP, Fiorentino A, Garrisi J, Munne S. 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. 72 Munne S, Magli C, Cohen J, et al. Positive outcome after preimplantation diagnosis of aneuploidy in human embryos. Hum Reprod (1999); 14:2191–9. 73 Gianaroli L, Magli MC, Ferraretti AP, Munne 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. 74 Gardner DK, Schoolcraft WB. No longer neglected: the human blastocyst. Hum Reprod (1998); 13:3289–92. 75 Cruz JR, Dubey AK, Patel J, Peak D, Hartog B, Gindoff PR. Is blastocyst transfer useful as an alternative treatment for patients with multiple in vitro fertilization failures? Fertil Steril (1999); 72:218–20. 76 Schoolcraft WB, Gardner DK, Lane M, Schlenker T, Hamilton F, Meldrum DR. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril (1999); 72:604–9. 77 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. 78 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. 79 Wiemer KE, Garrisi J, Steuerwald N, et al. Beneficial aspects of coculture with assisted hatching when applied to multiple-failure in-vitro fertilization patients. Hum Reprod (1996); 11:2429–33. 80 Hu Y, Maxson WS, Hoffman DI, Eager S, Dupre J. Coculture of human embryos with buffalo rat liver cells for women with decreased

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prognosis in in vitro fertilization. Am J Obstet Gynecol (1997); 177:358–63. 81 Bongso A, Ng SC, Sathananthan H, Lian NP, Rauff M, Ratnam S. Improved quality of human embryos when cocultured with human ampullary cells. Hum Reprod (1989); 4:706–13. 82 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. 83 Plachot M, Antoine JM, Alvarez S, et al. Granulosa cells improve human embryo development in vitro. Hum Reprod (1993); 8:2133–40. 84 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. 85 Cross JC, Werb Z, Fisher SJ. Implantation and the placenta: key pieces of the development puzzle. Science (1994); 266:1508–17. 86 Liu H-C, He Z-H, Mele CA, Veeck LL, Davis O, Rosenwaks Z. 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. 87 Tazuke SI, Giudice L. Growth factors and cytokines in the endometrium, embryonic development, and maternal embryonic interactions. Semin Reprod Endocrinol (1996); 14:231–45. 88 Jayot S, Parneix I, Verdaguer S, Discamps G, Audervert A, Emperaire J-C. Coculture of embryos on homologous endometrial cells in patients with repeated failures of implantation. Fertil Steril (1995); 63:109– 114. 89 Nieto NS, Watkins WB, Lopata A, Baker HWG, 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. 90 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. 91 Spandorfer SD, Liu H-C, Xu KP, Barmat LI, Davis OK, Rosenwaks Z. The day of the luteal phase endometrial biopsy is an important predictor of success when autologous endometrial coculture is utilized in IVF-ET (Abstr). Presented at the 55th Annual Meeting of the American Society for Reproductive Medicine, Toronto, CA, September 25–30, 1999. 92 Spandorfer SD, Clarke R, Bovis L, Liu H-C, Veeck L, Davis OK, Rosenwaks Z. Autologous endometrial coculture is associated with a high rate of success in patients under 36 years old with a history of a single IVF-ET failure secondary to poor embryo development.

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Presented at the 55th Annual Meeting of the American Society for Reproductive Medicine, Toronto, CA, September 25–30, 1999. 93 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. 94 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. 95 Van Voorhis BJ, Sparks AE, Syrop CH, Stovall DW. Ultrasoundguided aspiration of hydrosalpinges is associated with improved pregnancy and implantation rates after in-vitro fertilization cycles. Hum Reprod (1998); 13:736–9. 96 Raziel A, El RR, Wardimon J, Arad D, Langer R, Caspi E. Ultrasonographic diagnosis of post salpingectomy interstitial pregnancy. Case report and review of the literature. Acta Obstet Gynecol Scand (1989); 68:85–6. 97 Fisch B, Peled Y, Kaplan B, Zehavi S, Neri A. Abdominal pregnancy following in vitro fertilization in a patient with previous bilateral salpingectomy. Obstet Gynecol (1996); 88:642–3. 98 Cohen J, Malter H, Elsner C, Kort H, Massey J, Mayer MP. Immunosuppression supports implantation of zona pellucida dissected human embryos. Fertil Steril (1990); 53:662–5. 99 emeter P, Feichtinger W. Prednisolone supplementation to Clomid and/or gonadotrophin stimulation for invitro fertilization-a prospective randomized trial. Hum Reprod (1986); 1:441–4. 100 Catt JW, Ryan JP, Saunders DM, O’Neill C. Short-term corticosteroid treatment does not improve implantation for embryos derived from subzonal insertion of sperm. Fertil Steril (1991); 61:565– 6. 101 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. 102 Herschlag A, Paine T, Cooper GW, Scholl GM, Rawlinson K, Kvapil G. Monozygotic twinning associated with mechanical assisted hatching. Fertil Steril (1999); 71:144–6. 103 Harper JC, Coonen E, Handyside AH, Winston RM, Hopman AH, Delhanty JD. Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos. Prenatal Diag (1995); 15:41–9. 104 Munne S, Lee A. Rosenwaks Z, Grifo J, Cohen J. Diagnosis of major chromosome aneuploidies in human preimplantation embryos . Hum Reprod (1993); 8:2185–91. 105 Reubinoff BE, Shushan A. Preimplantation diagnosis in older patients. To biopsy or not to biopsy? Hum Reprod (1996); 11:2071–8.

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106 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. 107 Coulam CB, Krysa LW, Bustillo M. Intravenous immunoglobulin for in-vitro fertilization failure. Hum Reprod (1994;) 9:2265–9. 108 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. 109 Birkenfeld A, Mukaida T, et al. Incidence of autoimmune antibodies in failed embryo transfer cycles. Am J Reprod Immunol (1994); 31:65– 8. 110 Coulam CB, Kaider BD, Kaider AS, Janowicz P, Roussev RG. Antiphospholipid antibodies associated with implantation failure after IVF/ET. J Assist Reprod Genet (1997); 14:603–8. 111 Denis AL, Guido M, Adler RD, Bergh PA, Brenner C, Scott RT. Antiphospholipid antibodies and pregnancy rates and outcome in in vitro fertilization patients. Fertil Steril (1997); 67:1084–90. 112 Alikani M, Cohen J, Tomkin G, Garrisi J, Mack C, Scott RT. Human embryo fragmentation in vitro and its implications for pregnancy and implantation. Fertil Steril (1999); 71:836–42. 113 Cohen J, Scott R, Schimmel T, Levron J, Willadsen S. Birth of infant after transfer of anucleate donor oocyte cytoplasm into recipient eggs. Lancet (1997); 350:186–7. 114 Liu HC, Tseng L. Estradiol metabolism in isolated human endometrial epithelial gland and stromal cells. Endocrinology (1979); 104:1674–81.

46 Ultrasound in ART Marinko M Biljan

INTRODUCTION In the past 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 transfer2 have to a large extent contributed to the simplification of IVF procedures, which has facilitated easier, more economical and affordable treatment.3 This chapter will review various ultrasound techniques that are currently in use, the value of performing a baseline ultrasound scan before starting infertility treatment, its use in the assessment of pelvic morphology and prediction of patients’ response to ovarian stimulation. Also, 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, uterine and subendometrial perfusion in the evaluation of its receptivity will be highlighted.

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 early 1980s in infertility treatment, has now been largely made obsolete. To obtain an adequate image of pelvic structures by transabdominal scanner, the bowels have to be displaced from the pelvis by a full bladder. This approach has a number of disadvantages. Firstly, 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 artifact is created by the abdominal subcutaneous tissues, a precise characterization of pelvic structures is sometimes not

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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 peri-adnexal adhesions which limit the bowel displacement. More recently, the transvaginal approach for ultrasound scanning has been used. Since the pelvic structures are in close proximity of the vaginal vault, higher frequency ultrasound probes (for example 7MHz) 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 artifact associated with obesity. In a direct comparison of the two techniques, it has been found that the transvaginal approach allows increased visualization and resolution compared with the transabdominal approach.4,5 Transabdominal scanning is used in our center only in very rare cases, where ovaries are located high in the pelvis and are therefore not accessible to transvaginal scanning. DOPPLER ULTRASOUND In recent years the assessment of pelvic vascularization by means of Doppler ultrasound has become an integral part of ultrasound exam. Doppler ultrasound uses the physical characteristic of sound, which, when directed against a moving object, is 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 while flow away from the probe causes a negative frequency shift. The two types of Doppler equipment used in infertility work-up are the pulsed Doppler and color Doppler. Both of these are used in combination with standard ultrasound imaging. Pulsed Doppler ultrasound enables the frequencies from individual blood vessels to be displayed in 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. It 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 that interprets a positive frequency shift as a red color and a negative frequency shift as a blue color. These colors are superimposed onto the realtime 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

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changes in organ perfusion. Every major artery in the body has its own characteristic FVW. The maximum outline, that is, 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, for example the external iliac artery, supplying high resistance vascular beds. In contrast, high enddiastolic velocities are usually present in smaller arteries supplying 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 S/D (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

Fig 46.1 Pulsed Doppler ultrasound. Flow velocity waveform (FVW) of uterine artery is demonstrated.

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Fig 46.2 Color Doppler ultrasound enables clear visualization of vascular structures in the pelvis. Here, right uterine artery is clearly visualized. and end-diastolic 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 that are not clearly visualized and 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 volume6 and can be used even when there is an absence of diastolic velocities or reverse flow in the diastolic phase. The initial studies of Doppler ultrasound were performed using the transabdominal approach. However, as previously discussed, the transabdominal approach requires the presence of a distended bladder, which increases the distance between the Doppler probe and the vessels under investigation so that low pulse repetition frequencies, which are relatively inaccurate, have to be used. Another disadvantage of the abdominal approach is that the distended bladder may alter blood flow in the smaller arteries. Finally, patients can rarely tolerate an uncomfortably full bladder long enough for the Doppler study to be completed.7 In contrast, vaginal sonography obviates the need of a full bladder and the ultrasound probe is close to the vessel under investigation, so that the optimal pulse repetition frequency can be chosen. Steer et al7 have

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investigated the accuracy of transvaginal and transabdominal Doppler assessment of the uterine artery in subfertile population. They found transvaginal assessment easier to perform and significantly more reproducible. The two vessels that have been studied in relation to infertility have therefore been the uterine and ovarian arteries. With the development of power-Doppler ultrasound some attention has also been drawn to the importance of smaller blood vessels in reproductive organs, such as, the subendometrial8 and endometrial9 blood vessels in uterus and perifollicular blood supply.10–15 It has emerged that Doppler results can be significantly affected by patients’ activity prior to examination as well as the time of the day when the investigation is performed. In an interesting study, Dickey and colleagues16 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 enddiastolic flow increased. Zaidi et al17 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 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 approximately 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 techniques (ART) is of paramount importance. It enables a clinician to assess uterine, tubal, and ovarian morphological appearance, and detect abnormalities that may contribute to a patient’s infertility. Additionally, the assessment of ovarian volume, appearance, and vascularization enable a better prediction of patients’ response to ovulation induction medication. To avoid a distortion of ovarian volume caused by a developing follicle, a baseline scan is usually performed between day 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 media.18

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 maximal length

Fig 46.3 Standard form used to document results of baseline scan at the McGill Reproductive Center.

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from the cervix to the fundus, the length of the uterine cavity, and the maximal uterine thickness is measured. The angle between cervix and uterine body is assessed and documented. The length of uterine cavity and the angle between 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 a hard copy of the ultrasonic image of the uterus in its longitudinal plane, with measurements, and leave it in the patient 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.19 Additionally, a uterine septum has been related to high miscarriage20 and perhaps lower implantation rates,21 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 5mm 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.22 The accuracy of detection of a septate uterus can be improved with the use of saline installation.23 This is a relatively simple technique whereby saline is used as a non-echogenic 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 installation with 500mg Naproxen (Naprosyn, Roche, Canada) suppository 2 hours prior to the procedure. Recently, some researchers suggested a higher detection rate of uterine septae and better distinction between bicornuate uterus and uterine septum with the use of threedimensional (3D) ultrasound.24–26 However, because of the higher cost of 3D ultrasound machines, and only a marginal improvement in detection rate, at present 3D ultrasound does not seem to have a major diagnostic impact on clinical practice.

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 is demonstrated.

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Fig 46.5 This shows a uterine fibroid which is distorting the endometrial lining. Additional attention should be paid to the presence of uterine fibroids, especially submucous and perhaps intramural, which probably decrease implantation rate.27 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 that distort the uterine cavity, regardless of their size. OVARIES Examination of ovaries follows the assessment of the uterus. Ovaries are usually found by directing the ultrasound probe 2–3cm lateral to the cervix. When examining the ovaries, attention should be paid to ovarian size and structure, and its relation to 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.

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POLYCYSTIC OVARIES The precise diagnosis of polycystic ovaries depends on the findings of multiple follicular cysts and increased stroma in ovaries that are usually, but not always, enlarged (Fig 46.7).28 The advent of ultrasound scanning has shown that polycystic ovaries are much more common than was previously believed. Adams et al29 reported that polycystic ovaries were found in 26% of women with amenorrhea, 87% with oligomenorrhea, and 92% with idiopathic hirsutism. With regard to assisted conception, patients with polycystic ovaries are prone to develop ovarian hyperstimulation syndrome (OHSS). In a recent 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 these 15 patients.30 Moreover, a diagnosis of polycystic ovaries is perhaps of prognostic value with regard to

Fig 46.6 The ovary shown in two perpendicular planes. Assessment of the largest diameters in both planes allows a more accurate assessment of ovarian volume.

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Fig 46.7 Ultrasound image of an enlarged polycystic with multiple small cysts scattered around the periphery, and increased highly echogenic stroma. The volume of this ovary is 13.75cm2. the outcome of assisted conception. Engmann et al31 compared the outcome of 46 patients (97 cycles) with polycystic ovaries, but no signs of polycystic ovarian 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 three cycles of treatment patients with polycystic ovaries had significantly higher chances of achieving a pregnancy (odds ratio (OR)=1.69 95% confidence interval (Cl)=0.99– 2.90) and achieving a live birth (OR=1.82 95% Cl=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.32

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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). FUNCTIONAL OVARIAN CYSTS Functional ovarian cysts are defined as any intra-ovarian sonoluscent structure measuring >15mm in the mean diameter causing elevation of serum E2 above 150 pmol/L33 In patients undergoing ovulation induction, who have a functional ovarian cyst, in order to avoid the negative effect of that the estrogen producing ovarian cyst have on pituitary-ovarian axis, we normally advise that treatment is 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

Fig 46.8 A functional ovarian cyst. Note anechogenic contents and clearly defined edges of the cyst.

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Fig 46.9 Two endometriomas of the ovary showing multiple echoes within the cysts. cysts were detected, including high cancellation and low pregnancy rates,34–37 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.38–42 In one study, we have prospectively followed 51 patients during their IVF treatment.19 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 v 7 days), had significantly lower follicle stimulating hormone (FSH) levels at the time of initiation of gonadotropin therapy, required more ampoules of gonadotropin to achieve ovarian stimulation (45 v 41 ampoules), developed fewer follicles (13 v 17.5), and had lower cumulative embryo scores (28 v 36). However, there were no significant differences in the implantation (23.5% v 17.2%) and pregnancy rates (37.2% v 29.2%) between patients who developed cysts compared with those who did not. On the basis of 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.

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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 treatment, 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 INTRA-OVARIAN BLOOD FLOW Following the assessment of ovarian volume and structure, we proceed with the determination of ovarian stromal circulation. To do so, arteries within the 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.

Fig 46.10 Stromal blood flow in the polycystic ovary on day 2 of the menstrual cycle is

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12cm/sec2 in this image. Increased stromal blood flow is predictor of a good response to ovulation induction. FALLOPIAN TUBES MORPHOLOGY OF FALLOPIAN TUBES Normal Fallopian tubes are very fine structures that do not contain a significant quantity of fluid and are 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 past several years a number of researchers reported a reduction in pregnancy rates in patients with hydrosalpinx undergoing IVF treatment.43–47 Strandell et al recently reported results of the largest prospective randomized trial on the effect of salpingectomy on IVF treatment outcome. They showed that in the subgroup of patients who had an ultrasound-visible hydrosalpinx, salpingectomy prior to IVF resulted

Fig 46.11 This “sausage” shaped structure behind the right ovary is a hydrosalpinx. in a 3.5-fold increase in delivery rate (P=0.019). Additionally, in the subgroup of patients who had bilateral hydrosalpinges, removal of the

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Fallopian tubes prior to IVF resulted in a significant increase in implantation (25.6% v 12.3%, P=0.038), clinical pregnancy (45.7% v 22.5%, P=0.029) and live birth (40.0% v 17.5%, P=0.039) rates.48 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. Lessey et al49 demonstrated that αv3 endometrial integrins—were expressed at significantly lower levels in women with hydrosalpinges, and the level of αv3 endometrial integrins apparently returned to normal after surgical treatment of hydrosalpinges. It has been also indicated that hydrosalpingeal fluid has a direct toxic effect on murine embryos even at a concentration of 10%.50 Whatever the mechanism, there is compelling evidence that, at least in patients who have bilateral or ultrasoundvisible hydrosalpinges, their presence leads to decreased pregnancy rates in IVF.48 It therefore seems reasonable to recommend the patients to have either a distal salpingostomy to allow peritoneal drainage of intra-tubal 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 et al 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 micro-particles in galactose solution (Echovist, Berlex, Canada).51 This compared favorably with results of hysterosalpingography. The assessment of tubal patency and uterine cavity abnormalities is therefore possible under ultrasound control and has the potential to replace hysterosalpingography as the method of choice for screening tubal disease. THE VALUE OF THE BASELINE ULTRASOUND SCAN IN PREDICTING SUBSEQUENT RESPONSE TO OVULATION INDUCTION OVARIAN VOLUME AND STRUCTURE In the past several years several 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 colleagues52 examined ultrasonic images made on 188 patients undergoing assisted conception. Estimation of ovarian volume was based on two ultrasonic images taken in sagital and coronal planes. Patients who had part of an ovary removed were excluded from the study. Their results

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showed 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 that the majority of patients with large ovaries were in fact patients with PCO, who normally exhibit an exaggerated response to ovarian stimulation.32 Tomas et al studied the predictive value of ovarian volume, measured in two perpendicular planes, and the number of small follicles (2–5mm in diameter) seen at baseline on ovarian response.53 They divided the 166 patients studied into three groups according to the number of small follicles, inactive (<5 follicles), normal (5–15 follicles), and a PCO-like category with more than 15 follicles. These authors confirmed previous observations showing that patients with smaller ovaries and fewer small follicles on the initial ultrasound scan developed fewer follicles than patients with PCO. They also concluded that the number of small follicles at the beginning of the cycle may be more representative of the actual functional ovarian reserve than the patient’s age. In a separate study using a 3D ultrasound, Danninger and colleagues found that, even after excluding patients with PCO, there was a relation between the volume of ovaries and the likelihood of developing OHSS.54 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. OVARIAN STROMAL PERFUSION Until recently there had been no attempts to use color Doppler ultrasound in the assessment of ovarian reserve. However, recently Zaidi et al performed a study on 105 patients, 26 of which had ultrasonic features of PCOS, undergoing IVF treatment.55 In that study, both ovarian morphology and blood flow were assessed during the early follicular phase of IVF cycles. Poor ovarian response was defined as the development of six or fewer follicles, which was representative of 10% of patients exhibiting the worst response. This study showed a positive independent relation between the ovarian stromal peak systolic blood flow velocity (PSV) at the time of baseline ultrasound scan, and the subsequent follicular response. No relation was found between ovarian stromal PI and follicular response. It is therefore likely that women with greater ovarian stromal PSV have an increased intra-ovarian perfusion. Thus, in response to the same dose of exogenous gonadotropins, a larger amount is delivered to the target cells. Interestingly, it seems that women with polycystic ovaries have a higher ovarian stromal blood flow velocity not only at the baseline scan56 but during the entire menstrual cycle as well.57 Color Doppler therefore offers valuable information regarding the dose of gonadotropin required for successful ovarian stimulation in assisted

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conception. Further studies are required to establish a precise relationship between peak velocities and the required doses of gonadotropins. SUMMARY The baseline ultrasound and Doppler pelvic scan are an important part of the infertility investigation. 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, detection of PCO and ovarian cysts, and measurement of intra-ovarian flow allows, to a certain degree, prediction of a patient’s response to ovarian stimulation. Investigation of the fallopian tubes allows the detection of hydrosalpinges, which should probably be removed prior to treatment. Finally, the use of echogenic media allows the patency of fallopian tubes to be assessed.

MONITORING FOLLICULAR DEVELOPMENT IN ART TECHNIQUE OF FOLLICULAR MEASUREMENT, METHODS OF RECORDING, AND FREQUENCY OF MONITORING The ultrasound technique provides more accurate information of follicle number and size than can be obtained by serum estrogen determinations alone.58 Under optimal conditions a follicle in the ovary can be visualized from a diameter of 2–3mm. The follicles appear as echo-free structures amid 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 2–3mm per day. We record follicular growth on specially designed charts that allow us to see at a glance all the relevant characteristics of the cycle, including the number of developing follicles, dynamics of follicular growth, endometrial thickness, type of ovulation regimen, and quantity of medication used (Fig 46.13). 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 9 of stimulation with gonadotropins. The frequency of scanning thereafter depends on the patient’s response. Typically, patients with a rapid response would require no further ultrasound scans, while patients with a somehow slower response have further scans scheduled. 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 6 of ovulation induction and thereafter depending on their initial response.

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 averaged of four largest diameters is calculated.

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Fig 46.13 Standard form used to monitor follicular development at the McGill Reproductive Center. 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.59 In a retrospective study on more than 6000 follicles from 1109 patients undergoing IVF, Wittmaack et al investigated the effect of follicular size on collection, fertilization, and pregnancy rates.60 They found a relatively constant oocyte recovery in follicles measuring between 12.5mm and 24mm 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 24mm was 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 are allowed to reach at least 13mm in diameter. They therefore challenged the policy adopted by many centers where human chorionic gonadotropin (hCG) is given when 3 follicles

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reach 18mm, 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 GnRH agonist is used in an IVF program, Tan et al performed a randomized controlled trial involving 247 patients.61 In this study the first group of patients had hCG administered on the day when the largest follicle reached 18mm in diameter, two other follicles were larger than 14mm, and the serum concentrations of 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 greater than 14mm in diameter on the day of hCG administration. 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. Optimal oocyte recovery and fertilization rates can apparently be obtained from follicles between 14mm and 24mm in diameter. Oocyte recovery rates decrease after the follicles exceed 24mm 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.60 It would, therefore, be beneficial to have an additional non-invasive test of oocyte quality available prior to hCG administration. A rapid rise in blood flow velocity in the perifollicular and ovarian stromal blood vessels at the time of the luteinizing hormone (LH) surge has been reported.57 These changes may be a result of neoangiogenesis occurring during late follicular development. A marked increase in the peak systolic blood flow velocity around the follicle, in the presence of a relatively constant pulsatility index, could be a sign of follicle maturity and herald impending ovulation. Nargund et al studied the relation between follicular blood flow and the production of morphologically normal embryos.11,12 These authors investigated individual follicles, oocytes, and pre-implantation embryos, rather than pooling data. Interestingly, by using this approach, a very strong relation 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 ≥10cm/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 their vascularized surface.14,15 Oocytes obtained from highly vascularized follicles were of a higher quality and

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were more likely to fertilize and result in pregnancy. In a similarly designed study, Huey and colleagues confirmed that perivascular flow reflects developing competence of the corresponding oocyte.62 In another study, Coulam et al 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.13 In this group, only patients who had peak systolic perifollicular flow >10cm/s and more than 75% of the follicle vascularized achieved a pregnancy. It was concluded that in patients in whom no adequate vascularization is observed, cycle cancellation should be considered. From the available data, it appears that assessment of perifollicular vascular perfusion

Fig 46.14 Color Doppler ultrasound scan showing perifollicular blood flow. could lead to an improved timing of oocyte collection 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, owing to the large number of follicles, it may be difficult to determine the exact vascularization of each single follicle. In women who develop relatively few follicles, however, perifollicular blood flow measurements may prove to be very useful.

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SUMMARY Ultrasound monitoring of follicular growth is the most important tool in the assessment of progress in ovarian stimulation. With follicles that are less than 24mm in size, with increasing size the likelihood of obtaining mature oocyte increases. However, there is no difference in the quality of oocytes obtained from follicles between 18mm and 22mm in diameter. This allows more convenient and predictable planning of oocyte collection. Quantitative and qualitative assessment of perifollicular flow may allow for more accurate assessment of follicular competence. Follicles that have <75% of their surface perfused, or where PSV <10cm/s appear to contain an oocyte of unsatisfactory quality.

ENDOMETRIUM The introduction of sequential culture media and culturing embryos up to the blastocyst stage resulted in a major improvement in selection of embryos capable of implantation.63 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.64 The value of this invasive test is, however, rather restricted in assisted conception cycles owing to its relatively low predictive value and the concern of performing a biopsy during the treatment cycle itself because of the associated endometrial trauma and bleeding. In the past several years there has been a growing interest in assessment of endometrial receptivity by means of ultrasound scanning technology, and Doppler assessment of uterine and endometrial vascularization. ULTRASOUND Two anatomical parameters have been suggested for the evaluation of the endometrium by ultrasound; endometrial thickness and endometrial pattern. 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 et al suggested that endometrial thickness on the day before oocyte recovery,

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was significantly greater in pregnant than in non-pregnant women; and postulated that it may predict the likelihood of implantation.65 However, Glissant et al,66 Fleicher et al,67 and Welker et al68 found that the measurement of endometrial thickness had no predictive value for pregnancy. Moreover, Li et al70 reported no correlation between endometrial thickness, measured by abdominal ultrasound, and histological dating of the endometrium. In their study of endometrial thickness, Dickey and colleagues69 found an increased rate of early miscarriage in a group of patients with very thin (<6mm) or thick endometrium (>13mm). In a retrospective analysis, Weissman et al also reported decreased implantation and pregnancy rates, and perhaps increased miscarriage rates in patients whose endometrium was >14mm (Fig 46.16) at the time of hCG administration.71 Krampel and Feichtinger, however, found no correlation between endometrial thickness and the likelihood of miscarriage.72 Imoedemhe et al compared the endometrial thickness in three groups of patients who were treated with three different ovulation induction regimens.73 They found that the endometrial thickness in all three groups of patients was similar and comparable to that observed in a group of spontaneously ovulating, fertile control patients, despite

Fig 46.15 The endometrium is clearly visualized in this picture with a thickness of 11.0mm. It has a triple line appearance which is believed to represent optimal uterine receptivity.

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Fig 46.16 A very thick endometrium (15.5mm) is, perhaps, an indicator of diminished receptivity. significantly higher serum concentrations of estradiol 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 (threshold theory). Freidler et al reviewed 2665 assisted conception cycles from 25 reports.74 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 regarding the 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 et al reported an absence of pregnancies in donor insemination cycles where the endometrium did not reach at least 6mm in diameter.75 Similarly, in a group of oocyte recipients, no pregnancies were reported in women who had an endometrial thickness of less than 5mm in diameter, whereas several pregnancies occurred in patients with an endometrium thinner than 7.5mm.76 While the chances of pregnancy are decreased if the endometrium measures <5mm, it does not always preclude a pregnancy. Sundstrom described a successful pregnancy in

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patient whose endometrium measured only 4mm.77 At our center, we observed two pregnancies in patients in whom the endometrium measured only 4mm. One of these pregnancies continued, while the other resulted in a miscarriage at 11 weeks of gestation. ENDOMETRIAL PATTERN Endometrial pattern is defined as the relative echogenicity of the endometrium and the adjacent myometrium as shown 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 hypo-echogenic regions between the two outer lines and the central line may represent the functional layer of endometrium.78 If three regions are clearly visible, the endometrium is described as multilayered (or tri-phasic) (Fig 46.15). If the endometrium is more echogenic and its central line is blurred or non-existent, the endometrium is classified as non-multilayered.79 Endometrial thickness is unrelated to endometrial pattern.65 In a literature review of 13 studies74 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 the possibility of pregnancy. Many authors have demonstrated that pregnancies can occur in patients with a nonmultilayered pattern of endometrium, albeit at a lower frequency.70,80 The endometrial pattern does not seem to be influenced by the type of ovarian stimulation and it is of prognostic value in both fresh IVF, as well as frozen embryo transfer cycles. DOPPLER STUDIES UTERINE ARTERIES The uterine artery was the first vessel investigated in relation to implantation. Sterzik et al reported that the resistance index measured on the day of embryo transfer was significantly lower in patients who subsequently became pregnant compared with those who failed to achieve pregnancy.81 Steer et al used transvaginal color Doppler to study the uterine arterial blood flow in 82 women undergoing IVF on the day of embryo transfer.82 The PI was calculated and the patients were 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 compared with those who did not. Zaidi et al showed that an elevated PI of the uterine arteries maintains a similarly poor bad prognostic value, even if it is performed at the time of hCG administration.83 These findings were confirmed by other authors. Coulam et al found significantly more

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non-conceptional than conceptional cycles (P<0.001) in women where a uterine artery PI>3.3 was detected.84 Similarly, Bloechle et al reported a significantly lower PI and RI in patients who achieved pregnancy by IVFET following pituitary suppression with goserelin and subsequent stimulation with recombinant FSH.85 Battaglia et al reported a good correlation between PI, serum tromboxane levels and the chances of achieving a pregnancy.86 Interestingly, Tekay et al were unable to confirm data

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. reported by other groups.87 In their study, which included only 30 nonselected patients, no difference was found in uterine perfusion between pregnant and non-pregnant patients. This difference could be attributed to an inconsistency in patient preparation and timing of Doppler investigations.

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SUBENDOMETRIAL AND ENDOMETRIAL BLOOD FLOW With the introduction 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 uterine arteries. In the first study investigating the intrauterine circulation, Zaidi et al studied 96 women undergoing IVF treatment on the day of hCG administration by transvaginal ultrasonography with color and pulsed Doppler ultrasound.8 These authors observed no pregnancies in the group of patients where subendometrial color flow and intra-endometrial vascularization were absent. The importance of subendometrial flow was further investigated by Achiron et al.88 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. These authors concluded that hormone replacement enables normalization of subendometrial blood flow and creates a vascular status compatible with pregnancy. Yang et al9 adopted a semi-qualitative approach in the assessment of endometrial flow.9 They investigated 95 patients who had endometrial thickness >10mm on the day of hCG administration. These authors described endometrial power Doppler area (EPDA), defined as a part of the endometrium where vascular signal with velocities >5cm/s were detected. They found that patients in whom EPDA <5mm2 had significantly lower pregnancy and implantation rates. Interestingly, decreased EDPA was not reflected in impaired uterine PI, which in both group of patients was normal.

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Fig 46.18 Subendometrial blood flow is an additional parameter in the assessment of uterine receptivity. POSSIBLE TREATMENT FOR IMPAIRED UTERINE VASCULARIZATION The ability to predict implantation before the administration of hCG may allow the clinician the option to delay the administration of hCG until the uterine artery PI improves. An alternative approach would be to try to improve uterine perfusion pharmacologically, namely by the administration of glyceryl trinitrate (GTN). In a preliminary study Cacciatore et al reported, in a group of patients with increased uterine artery PI, a 20% increase in uterine blood flow following the administration of GTN throughout the normal menstrual cycle.89 It has been suggested that the administration of GTN may increase pregnancy rates in women with poor uterine perfusion. However, no randomized studies have been performed to address this issue. Rubinstein et al investigated the value of low-dose aspirin in an attempt to ameliorate pelvic vascularization.90 In a prospective randomized doubleblind trial with 298 patients, they administered 100mg aspirin daily starting in the cycle prior to IVF treatment to patients in the study group. The authors reported a significant decrease in uterine artery PI, increase in the number of recruited follicles and oocytes retrieved, implantation rate, and

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pregnancy rate in the study group. It seems that 100mg aspirin increases uterine perfusion and implantation, and therefore pending confirmation in a repeated study, should possibly be considered as a part of routine pretreatment in assisted conception cycles. SUMMARY An endometrial thickness of <7mm or >14mm, the absence of a multilayered endometrium and a 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 to distinguish between patients with normal and abnormal implantation potential. Glyceryl trinitrate or low-dose aspirin seem to have the potential of improving uterine vascularization and perhaps increase implantation and pregnancy rates in selected cases. However, before either of these preparations is recommended for routine use, additional studies are required.

FUTURE DIRECTIONS In the past 10 years we have witnessed an incredible improvement in the sensitivity of ultrasound technology. This has enabled us to measure follicles and endometrial parameters more precisely, and to assess blood flow not only in the 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 the next few years, additional improvements in the understanding and use of standard two-dimensional ultrasound technology, and that a more substantial and important role of three-dimensional gray scale and color Doppler ultrasound techniques will occur. Ultrasound will remain the most important non-invasive 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.

REFERENCES 1 Lenz S, Lauritsen JG. Ultrasonically guided percutaneous aspiration of human follicles under local anesthesia: a new method of collecting oocytes for in vitro fertilization . Fertil Steril (1982); 38(6):673–7.

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2 Lindheim SR, Cohen MA, Sauer MV. Ultrasound guided embryo transfer significantly improves pregnancy rates in women undergoing oocyte donation. Int J Gynecol Obstei (1999); 66(3):281–4. 3 Tan SL. Simplifying in-vitro fertilization therapy. Curr Opin Obstet Gynecolol (1994); 6(2): 111–4. (edit). 4 Andreotti RF, Thompson GH, Janowitz W, Shapiro AG, Zusmer NR. Endovaginal and transabdominal sonography of ovarian follicles. J Ultrasound Med (1989); 8(10):555–60. 5 Yee B, Barnes RB, Vargyas JM, Marrs RP. Correlation of transabdominal and transvaginal ultrasound measurements of follicle size and number with laparoscopic findings for in vitro fertilization. Fertil Steril (1987); 47(5):828–32. 6 Miles RD, Menke JA, Bashiru M, Colliver JA. Relationships of five Doppler measures with flow in an in vitro model and clinical findings in newborn infants . J Ultrasound Med (1987); 6(10):597–9. 7 Steer CV, Williams J, Zaidi J, Campbell S, Tan SL. Intra-observer, interobserver, interultrasound transducer and intercycle variation in colour Doppler assessment of uterine artery impedance. Hum Reprod (1995); 10(2):479–81. 8 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(3): 191–8. 9 Yang JH, Wu MY, Chen CD, Jiang MC, Ho HN, Yang YS. Association of endometrial blood flow as determined by a modified colour Doppler technique with subsequent outcome of in-vitro fertilization. Hum Reprod (1999); 14(6):1606–10. 10 Campbell S, Bourne TH, Waterstone J, et al. Transvaginal color blood flow imaging of the periovulatory follicle. Fertil Steril (1993); 60(3):433–8. 11 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); 12 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(1): 109–13. 13 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(8):1979–82. 14 Chui DK, Pugh ND, Walker SM, Gregory L, Shaw RW. Follicular vascularity—the predictive value of transvaginal power Doppler ultrasonography in an in-vitro fertilization programme: a preliminary study. Hum Reprod (1997); 15 Bhal PS, Pugh ND, Chui DK, Gregory L, Walker SM, Shaw, RW. The use of transvaginal power Doppler ultrasonography to evaluate the

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relationship between perifollicular vascularity and outcome in in-vitro fertilization treatment cycles. Hum Reprod (1999); 14(4):939–45. 16 Dickey RP, Hower JF, Matulich EM, Brown GT. Effect of standing on non-pregnant uterine flow. Ultrasound Obstet Gynecol (1994); 4:480– 7. 17 Zaidi J, Jurkovic D, Campbell S, Pittrof R, McGregor A, Tan SL. Description of circadian rhythm in uterine artery blood flow during the peri-ovulatory period. Hum Reprod (1995); 10(7):1642–6. 18 Balen FG, Allen CM, Siddle NC, Lees WR. Ultrasound contrast hysterosalpingography—evaluation as an outpatient procedure. Br J Radiol (1993); 66(787):592–9. 19 Homer HA, Li TC, Cooke ID. The septate uterus: a review of management and reproductive outcome. Fertil Steril 2000; 73:1–14. 20 Harger JH, Archer DF, Marchese SG, Muracca-Clemens M, Garver KL. Etiology of recurrent pregnancy losses and outcome of subsequent pregnancies. Obstet Gynecol (1983); 62(5):5 74–81. 21 Fedele L, Bianchi S, Marchini M, Franchi D, Tozzi L, Dorta M. Ultrastructural aspects of endometrium in infertile women with septate uterus. Fertil Steril (1996); 65(4): 750–2. 22 Pellerito JS, McCarthy SM, Doyle MB, Glickman MG, DeCherney AH. Diagnosis of uterine anomalies: relative accuracy of MR imaging, endovaginal sonography, and hysterosalpingography. Radiology (1992); 183(3):795–800. 23 de Crespigny L, Kuhn R, McGinnes D. Saline infusion sonohysterosalpingography, an underutilized technique. Aust NZ J Obstet Gynaecol (1997); 37(2):206–9. 24 Jurkovic D, Geipel A, Gruboeck K, Jauniaux L, Natucci M, Campbell S. Three-dimensional ultrasound for the assessment of uterine anatomy and detection of congenital anomalies: a comparison with hysterosalpingography and two-dimensional sonography. Ultrasound Obstet Gynecol (1995); 5(4):233–7. 25 Ayida G, Kennedy S, Barlow D, Chamberlain P. Contrast sonography for uterine cavity assessment: a comparison of conventional twodimensional with three-dimensional transvaginal ultrasound; a pilot study. Fertil Steril (1996); 66(5):848–50. 26 Raga F, Bonilla-Musoles F, Blanes J, Osborne NG. Congenital Mullerian anomalies: diagnostic accuracy of threedimensional ultrasound. Fertil Steril (1996); 65(3):523–8. 27 Eldar-Geva T, Meagher S, Healey DL, MacLachlan V, Breheney S, Wood C. Effect of intramural, subserosal, and submucosal uterine fibroids on the outcome of assisted reproductive treatment. Fertil Steril (1998); 70:687–91. 28 Adams J, Franks S, Polson DW, et al. Multifollicular ovaries: clinical and endocrine features and response to pulsatile gonadotropin releasing hormone. Lancet (1985); 2(8469–70):1375–9.

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29 Adams J, Polson DW, Franks S. Prevalence of polycystic ovaries in women with anovulation and idiopathic hirsutism. BMJ (1986); 293(6543):355–9. 30 MacDougall MJ, Tan SL, Jacobs HS. In-vitro fertilization and the ovarian hyperstimulation syndrome. Hum Reprod (1992); 7(5):597– 600. 31 Engmann L, Maconochie N, Sladkevicius P, Bekir J, Campbell S, Tan SL. The outcome of in-vitro fertilization treatment in women with sonographic evidence of polycystic ovarian morphology. Hum Reprod (1999); 32 MacDougall MJ, Tan SL, Balen A, Jacobs HS. A controlled study comparing patients with and without polycystic ovaries undergoing invitro fertilization. Hum Reprod (1993); 8(2):233–7. 33 Biljan MM, Mahutte NG, Dean N, Hemmings R, Bissonnette F, Tan SL. Effects of pre-treatment with an oral contraceptive pill on the time required to achieve pituitary suppression by GnRh analogues and subsequent implantation and pregnancy rates. Fertil Steril (1998); 70(6): 1063–9. 34 Thatcher SS, Jones E, DeCherney AH. Ovarian cysts decrease the success of controlled ovarian stimulation and in vitro fertilization . Fertil Steril (1989); 52(5):812–6. 35 Segal S, Shifren JL, Isaacson KB, et al. Effect of a baseline ovarian cyst on the outcome of in vitro fertilizationembryo transfer. Fertil Steril (1999); 71(2):274–7. 36 Keltz MD, Jones EE, Duleba AJ, Polcz T, Kennedy K, Olive DL. Baseline cyst formation after luteal phase gonadotropin-releasing hormone agonist administration is linked to poor in vitro fertilization outcome. Fertil Steril (1995); 64(3):568–72. 37 Ben-Rafael Z, Bider D, Menashe Y, Maymon R, Zolti M, Mashiach S. Follicular and luteal cysts after treatment with gonadotropin-releasing hormone analog for in vitro fertilization. Fertil Steril (1990); 53 (6): 1091–4. 38 Feldberg D, Ashkenazi J, Dicker D, Yeshaya A, Goldman GA, Goldman JA. Ovarian cyst formation: a complication of gonadotropinreleasing hormone agonist therapy. Fertil Steril (1989); 51(1):42–5. 39 Karande VC, Scott RT, Jones GS, Muasher SJ. Non-functional ovarian cysts do not affect ipsilateral or contralateral ovarian performance during in-vitro fertilization. Hum Reprod (1990); 5(4):431–3. 40 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(3):437–40. 41 Herman A, Ron-El R, Golan A, Nahum H, Soffer Y, Caspi E. Follicle cysts after menstrual versus midluteal administration of gonadotropinreleasing hormone analog in in vitro fertilization. Fertil Steril (1990); 53(5):854–8.

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42 Sampaio M, Serra V, Miro F, Calatayud C, Castellvi RM, Pellicer A. Development of ovarian cysts during gonadotrophin-releasing hormone agonists (GnRHa) administration. Hum Reprod (1991); 6(2):194–7. 43 Strandell A, Waldenstrom U, Nilsson L, Hamberger L. Hydrosalpinx reduces in-vitro fertilization/embryo transfer pregnancy rates. Hum Reprod (1994); 9(5):861–3. 44 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(10): 1935–8. 45 Kassabji M, Sims JA, Butler L, Muasher SJ. Reduced pregnancy outcome in patients with unilateral or bilateral hydrosalpinx after in vitro fertilization. E J Obst Gynecol Reprod Biol (1994); 56(2): 129– 32. 46 Vandromme J, Chasse E, Lejeune B, Van Rysselberge M, Delvigne A, Leroy F. Hydrosalpinges in in-vitro fertilization: an unfavourable prognostic feature. Hum Reprod (1995); 10(3):576–9. 47 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(1):122–5. 48 Strandell A, Linhard A, Waldenstrom U, Thorburn J, Janson PO, Hamberger L. Hydrosalpinx and IVF outcome: a prospective, randomized multicentre trial in Scandinavia on salpingectomy prior to IVF. Hum Reprod (1999); 14(11):2762–9. 49 Meyer WR, Castelbaum AJ, Somkuti S, et al. Hydrosalpinges adversely affect markers of endometrial receptivity. Hum Reprod (1997); 12(7):1393–8. 50 Mukherjee T, Copperman AB, McCaffrey C, Cook CA, Bustillo M, Obasaju, ME Hydrosalpinx fluid has embryotoxic effects on murine embryogenesis: a case for prophylactic salpingectomy. Fertil Steril (1996); 66(5):851–3. 51 Campbell S, Bourne TH, Tan SL, Collins WP. Hystero-salpingo contrast sonography (HyCoSy) and its future role within the investigations of infertility in Europe. Ultrasound Obstet Gynecol (1994); 4(245):253. 52 Syrop CH, Willhoite A, Van Voorhis BJ. Ovarian volume: a novel outcome predictor for assisted reproduction. Fertil Steril (1995); 64(6):1167–71. 53 Tomas C, Nuojua-Huttunen S, Martikainen H. Pretreatment transvaginal ultrasound examination predicts ovarian responsiveness to gonadotrophins in in-vitro fertilization. Hum Reprod (1997); 12(2):220–3. 54 Danninger B, Brunner M, Obruca A, Feichtinger W. Prediction of ovarian hyperstimulation syndrome of baseline ovarian volume prior to stimulation. Hum Reprod (1996); 11(8): 1597–9. 55 Zaidi J, Barber J, Kyei-Mensah A, Bekir J, Campbell S, Tan SL. Relationship of ovarian stromal blood flow at the baseline ultrasound

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scan to subsequent follicular response in an in vitro fertilization program. Obstet Gynecol (1996); 88(5):779–84. 56 Zaidi J, Campbell S, Pittrof R, et al. Ovarian stromal blood flow in women with poly cystic ovaries—a possible new marker for diagnosis? Hum Reprod (1995); 10(8): 1992–6. 57 Tan SL, Zaidi J, Campbell S, Doyle P, Collins W. Blood flow changes in the ovarian and uterine arteries during the normal menstrual cycle. Am J Obstet Gynecol (1996); 175(3 Pt 1):625–31. 58 Haning RV Jr, Austin CW, Kuzma DL, Shapiro SS, Zweibel, WJ. Ultrasound evaluation of estrogen monitoring for induction of ovulation with menotropins. Fertil Steril (1982); 37(5):627–32. 59 Edwards RG. Conception in the Human Female. (Academic Press: New York, 1980). 60 Wittmaack FM, Kreger DO, Blasco L, Tureck RW, Mastroianni L, Jr, Lessey BA. 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(6):1205–10. 61 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(6):1259–64. 62 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(4):707–12. 63 Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Schoolcraft WB. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril (1998); 69(1):84–8. 64 Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril (1997); 1:23. 65 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(3):446–50. 66 Glissant A, de Mouzon J, Frydman R. Ultrasound study of the endometrium during in vitro fertilization cycles. Fertil Steril (1985); 44(6):786–90. 67 Fleischer AC, Herbert CM, Sacks GA, Wentz AC, Entman SS, James AE Jr. Sonography of the endometrium during conception and nonconception cycles of in vitro fertilization and embryo transfer. Fertil Steril (1986); 46(3):442–7. 68 Welker BG, Gembruch U, Diedrich K, al-Hasani S, Krebs D. Transvaginal sonography of the endometrium during ovum pickup in stimulated cycles for in vitro fertilization. J Ultrasound Med (1989); 8(10):549–53.

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69 Dickey RP, Olar TT, Curole DN, Taylor SN, Rye PH. Endometrial pattern and thickness associated with pregnancy outcome after assisted reproduction technologies. Hum Reprod (1992); 7(3):418–21. 70 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): 1–5. 71 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(1):147–9. 72 Krampl E, Feichtinger W. Endometrial thickness and echo patterns. Hum Reprod (1993); 8(8): 1339 (letter; comment). 73 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(7):545–7. 74 Freidler 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(4):323–35. 75 Gonen Y, Calderon M, Direnfeld M, Abramovici H. The impact of sonographic assessment of the endometrium and meticulous hormonal monitoring during natural cycle in patients with failed donor artificial insemination. Ultrasound Obstet Gynecol (1991); 1:122–6. 76 Abdalla HI, Brooks AA, Johnson MR, Kirkland A, Thomas A, Studd JW. Endometrial thickness: a predictor of implantation in ovum recipients? Hum Reprod (1994); 9(2):363–5. 77 Sundstrom P. Establishment of a successful pregnancy following invitro fertilization with an endometrial thickness of no more than 4 mm. Hum Reprod (1998); 13(6): 1550–2. 78 Forrest TS, Elyaderani MK, Muilenburg MI, Bewtra C, Kable WT, Sullivan P. Cyclic endometrial changes: US assessment with histologic correlation. Radiology (1988); 167(1):233–7. 79 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(2):232–7. 80 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(4):815–22. 81 Sterzik K, Grab D, Sasse V, Hutter W, Rosenbusch B, Terinde R. Doppler sonographic findings and their correlation with implantation in an in vitro fertilization program. Fertil Steril (1989); 52(5):825–8. 82 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(2):372–6.

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83 Zaidi J, Pittrof R, Shaker A, Kyei-Mensah A, Campbell S, Tan SL. 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(2):377–81. 84 Coulam CB, Bustillo M, Soenksen DM, Britten S. Ultrasonographic predictors of implantation after assisted reproduction. Fertil Steril (1994); 62(5): 1004–10. 85 Bloechle M, Schreiner T, Kuchler I, Schurenkamper P, Lisse K. Colour Doppler assessment of ascendent 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(8):1772–7. 86 Battaglia C, Artini PG, Giulini S, et al. Colour Doppler changes and thromboxane production after ovarian stimulation with gonadotrophinreleasing hormone agonist. Hum Reprod (1997); 12(11):2477–82. 87 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(3):688–93. 88 Achiron R, Levran D, Sivan E, Lipitz S, Dor J, Mashiach S. Endometrial blood flow response to hormone replacement therapy in women with premature ovarian failure: a transvaginal Doppler study. Fertil Steril (1995); 63(3):550–4. 89 Cacciatore B, Tiitinen A, Ylikorkala O. Is it possible to improve uterine blood flow in infertile women? Ultrasound Obstet Gynecol (1996); 8:(Suppl 1)204. 90 Rubinstein M, Marazzi A, Polak de Fried E. Low-dose 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 placebocontrolled assay. Fertil Steril (1999); 71(5):825–9.

47 Epididymal and testicular sperm extraction: clinical aspects Herman Tournaye

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. Especially the introduction of intracytoplasmic sperm injection (ICSI) has completely changed the clinical approach towards male infertility. 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; epididymal or testicular spermatozoa too can be used for ICSI. Surgical retrieval of spermatozoa for ICSI has therefore become a routine technique in clinical andrology. Several techniques are available in order 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.

SHOULD I GO FOR SURGICAL SPERM RECOVERY? Most methods described for surgical sperm recovery are simple techniques. However, in some patients with azoospermia even such simple techniques are not indicated. When after appropriate analysis the diagnosis of azoospermia is made, a 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 hypogonadotropichypogonadism, then hormone replacement therapy must be proposed. Often the diagnosis of azoospermia is made without further centrifugation. Centrifugation at 1800×G for at least 5 minutes may reveal spermatozoa in the pellet which may be used for ICSI.1 In a series of 49 patients with non-obstructive azoospermia it was shown that in 35% of patients spermatozoa could be recovered from the ejaculate for ICSI.2 In cases of non-obstructive azoospermia it may therefore be worth while to perform centrifugation of an ejaculate before embarking on a surgical

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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 for obtaining spermatozoa for assisted reproduction in men with anejaculatory infertility. However, given the efficiency of assisted ejaculation in these men, surgical methods are only to be considered when penile vibrostimulation3 or electro-ejaculation4,5 have failed. Epididymal or testicular sperm recovery procedures are often proposed to anejaculatory patients because no penile vibrostimulation or electro-ejaculation is available. However, it is preferable to refer patients with anejaculation, especially patients with spinal cord injuries, to specialized services where assisted ejaculation can be performed. Vibro- or electro-stimulation are non-invasive techniques which 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.6,7 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 obstruction. Psychogenic anejaculation may be encountered unexpectedly during treatments with assisted reproductive technologies, for example, ICSI. Here too, assisted ejaculation may be useful, rather than surgical methods, in order to obtain spermatozoa if treatment by sildanefil citrate has failed to overcome the problem of a erectile dysfunction.8

METHODS FOR RETRIEVAL OF EPIDIDYMAL OR TESTICULAR SPERMATOZOA At present, different methods are available to obtain spermatozoa from the vas deferens, epididymis or testicular mass.9 The method of choice will depend merely on the surgical skills and the techniques available in a

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given setting. If sperm has to be retrieved on an outpatient basis, techniques should be adopted that are compatible with local or locoregional anaesthesia. Fig 47.1 shows the algorithm currently used in our setting. If no motile spermatozoa can be obtained after centrifugation, a sperm retrieval method has to be performed.

Fig 47.1 Algorithm for obstructive azoospermia. In case of obstructive azoospermia, several methods are available. If obstructive azoospermia 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 obstructive azoospermia, but also provide the possibility of performing reconstructive surgery. If no surgical correction is feasible than we prefer to perform a microsurgical epididymal sperm aspiration (MESA) during the exploration. The

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Fig 47.2 Algorithm for non-obstructive azoospermia. epididymal spermatozoa that are obtained can be easily cryopreserved for later use without jeopardizing the outcome after ICSI.10 If, however, a previous workup has shown that microsurgical reconstruction is not possible, then a percutaneous epididymal sperm aspiration (PESA) may be performed. Although there have been some concerns that this blind method may cause epididymal damage and fibrosis11 this issue is not important where reconstruction is not possible. After PESA, epididymal sperm may not always be obtained.12 In this case, testicular spermatozoa may be obtained either by open testicular biopsy or by fine needle aspiration of the testis. Both methods are similar in terms of outcome,13 but the numbers of sperm obtained after open biopsies are

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much higher. For this reason, open testicular biopsy may be preferred whenever cryopreservation is desired. Alternative methods of testicular aspiration have been described yielding higher numbers of spermatozoa.14,15 In these aspiration techniques, needles with a larger diameter are used in order to obtain tissue cylinders. Compared with fine needle aspiration, these alternative methods are less patient friendly and need local or locoregional anaesthesia. 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 47.2 shows our current algorithm for patients with non-obstructive azoospermia willing to undergo ICSI treatment. 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 testicular sperm retrieval procedure performed on the day of the oocyte retrieval or the day before. The excisional biopsy may be scheduled under local anaesthesia. 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.16,17 Because multiple biopsies may lead to extensive fibrosis and devascularization,18,19 multiple excisional biopsies may be taken under an operating microscope at ×40 and ×80 magnification.20 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.9 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 non-obstructive azoospermia is suspected from the clinical findings, a testicular biopsy is performed preferentially under general anaesthesia. 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. Care should be taken to take small tissue pieces and to 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 buffer21 and enzymatic digestion.22 Some authors have reported that scheduling the testicular recovery procedure one day before the ovum pickup23 or the use

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of motility stimulants, for example, pentoxifylline, may facilitate the retrieval of motile spermatozoa from the tissue.24 In about half of patients with non-obstructive azoospermia, no testicular spermatozoa will be found9,17,25 and no accurate parameters are available by which to predict finding sperm.17,26 Artificial insemination by donor or adoption may be proposed in such cases. A special subgroup of patients with non-obstructive azoospermia are patients with Klinefelter’s syndrome. Again, in half of these patients spermatozoa may be recovered for ICSI.27,28 Pregnancies have been obtained after ICSI with testicular spermatozoa from 47,XXY non-mosaic Klinefelter’s syndrome.28–30 However, it is important to combine ICSI with pre-implantation genetic diagnosis31 because of the risk of aneuploidy in the testicular spermatozoa.32 Less invasive methods have been proposed in order to obtain testicular spermatozoa from patients with non-obstructive azoospermia, i.e. testicular aspiration. However, several prospective controlled studies have shown that the retrieval rate is significantly lower than with excisional biopsies.9,33,34 Furthermore, in patients with a history of cryptorchidia, 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.35

REFERENCES 1 Tournaye H, Joris H, Verheyen,G, Camus M, Devroey P, Van Steirteghem A. Sperm parameters, globozoospermia, necrozoospermia and ICSI outcome. In: Filicori M, ed. Treatment of infertility: the new frontiers. Communications Media for Education, USA. 259–68. 2 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. 3 Brackett N. Semen retrieval by penile vibratory stimulation in men with spinal cord injury. Hum Reprod (1999) Update 5:216–22. 4 Bennett CJ, McCabe M, Ayers JWT, Moinipanah R, Randolph JF Jr, McGuire EJ, Seager SWJ. Electroejaculation of paraplegic males followed by pregnancies. Fertil Steril (1987); 48:1070–2. 5 Hultling C, Rosenlund B, Levi R, Fridstrom M, Sjoblom P, Hillensjo T. 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. 6 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.

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7 Hovatta O, Reima I, Foudila T, Butzow T, Johansson K, von Smitten K. Vas deferens aspiration and intracytoplasmic sperm injection of frozenthawed spermatozoa in a case of anejaculation in a diabetic man. Hum Reprod (1996); 11:334–5. 8 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. 9 Tournaye H. Surgical sperm recovery for intracytoplasmic sperm injection: which method is to be preferred? Hum Reprod (1999); 14 (Suppl. 2) 71–81. 10 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. 11 Girardi SK, Schlegel P. MESA: review of techniques, preoperative considerations and results. J Androl (1996); 17:5–9. 12 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. 13 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. 14 Morey AF, Deshon GE Jr, Rozanski TA, Dresner ML. Technique of biopty gun testis needle biopsy. Urology (1993); 42:325–6. 15 Hovatta O, Moilanen J, Von Smitten K, Reima I. Testicular needle biopsy, open biopsy, epididymal aspiration and intracytoplasmic sperm injection in obstructive azoospermia. Hum Reprod (1995); 10:2595–9. 16 Tournaye H, Camus M, Goossens A, et al. Recent concepts in the management of infertility because of nonobstructive azoospermia. Hum Reprod (1995); (Suppl. 1) 115–9 17 Ezeh UIO, Moore HDM, Cooke ID. Correlation of testicular sperm extraction with morphological, biophysical and endocrine profiles in men with azoospermia due to primary gonadal failure. Hum Reprod (1998); 13:3066–74. 18 Schlegel P, Su LM. Physiological consequences of testicular sperm extraction. Hum Reprod (1997); 12:1688–92. 19 Ron-El R, Strauss S, Friedler S, Strassburger D, Komarovsky D, Raziel A. Serial sonography and colour flow Doppler imaging following testicular and epididymal sperm extraction. Hum Reprod (1998); 13:3390–3. 20 Schlegel PN, Li PS. Microdissection TESE: sperm retrieval in nonobstructive azoospermia. Hum Reprod (1998); Update; 4:439. 21 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.

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22 Crabbé E, Verheyen G, Tournaye H, Van Steirteghem A. The use of enzymatic procedures to recover testicular sperm. Hum Reprod (1997); 12:1682–7. 23 Angelopoulos T, Adler A, Krey L, Licciardi F, Noyes N, McCullough A. Enhancement or initiation of testicular sperm motility by in vitro culture of testicular tissue. Fertil Steril (1999); 71:240–3. 24 Tasdemir I, Tasdemir M, Tavukcuoglu S. Effect of pentoxifylline on immotile testicular spermatozoa. J Assisted Reprod Genet (1998); 15:90–2. 25 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. 26 Tournaye H, Verheyen G, Nagy P, et al. Are there any predictive factors for successful testicular sperm recovery? Hum Reprod (1997); 12:80–6. 27 Tournaye H, Staessen C, Liebaers I et al. Testicular sperm recovery in 47, XXY Klinefelter patients. Hum Reprod (1996); 11:1644–9. 28 Tournaye H, Camus M, Vandervorst M, et al. Sperm retrieval for ICSI. Int J Andrology (1997); 20(Suppl. 3)69–73. 29 Palermo GD, Schlegel PN, Scott Sils E. Births after intracytoplasmic sperm injection of sperm obtained by testicular sperm extraction from men with non-mosaic Klinefelter’s syndrome. N Engl J Med (1998); 338:588–90. 30 Ron-El R, Friedler S, Strassburger D, Komarovsky D, Schachter M, Raziel A. Birth of a healthy neonate following the intracytoplasmic injection of testicular spermatozoa from a patient with Klinefelter’s syndrome. Hum Reprod (1999); 14:368–70. 31 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. 32 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. 33 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. 34 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. 35 Novero V, Goossens A, Tournaye H, Silber S, Van Steirteghem A, Devroey P. Seminoma discovered in two males undergoing successful testicular sperm extraction for intracytoplasmic sperm injection. Fertil Steril (1996); 65:1015–54.

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APPENDIX

PROTOCOL: PERCUTANEOUS EPIDIDYMAL SPERM ASPIRATION (PESA) INDICATION All cases of obstructive azoospermia with normal spermatogenesis, for example congenital absence of the vas deferens, failed vasectomy reversal. (CBAVD patients: read caveat in MESA section). PATIENT PREPARATION Patient is fully draped with the operation site obscured to the patient. Patient wears a top only. Operation site cleansed with antiseptic solution (for example, MAC, Zeneca: hospital antiseptic concentrate. Contains chlorhexidine). Penis held up out of the way with a swab fixed underneath the drape. A drape with a small hole of 5cm in diameter in the middle covers the operation site. The testes are gently pulled through to be in the field of the procedure. Local anesthetic—1–2ml of 2% lignocaine (without adrenaline)—is injected in the spermatic cord in order to obtain locoregional anaesthesia and into the scrotal skin. THE PESA PROCEDURE A 18G needle is used. Attached is a 10ml 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 1.5ml 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.

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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. Pethidin hydrochloride 1mg/kg IM and midazolam 2.5mg IM may be given. Patient has to empty the bladder before surgery. PATIENT CARE POST OPERATION The man is told that there may be some pain, but it should be minimal. Paracetamol can be taken. If more is required then he should contact the clinic. REQUIREMENTS

A runner Drape with central hole Cleaning solution—not betadine Syringe 10cc (BS-10 ES Terumo) Micro test tube 1.5ml (Eppendorf 3810) (to be washed and sterilized first) with medium Modified Earle’s Balance Salt Solution+HEPES+0.4 Heparine Novo + 2.25% Human Serum Albumin Gauze squares 10×10 (35813 Hartmann) PROTOCOL: FINE NEEDLE ASPIRATION (FNA) OF TESTIS FOR SPERM RETRIEVAL INDICATION All cases of obstructive azoospermia with normal spermatogenesis, for example, congenital absence of the vas deferens, failed vasectomy reversal. (CBAVD patients: read caveat in MESA section). PATIENT PREPARATION Patient is fully draped with the operation site obscured to the patient. Patient wears a top only. Operation site cleansed with antiseptic solution (HAC, Zeneca: hospital antiseptic concentrate. Contains chlorhexidine). Penis held up out of the way with a swab fixed underneath the drape. A drape with a small hole of 5cm in diameter in the middle covers the operation site. The testes are gently pulled through to be in the field of the

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procedure. Local anesthetic—1–2ml of 2% lignocaine (without adrenaline)—may be injected into the scrotal skin and below to the tunica. THE FNA PROCEDURE A 21G ¾″ butterfly needle is used. Attached is a 20ml syringe. A small amount of culture medium is drawn up into the tubing and the majority expelled until only about 1–2mm 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 20ml 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 20ml syringe (must have rubber stop which may never be in contact with the medium) is removed and a 1ml syringe with the plunger partially withdrawn is attached. Otherwise the 20ml syringe may be used. The embryologist/nurse brings a dish with 9 droplets of culture medium placed (one 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, then an open biopsy under local anesthetic should be performed. DRESSING AFTER Gauze squares and disposable underpants.

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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. Pethidin hydrochloride 1mg/kg IM and midazolam 2.5mg IM may be given. Patient has to empty the bladder before surgery. PATIENT CARE POST OPERATION The man is told that there may be some pain, but it should be minimal. Paracetamol can be taken. If more is required then he should contact the clinic. REQUIREMENTS

A runner Drape with central hole Cleaning solution— not betadine Syringe 20cc Surflo Winged Infusionset flushed with medium Syringe 1cc Gauze squares 10×10

(BS-20 ES Terumo) CE 0197 21G×¾″ (SV-21BL Terumo) (modified Earle’s balance salt solution + HEPES+0,4 Heparine Novo+2.25% human serum albumin) (Air-Tite K-ATS-1000 Cook) (35813 Hartmann)

TO TRANSPORT SPERM

Tissue culture dishes (3200 Falcon Becton Dickinson) with droplets medium (modified Earle’s balance salt solution+HEPES+0.4 Heparine Novo+2.25% human serum albumin) PROTOCOL: 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).

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PATIENT PREPARATION Patient is fully draped with the operation site obscured to the patient. Patient wears a top only. Operation site cleansed with antiseptic solution (HAC, Zeneca: hospital antiseptic concentrate. Contains chlorhexidine). Penis held up out of the way with a swab fixed underneath the drape. A drape with a small hole of 5cm in diameter in the middle covers the operation site. The testes are gently pulled through to be in the field of the procedure. PROCEDURE Approximately 5ml lignocaine (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 1–2cm incision is then made into the scrotum and down through the tissue made edematous by the lignocaine 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. By the use of a curved pair of Mayo scissors a small sample is excised and placed into a Petri dish filled with sperm preparation medium, for example, 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 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. REQUIREMENTS

An assistant and a runner Monopolar pencil with needle and Cord Tubeholder (1×) to fix cords on drape (pencilcord off foot end) Needleholder Mayo-Hegar Straight Mayo scissors Adlerkreutz pincet Allis forceps Kryle forceps Micro Adson pincet (2×) Micro Adson pincet (2×)

(E 2502 Valleylab) (708130 Mölnlycke)

(20–642–16 Martin) (11 180 15 Martin) (12–366–15 Martin) (30–134–15 Martin) (13–341–14 Martin) (12–404–12 Martin) (12–406–12 Martin)

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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 nr 15 (0505 Swann-Morton) 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 balance salt solution+Hepes+0.4 Heparine Novo+2.25% Human Serum Albumin) LOCAL ANESTHESIA

Syringe 20cc CE 0197 Needle 18 G Needle 26 G Xylocaïne 2%

(BS-20 ES Terumo) (NN 1838 S Terumo) (NN 2613 R Terumo) (Astra Pharmaceuticals)

PRE-OP AND POST-OP CARE AS FOR FNA 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 paracetamol.

PROTOCOL: TESTICULAR BIOPSY UNDER GENERAL ANESTHESIA INDICATION All cases of non-obstructive azoospermia (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 identify the best testis to explore. This is done by reading any previous histology reports, feeling the testis for size and consistency. If the testis is high or retracted, then the chance of retrieving spermatozoa is less.

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PATIENT PREPARATION Patient is fully draped with the operation site obscured to the patient. Patient wears a top only. Operation site cleansed with antiseptic solution (HAC, Zeneca: hospital antiseptic concentrate. Contains chlorhexidine). Penis held up out of the way with a swab fixed underneath the drape. A drape with a small hole of 5cm in diameter in the middle covers the operation site. The testes are gently pulled through to be in the field of the procedure. PROCEDURE BIOPSIES TAKEN AT RANDOM As for under local anesthetic. 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 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, for example, 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. REQUIREMENTS AND PRE-OP AND POST-OP CARE See open biopsy under local anesthesia and microsurgical epididymal sperm aspiration.

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PROTOCOL: MICROSURGICAL EPIDIDYMAL SPERM ASPIRATION (MESA) INDICATION Patients with obstructive azoospermia with normal spermatogenesis who wish to have epididymal sperm cryopreserved. The main drawback of MESA is that it is an invasive and expensive procedure requiring 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 Patient is fully draped with the operation site obscured to the patient. Patient wears a top only. Operation site cleansed with antiseptic solution (HAC, Zeneca: hospital antiseptic concentrate. Contains chlorhexidine). Penis held up out of the way with a swab fixed underneath the drape. Four drapes cover the operation site leaving the scrotum uncovered. MESA PROCEDURE MESA can be performed during any scrotal exploration taking place even long before the ICSI treatment is scheduled or in a satellite centre, for example, 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 1ml syringe filled with 0.1ml HEPES-buffered Earle’s medium supplemented with

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0.4% human serum albumin. The aspirated epididymal fluid is then transferred into a Falcon test tube, filled with 0.9ml 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 min) 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. 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-Meuller) Jeweller’s forceps (3×) (E 1947 Storz) (72BD330Aesculaep) Curved blund 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 nr 15 (0505 Swann-Morton) Knife handle with blades nr 11 (0503 Swann-Morton) NaCl 0.9% 500ml (B1323 Baxter) with 2500 U.I. heparine Novo (Heparine Novo Nordisk Pharma) Syringes 20cc (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)

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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=200mm (M 382162 Leica)

48 Gamete intrafallopian transfer (GIFT) Machelle M Seibel

Gamete intrafallopian transfer (GIFT) has emerged as one of the major forms of assisted reproductive technology (ART). It began in 1979 with a case report in which clomiphene citrate, 50mg, was given to a woman on cycle days 5 to 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 After tubal ligation, 55 monkeys mated before the day of anticipated ovulation and laparoscopic oocyte retrieval performed within 12 hours of ovulation. The oocyte was transferred to the ipsilateral fallopian tube proximal to the ligation and the monkeys were mated again. Five (16%) of the 31 monkeys conceived and delivered normal offspring. This study demonstrated the potential application of gamete transfer after surgical tubal occlusion and established a basis for additional clinical studies. 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 to 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 silicone polymer sheath so that neither contraception nor masturbation was used.4 The first reported transfer of gametes in patients with unexplained infertility was by Asch et al.5 To ensure that fertilization occurred within

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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, gamete intrafallopian transfer (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. The primary contemporary significance of GIFT is religious and personal. As in the past, it still requires less equipment and less complexity than IVF and therefore remains an important procedure.

PATIENT SELECTION Patients who undergo GIFT should have completed a full Infertility evaluation including semen analysis, hysterosalpingogram (HSG), timed endometrial biopsy, and other tests appropriate to the couple’s history. Diagnostic laparoscopy is often performed prior to undergoing GIFT, but some couples prefer to combine the laparoscopy with either GIFT or IVF to increase their chances of conceiving. It is always necessary to address the couple’s insurance coverage, including restrictions that could be affected by combining these techniques.6 It is also important to consider whether or not the ovarian stimulation will interfere with visualization of the pelvic structures, or if unexpected scarring will compromise the GIFT procedure. Having the ability to provide IVF as a back up becomes extremely important in such instances. Theoretical hazards such as the impact of the laser plume, or increased levels of carbon dioxide for a prolonged time period on fertilization have not been completely analyzed. There are no statistical data that speaks to the potential benefit or risk of combining these procedures. 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, stage I and II endometriosis, cervical factor infertility, and oligospermia.7 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. This area is controversial as some groups will exclude patients with a history of tubal

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disease or tubal surgery while others will accept patients with adnexal and peritubal adhesions. Because the rate of ectopic pregnancy among patients undergoing GIFT is less than 1%,8 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. The protocols used for GIFT are quite variable and include a variety of ovulatory inducing agents and combinations including clomiphene citrate alone or in combination with human menopausal gonadotropins (hMG) and follicle stimulating hormone (FSH) and hMG.9 The most common protocol is pituitary desensitization with a gonadotropin releasing hormone (GnRH) agonist followed by either FSH or hMG. As with IVF, no one protocol has proven to be more effective than another, although long term gonadotropin releasing hormone agonist protocols appear to yield higher pregnancy rates than short regimens.10 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 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 listed.

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Because of concerns over disease transmission and the fact that better preparations exist today, media containing fetal cord serum have been omitted. Ham’s F-10 with 7.5% patient serum containing penicillin G, 75mg/l; streptomycin sulfate, 75mg/l; calcium lactate, 252mg/l; and sodium bicarbonate, 2.1g/l at a pH of 7.35 and osmolarity of 280 to 285 mOsm—Sigma Chemical, St Louis Earle’s medium—Sigma Chemical, St Louis Modified human tubal fluid (MHTF)—Irvine Scientific HEPES buffered media if not using a CO2 incubator or isolette—Irvine Scientific 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 at 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. Established criteria for the Society for Assisted Reproductive Technologies (SART) and the World Health Organization (WHO) differ as to their definition of male factor infertility. 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 using conscious sedation 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. Those authors first perform transvaginal ultrasound guided retrieval followed by GIFT using a 5mm laparoscope and two 3mm trocars with local anesthesia and intravenous sedation.11 There is a theoretical benefit to using ultrasound for the oocyte retrieval as it limits the exposure of the oocytes to the carbon dioxide necessary to achieve pneumoperitoneum. However, pregnancy data do

Fig 48.1 Laparoscopic oocyte retrieval (inset shows needle entering follicle).

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Fig 48.2 Ultrasound-guided transvaginal oocyte retrieval (inset shows needle entering follicle).

Fig 48.3 GIFT via laparascopy. 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. I 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 2–3cm

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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 in to view if they recede following aspiration of the larger follicles. Following retrieval, the oocytes are placed into culture media to assess maturity. Some of the culture medium used are listed below. IVF media from Zander IVF Menezo B2 medium with 50% inactivated maternal serum—CCD International Follicular fluid Ham’s F-10—Sigma Chemical, St Louis Ham’s F-10 with 50% maternal serum—Sigma Chemical, St Louis Earle’s medium—Sigma Chemical, St Louis Human tubal fluid (HTF) medium—Irvine Scientific B2 medium—CCD International As shown above, clear follicular fluid from the current case can be used instead of culture medium. No particular method has proven more effective than the other has although it has suggested that outcomes might be higher when follicular fluid is used to capacitate sperm and as a transfer medium rather than Ham’s F-10.12 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, even the authors of that report recommended IVF in preference to GIFT for sperm counts below 1.5×106. TRANSFER There are a number of transfer catheters designed specifically for GIFT that include: 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 Barnaby, Baltimore, MD); Cook Catheter (no. NRT 5.0-VT-50-PNS-GIFT, Cook, Melbourne, Australia). All should be quality assured for gamete toxicity using the human sperm survival assay and/or the two cell mouse bioassay prior to use. 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 5mm air bubble should separate the eggs and sperm so that fertilization cannot begin before

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transfer. However, at least one report suggests that results may be higher if the gametes are allowed to mix prior to transfer.13 If both tubes are cannulated, it is advisable to use different catheters for each. Typically, 10000 to 100000 motile sperm per egg are transferred. However, this number may be increased to 200000 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. In one report, a 52.7% pregnancy rate per cycle was obtained when three oocytes were transferred versus 30.7% when only two were transferred.14 Another study15 performed retrospectively on 399 cycles found a three times higher clinical pregnancy

Fig 48.4 GIFT. Gametes are separated by air or medium. 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.16 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 2–3cm is usually sufficient. If a metal cannula is used, it can be inserted 1–2cm and the transfer catheter advanced another 1–2cm. Often one can see the ampulla swell slightly following the 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=2.39).17 While it is intriguing to speculate why this might occur, it must be realized that other

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investigators have found that pregnancy occurred in 40.3% of cycles were used compared with 21.6% when only one tube was used.16 In order to avoid laparoscopy and general anesthesia, some authors have suggested using hysteroscopy to transfer the gametes.18 Under conscious sedation, Possati et al19 treated 27 patients with transvaginal ultrasound guided oocyte retrieval followed by flexible hysteroscopic gamete transfer using a 30° hysteroscope that had a 4mm outer diameter sheath and CO2 as a distention medium. Atropine 0.5cc intramuscularly was administered 30 minutes prior to the procedure. The catheter was advanced 2–4cm into the tubal lumen and the carbon dioxide distention stopped 1 minute prior to injecting the gametes. A pregnancy rate of 25.5% per cycle was reported.

Fig 48.5 Gamete uterine transfer. 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 3cm or 6cm. 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.20 Others have reported transvaginal ultrasound guided GIFT with variable results.21 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 wide spread appeal despite its inherent appeal. There are also several reports of placing gametes directly into the uterine cavity (gamete uterine transfer or GUT) following transvaginal

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oocyte retrieval (Fig 48.5). The procedure is similar to IUI and therefore requires limited technical experience or equipment. Success rates of 15% have been reported but series sizes are small.6 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 survey. The decline is in large part due to the need for general anesthesia required for GIFT coupled with the fact that success rates for IVF are roughly comparable to those of GIFT. Data for 1997, the most recent data available, do not separate GIFT from other forms of assisted reproduction due to the small percentages they represent. The overall live birth rates per 100 cycles for ART are 30.7% for women under 35 years, 25.5% for women ages 35 to 37, 17.1% for women ages 38 to 40, and 7.6% for women over the age of 40. The National Summary suggests that success rates for GIFT be calculated by assuming 3.2% of these data were due to GIFT in 1997. Despite the fact that GIFT requires gametes to be placed directly into the fallopian tubes, ectopic pregnancy rates are only 0.3%. These statistics are based on 55002 started cycles, 17054 deliveries, and 24582 babies for all ART procedures. Multiple births occur in roughly one in three deliveries. The twin pregnancy rate is 25.9 and triplets or more occur in 5.3% of deliveries. As with other forms of assisted reproduction, women older than 40 years experience worse outcomes and higher cancellation rates. However, one group recommended GIFT rather than donor oocyte IVF for women aged 40 to 42 with good ovarian reserve because it was less expensive (mean cost per infant $22924 versus $30457 for donor oocyte IVF).22 This of course must be established on a case by case basis. The cause of infertility leading to GIFT also bears 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 chlamydial trachomatis IgG antibodies is also associated with a significantly lower implantation rate and a tendency toward a higher early pregnancy loss.23 Other investigators have found an association between the type of light source used at laparoscopy and pregnancy rates. Those treated with a

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halogen light source had a pregnancy rate of 50% in 22 cycles compared with 9% in 12 cycles using a xenon light source.24 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.25 Patients may, however, fare better with epidural anesthesia versus general anesthesia (40.9% v 31%),26 although at least one report could not demonstrate a difference in success rates whether GIFT was performed under local anesthesia with air versus carbon dioxide pneumoperitoneum.27 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.16,28 In one large study of 1826 GIFT cycles,29 women older than 40 were found to have a higher rate of cancellation and a significantly lower delivery rate (12.5%). Women aged 44–45 years of age had a pregnancy rate of 4.2%. As with IVF, many centers obtain no pregnancies among women greater than 43 years. For this reason, it is common practice to transfer a greater number of oocytes into women over 40 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 achieved better results than IUI. In one randomized study30 of 200 couples receiving either GIFT or hMG followed by IUI or intercourse, the GIFT pregnancy rate was 26.7% versus 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 study31 evaluated cumulative pregnancy rates for GIFT and pronuclear stage transfer (PROST) used for male factor infertility versus 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. One study32 of 2941 patients evaluated the pregnancy outcome for successful GIFT patients undergoing a subsequent procedure. The initial pregnancy rate was 31%, 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. This 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.

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MODIFICATIONS OF GAMETE INTRAFALLOPIAN TRANSFER Since its inception, several variations of GIFT have evolved. Some of the incentive for these modifications resulted from a desire to reduce the need for general anesthesia. However, also of major importance was a need to assess whether or not fertilization occurred. This was accomplished by substituting the transfer of gametes with the transfer of pronuclear stage embryos (PROST), zygotes (zygote intrafallopian transfer or ZIFT), and multicell embryos (tubal embryo transfer or TET). The disadvantage of these procedures is that they require two anesthesias, one for the oocyte retrieval and a second for the transfer. Success rates are generally comparable to GIFT and, as demonstrated in metaanalysis comparing intrauterine with tubal embryo transfer,33 offer no outcome advantage. Overall, the percentage of all ART cycles attributed to ZIFT is 2% in 1995 and 1996, and 1.6% in 1997. Because they include in vitro fertilization and offer little if any advantage over IVF, I believe that these procedures will continue to decline in use over time.

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, many 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, the percentage of ART cycles attributed to GIFT has declined by nearly 50% from 1995 to 1997, the last three years for which data are available. It is likely that, over time, GIFT will become an even smaller percentage of ART.34 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. Simplified models of GIFT such as gamete uterine transfer may in the future increase the appeal of this procedure and widen its utilization.

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REFERENCES 1 Shettles LB. Ova harvest with in vivo fertilization. Am J Obstet Gynecol (1979); 133:845. 2 Kreitman O, Hodgen GD. Low tubal ovum transfer: An alternative to in vitro fertilization. Fertil Steril (1980); 34:374. 3 Tesarik J, Pilka L, Dvorak M, et al. Oocyte recovery in vitro insemination and transfer into the oviduct after its microsurgical repair at a single laparotomy. Fertil Steril (1983); 39:472. 4 McLaughlin DS, Troike DE, Tegenkamp TR, et al. Tubal ovum transfer: A Catholic approved alternative to in vitro fertilization. Lancet (1987); 1:214. 5 Asch RN, Ellsworth LR, Balmaceda JP, Wong PC. Pregnancy after translaparoscopic gamete intrafallopian transfer. Lancet (1984); 2:134. 6 Dlugi AM, Mersol-Barg M, Seibel MM. Gamete intrafallopian transfer. In: Seibel MM, ed. Infertility: A Comprehensive Text. Appleton and Lange; Norwalk: Conn, 1997:687. 7 Ranieri M, Beckett VA, Marchant S, Kinis A, Serhal P. Gamete intrafallopian transfer or in-vitro fertilization after failed ovarian stimulation and intrauterine insemination in unexplained infertility? Hum Reprod (1995); 10:2023. 8 National Summary and Fertility Clinic Reports. 1997 Assisted Reproductive Technology Success Rates. US Department of Health and Human Services, Centers for Disease Control and Prevention. December 1999. 9 Campo S, Garcea N. Efficacy assessment of highly purified folliclestimulating hormone alone or in combination with human menopausal gonadotropin during pituitary suppression in patients undergoing GIFT for unexplained infertility. Gynecol Endocrinol (1998); 12:161–6. 10 Cramer DW, Barbieri RL, Hornstein MD, et al. Gonadotropinreleasing hormone agonist use in assisted reproduction cycles: The influence of long and short regimens on pregnancy rates. Fertil Steril (1999); 72:83–9. 11 Milki AA, Tazuke SI. Office laparoscopy under local anesthesia for gamete intrafallopian transfer: technique and tolerance. Fertil Steril (1997); 68:128–32. 12 Fakih H, Vijayakumar R. Improved pregnancy rate and outcome with gamete intrafallopian transfer when follicular fluid is used as a sperm capacitation and gamete transfer medium. Fertil Steril (1990); 53:515. 13 Ali J, Joshi HN, Al-Badr M, et al. Ensuring contact between gametes immediately prior to transfer improves the efficiency of the gamete intrafallopian transfer procedure. Med Science Res (1998); 26:379–80. 14 Corson SL, Batzer F, Eisenberg E, et al. Early experience with the GIFT procedure. J Reprod Med (1986); 31:219.

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15 Penzias AS, Alper MM, Oskowitz SP, Berger MJ, Thompson IE. Gamete intrafallopian transfer: Assessment of the optimal number of oocytes to transfer. Fertil Steril (1991); 55:311. 16 Guzick DS, Yao YAS, Berga SL, et al. Endometriosis impairs the efficacy of gamete intrafallopian transfer: Results of a case-control study. Fertil Steril (1994); 62:1186. 17 Ransom MX, Corsan GH, Garcia AJ, Dogerty KA, Kemmann E. Tubal selection for gamete intrafallopian transfer. Fertil Steril (1994); 61:386. 18 Risquez F, Boyer P, Rolet F, et al. Retrograde tubal transfer of human embryos. Hum Reprod (1990); 5:185. 19 Possati G, Seracchiolo R, Melega C, Pareschi A, Maccolini R, Flamigni C. Gamete intrafallopian transfer by hysteroscopy as an alternative treatment for infertility. Fertil Steril (1991); 56:496. 20 Porcu E, Dal Prato L, Seracchioli R, Petracchi S, Fabbri R, Flamigni C. Births after transcervical gamete intrafallopian transfer with a falloposcopic delivery system. Fertil Steril (1997); 67:1175–7. 21 Jansen RPS, Anderson JC. Nonsurgical gamete intrafallopian transfer. Semin Reprod Endocrinol (1995); 13:72. 22 Silva PD, Olson KL, Meisch JK, Silva DE. Gamete intrafallopian transfer: A cost-effective alternative to donor oocyte in vitro fertilization in women aged 40–42 years. J Reprod Med (1998); 43:1019–22. 23 Sharara FI, Queenan JT Jr. Elevated serum Chlamydia trachomatis IgG antibodies. Association with decreased implantation rates in GIFT. J Reprod Med (1999); 44:581–6. 24 Evans J, Wells C, Hood K. A possible effect of different light sources on pregnancy rates following gamete intrafallopian transfer. Hum Reprod (1999); 14:80–2. 25 Beilin Y, Bodian CA, Mukherjee T, et al. The use of propofol, nitrous oxide, or isoflurane does not affect the reproductive success rate following gamete intrafallopian transfer: a multicenter pilot trial/survey. Anesthesiology (1999); 90:36–41. 26 Chung PH, Yeko TR, Mayer JC, Vila H Jr., Welden SW, Maroulis GB. J Reprod Med (1998); 43:681–6. 27 Milki AA, Tazuke SI. Comparison of carbon dioxide and air pneumoperitoneum for gamete intrafallopian transfer under conscious sedation and local anesthesia Fertil Steril (1998); 69:552–4. 28 Chang MY, Chiang CH, Hsieh TT, Soong YK, Hsu KH. The influence of endometriosis on the success of gamete intrafallopian transfer. J Assist Reprod Genet (1997); 14:76–82. 29 Bopp BE, Alper MM, Thompson IE, Mortola J. Success rates with gamete intrafallopian transfer and in vitro fertilization in women of advanced maternal age. Fertil Steril (1995); 63:1278. 30 Wessels PH, Cronje HS, Oosthuizen AP, Trumpelmann MD, Grobler S, Hamlett DK. Cost-effectiveness of gamete intrafallopian transfer in

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comparison with induction of ovulation with gonadotropins in the treatment of female infertility: A clinical trial. Fertil Steril (1992); 57:163. 31 Robinson D, Syrop CH, Hammitt DG. After superovulationintrauterine insemination fails: The prognosis for treatment by gamete intrafallopian transfer/pronuclear stage transfer. Fertil Steril (1992); 57:606. 32 Molloy D, Doody ML, Breen T. Second time around: A study of patients seeking second assisted reproduction pregnancies. Fertil Steril (1995); 64:546. 33 Tournaye H. Tubal embryo transfer improves pregnancy rate. Hum Reprod (1997); 12:630–1. 34 Castelbaum AJ, Freedman MF. Is there a role for gamete intrafallopian transfer and other tubal insemination procedures? Current Opin Obstet Gynecol (1998); 10:239–42.

49 Zygote intrafallopian transfer (ZIFT) Ariel Weissman, Jacob Farhi, David Levran

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.1 Soon thereafter, Devroey et al described the first successful zygote intrafallopian transfer (ZIFT) in humans.2 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 with UET, ZIFT was gradually incorporated into clinical practice. In 1991, ZIFT comprised 6.4% of all ART cycles in the US and Canada.3 Subsequently, with the publication of randomized clinical trials that failed to show a clear advantage for ZIFT compared with UET, along with the introduction of intracytoplasmic sperm injection (ICSI) as a powerful clinical tool for the treatment of male factor infertility,4 the use of ZIFT has declined (Fig 49.1). In 1996, ZIFT comprised only 1.8% of ART cycles in the US, corresponding to 1,200 procedures.5 Nevertheless, throughout 1989–96, delivery rates per retrieval in North America have been consistently superior with ZIFT compared with UET (Fig 49.2).3,5–11 In 1996, for example, 30.9% of all ZIFT cycles resulted in delivery,

Fig 49.1

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Percentage of ZIFT of all ART cycles in the United States and Canada, 1989–1996* (data from references 3, 5–11). * 1996, US only

Fig 49.2 Delivery rates per retrieval after IVFET and IVF-ZIFT in the US and Canada, 1989–1996* (data from references 3, 5– 11). * 1996, US only compared with only 26% of retrievals followed by UET. It should be stated, however, that information from national statistics is difficult to evaluate. It is compiled from data originating from different operators and laboratories, and from heterogeneous patient profiles that may differ by age distributions, diagnostic categories, treatment protocols, and the number and stage of zygotes/embryos transferred per patient. Nevertheless, the consistently higher delivery rates observed with ZIFT simply suggest that “there is something about ZIFT” that should be explored further. Our objective in this chapter is 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 Several techniques, alternative to UET, have been introduced for the treatment of nontubal infertility by ART. These include gamete intrafallopian transfer (GIFT) and several techniques for tubal embryo

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transfer, which 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 2–8 cell stage the procedure is known as TET (tubal embryo transfer). In the literature all the latter three are commonly referred to as ZIFT. The GIFT procedure is beyond the scope of this chapter and is covered in detail in Chapter 48. This chapter therefore focuses on 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 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,12 and commercial culture media that attempt to mimic the tubal milieu are widely used. One of the indications for tubal transfers may be a sub-optimal in vitro culture system.13 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.13 An in vivo role has been suggested for the Fallopian tube in preventing zona hardening, especially in couples with advanced female partner age.14 Several investigators have demonstrated that coculture on oviduct epithelial cells is efficient in promoting preimplantation embryo development.15–18 The specificity of human tubal cells secretions for embryo development is challenged by the development of coculture techniques involving human endometrial cells19 or non-human genital tract cell layers yielding favorable results.20 In addition, advances in culture media composition and laboratory conditions now allow for in

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vitro embryo culture to the blastocyst stage. Retrospective analysis of results obtained with ZIFT versus UET at the blastocyst stage after coculture in an unselected population yielded comparable ongoing pregnancy rates per transfer, at the range of 28% for both groups.21 Furthermore, culture protocols using artificially prepared sequential media support human blastocyst development and implantation irrespective of coculture use.22 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 then be sought. One of the potential advantages of ZIFT over UET is a more appropriate mode and timing of embryo entry 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 methylen blue in 57% of transfers23 and movement of x-ray contrast medium towards the Fallopian tubes and cervix/vagina in 38.2% and 20.6%, respectively.24 Furthermore, embryos have been found in the vagina after UET,25,26 and some UET techniques are more frequently associated with ectopic pregnancy.27,28 There is evidence that a greater frequency of contractions on the day of embryo transfer is associated with a reduced pregnancy rate. Fanchin et al have found a stepwise decrease in clinical and ongoing pregnancy rates as well as in implantation rates from the lowest to the highest junctional zone contractions frequency groups at the time of UET.29 Furthermore, interference with the endometrium by the transfer catheter at the time of UET may change the junctional zone contractions pattern and affect implantation in a mechanical way. Lensy et al,30 using a model of Echovist bolus during mock transfer, have demonstrated that difficult procedures generated strong random waves in the fundal area and waves from fundus to cervix which relocated the Echovist in the majority of cases. In contrast, easy mock transfers did not change endometrial mechanical activity, and the Echovist remained in the upper part of the uterine cavity for more than 45 minutes. Thus, the mechanical activity of the uterus may depend on physical stimulation by the transfer catheter, which is capable of relocating intrauterine embryos. These ill effects are probably prevented with ZIFT. In addition, endometrial activity is minimal and progressively decreases during the luteal phase.30,31 Fanchin et al observed a significant decrease in uterine contractions frequency from the day of human chorionic gonadotropin (hCG) onward, with a marked decline between the days of 2–4 cell embryo and blastocyst transfers, on hCG+4 and hCG+7, respectively. Thus, high frequency

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junctional zone contractions at the time of 2–4 cell embryo transfers reduce conventional UET outcome, probably by fostering embryo expulsion from the uterine cavity. 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. Another potential problem limiting the success of UET procedures may be related to the presence of cervical microorganisms on embryo transfer catheters. It has recently been shown that implantation and clinical pregnancy rates were significantly lower in women with positive microbial catheter tip cultures.32,33 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.33 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 49.1.

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 compared with uterine embryo transfer. Retrospective reports on ZIFT showed pregnancy rates per transfer ranging from 37% to 53% compared with only 12% to 28% for UET34–39 Subsequently,

Table 49.1. 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 micro-trauma 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

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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 blastocysts transfer. several randomized controlled trials were conducted in order to evaluate the efficacy of ZIFT versus standard UET for the treatment of non-tubal factor infertility. Only four randomized prospective clinical trials were ever published.40–43 Tanbo et al41 reported clinical pregnancies after 38% of UET and 26% of ZIFT cycles, with corresponding pregnancy rates per transfer of 45.7% and 37.9% for UET and ZIFT respectively. Tournaye et al43 reported clinical pregnancy rates per started cycle of 12.8% and 11.4% after pronuclear ZIFT and UET, respectively. As their study population was composed of male factor infertility patients, complete failed fertilization occurred in 38% of cycles. Pregnancy rates per transfer were 21.7% and 26.5%, for UET and ZIFT, respectively (not significant). Toth et al42 conducted a prospective, non-randomized trial comparing pronuclear ZIFT and UET and reported clinical pregnancy rates per transfer of 29.4% and 34.1% for ZIFT and UET, respectively. Finally, Fluker et al40 compared prospectively the outcome of ZIFT of cleavage stage embryos with UET. Clinical pregnancy rates per retrieval were 12% and 26.5% for ZIFT and UET, respectively, and the corresponding rates per transfer were 14.3% and 29%, respectively. It should be mentioned that all of the above prospective studies suffer from major drawbacks, which render their conclusions of questionable value. The four studies vary in terms of patient inclusion criteria, randomization methods, stimulation protocols, stage and number of embryos transferred, and main outcome measures reported (Table 49.2). Owing to heterogeneity in study design a valid comparison between studies cannot be made. Furthermore, all four studies either lack power calculation,41,42 or initial sample size requirements have not been met.40,43 As discussed by Toth et al,42 in order to achieve a 20% increase in the clinical pregnancy rate (for example, from 30% to 36%) a sample size >900 patients would be required. Overall, all four prospective trials failed to demonstrate any difference in the rates of implantation, clinical pregnancy, ongoing pregnancy, and miscarriage rate between ZIFT and standard UET. A meta-analysis using data from the three randomized prospective trials (Toth et al study42 was non-randomized and therefore

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excluded) has shown that cycle outcome with either ZIFT or UET is comparable, with a common odds ratio of 0.67 and a wide 95% confidence interval of 0.25 to 1.79 (Table 49.3). Nevertheless, considering the heterogeneity and sample size of the randomized trials, it can be concluded that the clinical efficacy of ZIFT has been never critically evaluated.

THE ZIFT PROCEDURE ZIFT normally requires general anesthesia and endotracheal intubation. Intrafallopian transfer with local anesthesia and continuous sedation has also been described.45 ZIFT is

Table 49.2. A summary of randomized trials comparing ZIFT with UET: patients, protocols and outcome.

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Table 49.3. Meta-analysis of randomized controlled trials on ZIFT versus UET (Courtesy of Dr Salim Daya). Author Odds ratio 95% confidence interval Tanbo et al (41) 0.58 0.23 to 1.45 Tournaye et al (43) 1.01 0.36 to 2.84 Fluker et al (40) 0.42 0.12 to 1.48 All trials 0.67 0.25 to 1.79 performed 18–48 hours after oocyte aspiration using a three puncture video-laparoscopic technique. After introducing the umbilical trocar and optical equipment, the abdominal cavity is surveyed. The sanguinous fluid, containing blood, follicular fluid, and pelvic fluid, is aspirated through a 5mm 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 3.5 FR 35cm Teflon catheter (Patton Laparoscopic Catheter Set, Cook Ob/Gyn, Spencer, IN, USA) is loaded with 10–20µl medium containing the zygotes or embryos. Using a third para-umbilical puncture with a dedicated trocar stylet (Cook Ob/Gyn, Spencer, IN, USA), the catheter tip is gently introduced about 3cm 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 (based on number of blastomers, even size and lack of fragmentation), 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 et al have suggested that the morphology of the human zygote at 16–18 hours after insemination can be used as a positive predictor for the outcome of day one, zygote UET.46 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

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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 pre-embryos 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 after 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 obese patients. Initial attempts at transcervical retrograde catheterization were made under ultrasound guidance, using modified embryo transfer catheters.47,48 Later, blind tactile tubal catheterization procedures with gametes49 and embryos50 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,51 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 non-surgical and laparoscopic GIFT were compared.52 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 resulting from pelvic adhesions is expected. The published experience with hysteroscopic transfer procedures is rather limited.53 The potential adverse effects of carbon dioxide exposure on gametes and embryos, and blunt endometrial trauma have limited their use. Overall, efforts to develop transcervical tubal transfer methods have not been translated into pregnancy rates higher than those achieved with UET or laparoscopic ZIFT. Consequently, this approach has been almost completely abandoned. CURRENT POTENTIAL INDICATIONS FOR ZIFT

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Originally, ZIFT was advocated for all classes of non-tubal factor infertility. Most 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 an almost double increase in the delivery rate per retrieval from only 14% in 19896 to 26% in 1996.5 Currently, delivery rates per retrieval are comparable for most categories of infertility.5 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.13,21,44,54 Who, in turn, are the patients that may benefit from ZIFT at the beginning of the third millennium? (1) 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.55 Thus, couples with repeated implantation failure (RIF) after UET represent one of the greatest challenges for the caring physician. Our own experience over the past few years consistently shows that patients with multiple failed attempts of UET are the most likely to benefit form ZIFT. RIF patients may initially present with any of the known infertility categories. Often, they would have normal clinical and laboratory cycle parameters during treatment. Although the presumed cause of infertility seems to be partly overcome by achieving good quality embryos, replacing them into the uterine cavity repeatedly fails to result in implantation and conception. The etiology for RIF is obscure in the vast majority of cases. A variety of etiologies and interventions have been advocated for couples with RIF. None 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 recently published case control study, summarizing the experience of two Israeli ART centers, the outcome of ZIFT and UET in RIF patients was compared.56 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. Patients who underwent ZIFT had a mean of 4.8±1.6 zygotes transferred, and

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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%) 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. Eighty-one patients, aged 26–46 years, with mean number of 8.4±3.4 previous failed attempts underwent a total of 112 ZIFT cycles. Clinical pregnancy rate per transfer was 35.7%, with a live birth rate per transfer of 32%. The cumulative pregnancy and live birth rates were 49.4% and 44.4%, respectively. This is remarkable because cycle outcome in the poor prognosis group of RIF patients became extremely favorable with ZIFT, comparable to the results achieved for patients with a good prognosis, who have just embarked on IVF-ET therapy in leading centers worldwide. Our results are encouraging, as they confirm the efficacy and reproducibility of ZIFT as a powerful clinical tool for the treatment of RIF. The potential advantages of ZIFT have been outlined above (Table 49.1). 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 good prognosis 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 after UET,25,26 is avoided. 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.57 Furthermore, transcervical transfer catheters may induce junctional zone contractions30 and inoculation of the uterine cavity with cervical microorganisms,32,33 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. (2) 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,58 ZIFT may spare the patient and

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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.59,60 (3) ZIFT FOR ADVANCED MATERNAL AGE Pregnancy and implantation rates decline progressively with advanced maternal age.3,5–11,61 Older patients commonly suffer from low ovarian reserve with poor response to stimulation, as well as poor egg quality with low implantation potential of resultant embryos. 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 UET and ZIFT cycles in women over 40 years of age.62 Seventy-seven consecutive UET cycles were compared with 50 consecutive ZIFT cycles performed during the same period. Laparoscopic ZIFT was performed 24–48 hours after egg retrieval, and UET was performed 48 hours after egg retrieval. A similar number of zygotes/embryos was 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 al39 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 through 39, approaching a 40% delivery rate overall for women aged 35 to 39 years. Too few cases were completed for the age group over 40 years for valid statistical analysis. In contrast, Balmaceda et al63 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. We recently summarized our experience with ZIFT in RIF patients, looking at cycle outcome by age group (unpublished). One hundred and twelve cycles in 81 patients were included in the analysis. It appears that pregnancy rates are fairly constant up to the age of 39 years. Pregnancy rates per procedure were 43.4% and 39.4% for the age groups of <35 years and 35–39 years, respectively. Clinical pregnancy rate in patients >39 years of age was 12% (3/25 cycles), demonstrating only a marginal benefit for ZIFT at this age group. Clearly, more studies are warranted in order to determine the value of ZIFT in patients with advanced maternal age.

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(4) ZIFT AFTER ICSI While the ZIFT procedure was originally advocated for couples with male factor infertility undergoing IVF, the introduction of ICSI changed dramatically 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 on the added value of ZIFT in cycles where fertilization was achieved by means of ICSI. Boldt et al64 analyzed whether the mode of embryo transfer (ZIFT v 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.65 One hundred and fifty-one ZIFT cycles were compared with 548 UET cycles. Clinical pregnancy, live birth, and implantation rates were all significantly higher for the ICSI-ZIFT group (34.4%, 27.2%, and 15.1%, respectively) compared with the ICSI-UET group (14.2%, 11%, and 6.6%, respectively). Fifty-one of the patients underwent both ZIFT and UET. Here again, clinical pregnancy, live birth, and implantation rates were all significantly higher for the ZIFT group (34.6%, 23.1%, and 11.9%) compared with the UET group (13.4%, 10.4%, and 6.6%, respectively). These data suggest that, at least under certain conditions, ZIFT may be beneficial as the method of transfer of ICSI-derived embryos. The clinical circumstances under which ZIFT should be recommended following ICSI should be further investigated. (5) 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.66 A close look at the 1996 US Registry National Summary and Fertility Clinics Reports reveals that for women under 35 years of age, live birth rate for fresh embryo transfer was 33.6%, whereas the corresponding rate following frozen-thawed transfers was only 18.2%.5 Similarly, with the use of donor eggs, corresponding figures for fresh and frozen-thawed transfers in women >39 years of age were 38.9% and 20.6%, respectively.5 Thus, cryopreservation adversely effects the implantation potential of embryos. 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

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environment may be more conductive 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 is indeed the case. In a retrospective study, Frederick et al67 reported their experience with 54 tubal transfers of frozen-thawed embryos. Embryos were frozen at the pronuclear or 2 cell stage. 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.68 Nineteen patients had 20 UET cycles, and 10 patients underwent ZIFT. The pregnancy rate per transfer was 20% in the UET group and 40% in the ZIFT group. After excluding 10 women in the UET group who had fewer than three embryos transferred, the pregnancy rates were similar in the two groups, 30% in the UET group, and 40% in the ZIFT group. Finally, in a small prospective study reported by Van Voorhis et al,69 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% v 10%), clinical (68% v 24%) and ongoing pregnancy rates (58% v 19%) compared with UET. It was concluded that tubal transfer of cryopreserved embryos is highly effective and offers an improved pregnancy rate 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 further explored.

CLINICAL AND TECHNICAL ISSUES RELATED TO ZIFT PROCEDURES PRONUCLEAR VERSUS CLEAVAGE STAGE TUBAL EMBRYO TRANSFER As mentioned above, ZIFT was originally described with pronuclear-stage embryo transfer into the Fallopian tube.2 Subsequently, a variant in which day 2 embryos are replaced in the tubal lumen (tubal embryo transfer; TET) was introduced.70 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 al,71 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.

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We have recently analyzed the results from our center (unpublished), where we normally perform ZIFT at the pronuclear stage. Cleavage stage tubal transfers are only performed in order to avoid weekend procedures or based on physician availability. Over a 2 year period, 120 transfers were performed in 86 patients at the pronuclear stage, and 42 transfers were performed in 27 patients at the 2–4 cell stage. All patients had multiple failures of UET, with a mean of 7.1 and 8.7 failed cycles in the cleavage and 2–4 cell stage groups, respectively. Clinical pregnancy rates per transfer were 38.3% and 40.5%, in the pronuclear stage and cleavage stage groups, respectively. A similar implantation rate of 12% was observed in both groups. It is suggested from our results that the increased implantation and pregnancy rates observed with ZIFT in patients with repeated failed UET cannot be simply explained by poor culture conditions in the lab, since extended culture to the 2–4 cell stage does not compromise the success of ZIFT. Pronuclear stage and cleavage stage ZIFT thus seem to be comparable. 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 than have been selected based on morphologic criteria and cleavage rate on day 2 or 3 UET. However, if implantation rates would be comparable with ZIFT to those achieved with UET than the multiple pregnancy rate with ZIFT would be unacceptably high. In a retrospective study, summarizing two years’ experience, Bollen et al reported results after transferring three oocytes, three zygotes and three embryos in GIFT, ZIFT, and UET, respectively.37 Implantation and clinical pregnancy rates were significantly higher for ZIFT (18.2% and 38.5%) compared with 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 al42 and Tanbo et al,41 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 al38 were able to reduce the multiple 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.72 For this reason, it has been our practice to replace five to six zygotes during ZIFT in patients

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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.56 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%), compared with 38% with transfer of six zygotes and 26% with transfer of four. We normally transfer five zygotes to RIF patients who are considered “good prognosis”—young—have produced multiple eggs and zygotes, and have good quality zygotes to select for transfer.46 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 of 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.56 Of 112 cycles, 38 pregnancies were achieved, 15 of them being multiple (40%). There were 10 sets of twins, two triplets, two quadruplets, and one quintuplet. Thus, 13% of all pregnancies were high order multiples. In 70% of all multiple pregnancies and 80% of high order multiples six zygotes were replaced. Consequently, our policy has been changed, and we only transfer a maximum of five zygotes in RIF patients undergoing ZIFT. 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 sub-class of tubal factor infertility. As early as 1989, Palermo et al advised the use of the ZIFT procedure “…if at least one healthy Fallopian tube is present”.73 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 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.37 Although the proportion of patients with a

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single healthy tube was not reported, there were no ectopic pregnancies in the entire ZIFT group. In a non-randomized study, Pool et al39 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% v 8%) and ongoing pregnancy/delivery rates (34% v 15.8%), with ZIFT versus 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 versus bilateral tubal patency.74 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 later group ZIFT could not be performed because of 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% v 9.4%) and clinical pregnancy rates (37.1% v 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, but certainly not increased with zygote transfer into a single patent tube, as compared with patients with bilateral tubal patency. Thus, whenever indicated, ZIFT may be offered to patients with a single patent tube. ZIFT VERSUS 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.75,76 Menezo and Janny21 reported their results of a retrospective analysis comparing ZIFT with uterine transfer at the blastocyst stage after coculture in an unselected population. A total of 137 ZIFT procedures were compared with 217 blastocyst transfers. While significantly more zygotes than blastocysts were transferred (2.6±0.78 v 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 have recently reported a retrospective comparison of day 3 versus blastocyst stage transfer in RIF patients.77 Twenty-two

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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 who underwent blastocyst transfer. Clinical pregnancy and implantation rates were significantly higher with blastocyst transfer (40% and 11.3% v 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).78 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—a mean of 4.1 and 8.1 previous failures by Cruz et al and us, 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.56 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 in 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.

SUMMARY AND CONCLUSIONS Despite the lack of convincing prospective data to support the tubal transfer of zygotes or embryos after IVF, there may be clinical conditions

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where the ZIFT procedure would be beneficial. Currently, the most valid indication for ZIFT seems 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 has 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, that 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, after ICSI and for frozen thawed embryo transfers. All later indications should be evaluated by properly designed prospective studies. Results with ZIFT using either pronuclear or cleavage stage embryos are comparable, so that ZIFT seems to be equally effective on day one or two after 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 seems to remain an effective treatment modality for selected infertile couples. Fifteen years after its first successful application, there is 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.

REFERENCES 1 Balmaceda JP, Pool TB, Arana JB, Heitman S, Asch RH. Successful in vitro fertilization and embryo transfer in cynomolgus monkeys. Fertil Steril (1984); 42:791–5. 2 Devroey P, Braeckmans P, Smitz J, et al. Pregnancy after translaparoscopic zygote intrafallopian transfer in a patient with sperm antibodies. Lancet (1986); 1:1329. 3 Assisted reproductive technology in the United States and Canada: 1991 results from the Society for Assisted Reproductive Technology generated from the American Fertility Society Registry. Fertil Steril (1993); 59:956–62.

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4 Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet (1992); 340:17–8. 5 Assisted reproductive technology in the United States: 1996 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril (1999); 71:798–807. 6 In vitro fertilization-embryo transfer (IVF-ET) in the United States: 1989 results from the IVF-ET Registry. Medical Research International, Society for Assisted Reproductive Technology, The American Fertility Society. Fertil Steril (1991); 55:14–22. 7 In vitro fertilization-embryo transfer (IVF-ET) in the United States: 1990 results from the IVF-ET Registry. Medical Research International. Society for Assisted Reproductive Technology (SART), The American Fertility Society. Fertil Steril (1992); 57:15–24. 8 Assisted reproductive technology in the United States and Canada: 1992 results generated from the American Fertility Society/Society for Assisted Reproductive Technology Registry. Fertil Steril (1994); 62:1121–8. 9 Assisted reproductive technology in the United States and Canada: 1993 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril (1995); 64:13–21. 10 Assisted reproductive technology in the United States and Canada: 1994 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril (1996); 66:697–705. 11 Assisted reproductive technology in the United States and Canada: 1995 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril (1998); 69:389–98. 12 Dickens CJ, Maguiness SD, Comer MT, Palmer A, Rutherford AJ, Leese HJ. Human tubal fluid: formation and composition during vascular perfusion of the fallopian tube. Hum Reprod (1995); 10:505– 8. 13 Tournaye H, Camus M, Ubaldi F, Clasen K, Van Steirtgeghem A, Devroey P. Tubal transfer: a forgotten ART? Is there still an important role for tubal transfer procedures? Hum Reprod (1996); 11:1815–8. 14 Cohen J. Assisted hatching: indications and techniques. Acta Eur Fertil (1993); 24:215–9. 15 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. 16 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.

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17 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. 18 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. 19 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. 20 Menezo YJ, Guerin F, Czyba JC. Improvement of human early embryo development in vitro by coculture on monolayers of Vero cells. Bio Reprod (1990); 42:301–6. 21 Menezo YJ, Janny L. Is there a rationale for tubal transfer in human ART? Hum Reprod (1996); 11:1818–20. 22 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. 23 Mansour RT, Aboulghar MA, Serour GI, Amin YM. Dummy embryo transfer using methylene blue dye. Hum Reprod (1994); 9:1257–9. 24 Knutzen V, Stratton CJ, Sher G, McNamee PI, Huang TT, Soto-Albors C. 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. 25 Poindexter AN, Thompson DJ, Gibbons WE, Findley WE, Dodson MG, Young RL. Residual embryos in failed embryo transfer. Fertil Steril (1986); 46:262–7. 26 Schulman JD. Delayed expulsion of transfer fluid after IVF/ET. Lancet (1986); 1:44. 27 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. 28 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. 29 Fanchin R, Righini C, Olivennes F, Taylor S, de Ziegler D, Frydman R. Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization. Hum Reprod (1998); 13:1968–74. 30 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. 31 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. 32 Fanchin R, Harmas A, Benaoudia F, Lundkvist U, Olivennes F, Frydman R. Microbial flora of the cervix assessed at the time of

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embryo transfer adversely affects in vitro fertilization outcome. Fertil Steril (1998); 70:866–70. 33 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. 34 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. 35 Hammitt DG, Syrop CH, Hahn SJ, Walker DL, Butkowski CR, Donovan JE. Comparison of concurrent pregnancy rates for in-vitro fertilization—embryo transfer, pronuclear stage embryo transfer and gameta intra-fallopian transfer. Hum Reprod (1990); 5:947–54. 36 Yovich JL, Yovich JM, Edirisinghe WR. The relative chance of pregnancy following tubal or uterine transfer procedures. Fertil Steril (1988); 49:858–64. 37 Bollen N, Camus M, Staessen C, Tournaye H, Devroey P, Van Steirteghem AC. The incidence of multiple pregnancy after in vitro fertilization and embryo transfer, gamete, or zygote intrafallopian transfer. Fertil Steril (1991); 55:314–8. 38 Devroey P, Staessen C, Camus M, De Grauwe E, Wisanto A, Van Steirteghem AC. Zygote intrafallopian transfer as a successful treatment for unexplained infertility. Fertil Steril (1989); 52:246–9. 39 Pool TB, Ellsworth LR, Garza JR, Martin JE, Miller SS, Atiee SH. Zygote intrafallopian transfer as a treatment for nontubal infertility: a 2-year study. Fertil Steril (1990); 54:482–8. 40 Fluker MR, Zouves CG, Bebbington MW. A prospective randomized comparison of zygote intrafallopian transfer and in vitro fertilizationembryo transfer for nontubal factor infertility. Fertil Steril (1993); 60:515–9. 41 Tanbo T, Dale PO, Abyholm T. Assisted fertilization in infertile women with patent fallopian tubes. A comparison of in-vitro fertilization, gamete intra-fallopian transfer and tubal embryo stage transfer. Hum Reprod (1990); 5:266–70. 42 Toth TL, Oehninger S, Toner JP, Brzyski RG, Acosta AA, Muasher SJ. Embryo transfer to the uterus or the fallopian tube after in vitro fertilization yields similar results. Fertil Steril (1992); 57:1110–3. 43 Tournaye H, Devroey P, Camus M, Valkenburg M, Bollen N, Van Steirteghem AC. 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. 44 Tournaye H. Tubal embryo transfer improves pregnancy rate. Hum Reprod (1997); 12:630–31. 45 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.

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46 Scott LA, Smith S. The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum Reprod (1998); 13:1003–13. 47 Jansen RP, Anderson JC, Sutherland PD. Nonoperative embryo transfer to the fallopian tube. N Engl J Med (1988); 319:288–91. 48 Jansen RP, Anderson JC. Catheterisation of the fallopian tubes from the vagina. Lancet (1987); 2:309–10. 49 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. 50 Diedrich K, Bauer O, Werner A, van der Ven H, al-Hasani S, Krebs D. Transvaginal intratubal embryo transfer: a new treatment of male infertility. Hum Reprod (1991); 6:672–5. 51 Scholtes MC, Roozenburg BJ, Verhoeff A, Zeilmaker GH. A randomized study of transcervical intrafallopian transfer of pronucleate embryos controlled by ultrasound versus intrauterine transfer of fourto eight-cell embryos. Fertil Steril (1994); 61:102–4. 52 Jansen RP, Anderson JC. Transvaginal versus laparoscopic gamete intrafallopian transfer: a case-controlled retrospective comparison. Fertil Steril (1993); 59:836–40. 53 Seracchioli R, Possati G, Bafaro G, et al. Hysteroscopic gamete intrafallopian transfer: a good alternative, in selected cases, to laparoscopic intra-fallopian transfer. Hum Reprod (1991); 6:1388–90. 54 Chen CD, Ho HN, Yang YS. Tubal embryo transfer improves pregnancy rate. Hum Reprod (1997); 12:629–31. 55 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. 56 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. 57 Fanchin R, Righini C, Ayoubi JM, et al. Contraction (UC) frequency decreases at the time of blastocyst transfer. Conjoint Annual Meeting of the American Society of Reproductive Medicine and Canadian Fertility and Andrology Society, Toronto, Ontario, Canada, September 25–30, 1999. Fertil Steril (1999); 72 (Suppl 1): S36. 58 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. 59 Thijssen RF, Hollanders JM, Willemsen WN, van der Heyden PM, van Dongen PW, Rolland R. Successful pregnancy after ZIFT in a patient with congenital cervical atresia. Obstet Gynecol (1990); 76:902–4. 60 Fluker MR, Bebbington MW, Munro MG. Successful pregnancy following zygote intrafallopian transfer for congenital cervical hypoplasia. Obstet Gynecol (1994); 84:659–61. 61 In vitro fertilization-embryo transfer in the United States: 1988 results from the IVF-ET Registry. Medical Research International. Society for

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Assisted Reproductive Technology. American Fertility Society. Fertil Steril (1990); 53:13–20. 62 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: J Assist Reprod Genet, Ninth World Congress on In Vitro Fertilization and Assisted Reproduction, Vienna, Austria, April 3–7, 1995. Vol. 12. 63 Balmaceda JP, Gonzales J, Bernardini L. Gamete and zygote intrafallopian transfers and related techniques. Curr Opin Obstet Gynecol (1992); 4:743–9. 64 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. 65 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. 66 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. 67 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. 68 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. 69 Van Voorhis BJ, Syrop CH, Vincent RD, Jr, Chestnut DH, Sparks AE, Chapler FK. Tubal versus uterine transfer of cryopreserved embryos: a prospective randomized trial. Fertil Steril (1995); 63:578–83. 70 Balmaceda JP, Gastaldi C, Remohi J, Borrero C, Ord T, Asch RH. Tubal embryo transfer as a treatment for infertility due to male factor. Fertil Steril (1988); 50:476–9. 71 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. 72 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. 73 Palermo G, Devroey P, Camus M, et al. Zygote intra-fallopian transfer as an alternative treatment for male infertility. Hum Reprod (1989); 4:412–5. 74 Farhi J, Weissman A, Nahum H, Levran D. Zygote intrafallopian transfer in tubal factor patients with repeated failure of implantation in IVF-ET. Fertil Steril (in press).

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75 Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod (1998); 13:3434–40. 76 Schoolcraft WB, Gardner DK, Lane M, Schlenker T, Hamilton F, Meldrum DR. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril (1999); 72:604–9. 77 Cruz JR, Dubey AK, Patel J, Peak D, Hartog B, Gindoff PR. Is blastocyst transfer useful as an alternative treatment for patients with multiple in vitro fertilization failures? Fertil Steril (1999); 72:218–20. 78 Levran D, Weissman A, Farhi J, Nahum H, Zakut H, Glezerman M. The management of patients with repeated implantation failure: a randomized prospective trial. Conjoint Annual Meeting of the American Society of Reproductive Medicine and Canadian Fertility and Andrology Society, Toronto, Ontario, Canada, September 25–30, 1999. Fertil Steril (1999); 72 (Suppl 1): S30.

50 Embryo transfer William B Schoolcraft

INTRODUCTION The main variables affecting pregnancy and implantation rates are uterine receptivity, embryo quality, and transfer efficiency. Historically, much less effort has been placed on assessing or maximizing embryo transfer procedures compared to the other aspects of in vitro fertilization. Embryo transfer is usually performed blindly, with no attempt to document the variables which might adversely impact pregnancy rates. Physicians too often underestimate the importance of the embryo transfer technique and are unwilling to modify their own personal habits or catheter choices.

VARIABLES AFFECTING EMBRYO TRANSFER SUCCESS TRIAL TRANSFER The ultimate goal of a successful embryo transfer is to deliver the embryos atraumatically to the uterine fundus in a location where implantation is maximized. A trial transfer in a cycle preceding IVF for the purpose of measuring the uterine cavity depth and direction appears to be of value. Mansour1 evaluated 335 patients randomized to a precycle trial transfer or no trial transfer. Embryo transfer was found to be difficult in 50 (29.8%) of cases where no trial transfer was performed, compared to no difficult embryo transfers in the trial transfer group. For the trial transfer group, the pregnancy and implantation rates were 22.8% and 7.2%, respectively, compared with a 13.1% pregnancy rate and a 4.3% implantation in the no trial transfer group. Owing to the great variability in cervical and uterine anatomy, a trial or practice embryo transfer is beneficial. The direction of the cervix and uterus can be mapped, the depth of the cavity recorded, and any stenosis can be addressed. In the event of a difficult trial transfer, we have found it helpful to place a laminaria approximately one month prior to the planned in vitro fertilization-embryo transfer (IVF-ET) cycle. THE PROBLEM OF CERVICAL MUCUS Mucus plugging of the catheter tip can cause retained embryos, damage to the embryos (especially with assisted hatching), and improper embryo

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placement. Mansour1 found that prior aspiration of cervical mucus led to visualization of methylene blue in the cervix in 23% of mock embryo transfers. Without aspiration, however, 57% of patients showed methylene blue in the cervix with a mock transfer. Cervical mucus may also be a source of contamination of the endometrial cavity and the embryos. Egbase2 found cervical mucus to be culture positive in 71% of cases. This in turn led to a positive culture of the catheter tip in 49% of patients. The clinical pregnancy rate was 29.6% in the catheter tip positive patients compared to a 57% pregnancy rate when the tip was culture negative. MacNamee (personal communication) evaluated a vigorous cervical lavage technique before embryo transfer to remove all visible mucus. In a retrospective study, patients undergoing vigorous lavage had a 55.5% pregnancy rate and a 26% implantation rate compared with a 41.7% pregnancy rate and a 10.4% implantation in control patients. RETAINED AND EXPELLED EMBRYOS Uterine contractions in the early luteal phase are generally cervicofundal. This may account for some of the ectopic pregnancies seen after IVF. Alterations in the normal contraction pattern may cause expulsion of the embryos into the cervix. Poindexter3 found that four (8.7%) of 46 patients had embryos in the cervix or on the speculum after reportedly routine transfers. Embryos may be retained in the catheter owing to plugging of the tip with mucus or uterine tissue. Inadequate transfer volume or failure to place embryos toward the top of the embryo fluid column may also cause retention. Visser4 found a decrease in pregnancy rates from 20% to 3% when embryos were retained.

CATHETER TYPE Variations in catheter design include stiff versus soft materials, end and side openings, the presence of an outer sheath, malleability, and quality of the materials and finish. Although stiff catheters and the use of a rigid outer sheath make catheter placement easier, they may result in more bleeding, trauma, mucus plugging, and stimulation of uterine contractions. A study evaluating intrauterine insemination with ultrasound monitoring found a disruption of the endometrium in 50% of women where a Tomcat catheter was used compared to only 12.5% with the use of the Wallace catheter.5 Soft catheters allow the tip to follow the contour of the cervical and uterine axis and minimize trauma to the endometrium. Variations of soft catheters are now preferred by most programs. The benefit of one catheter over another is controversial. Wisanto studied three catheter types in 400 patients retrospectively.6 The

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pregnancy rate was as follows: Frydman (32%), Wallace (19%), and TDT (19%). In contrast, Al-shawaf found no difference between the Frydman (31%) and the Wallace (30%).7 Similar findings were described in a study from Englert.8 The change from the Tomcat to the Wallace catheter in our program was associated with an improvement in pregnancy and implantation rates, as well as fewer catheters exhibiting blood, mucus or retained embryos. Similar findings were reported in a study by Penzias.9 LOADING OF THE CATHETER The syringe used with a transfer catheter should be tested to confirm that there is no embryo toxicity associated with it. The operator must be familiar with the properties of the syringe. Some syringes must be squeezed in a controlled fashion so as not to “pop” the embryos with such force that they are damaged or thrust into fallopian tube. Also, syringes may have a degree of “recoil” to them; if the plunger is released, embryos can actually be reaspirated back into the catheter due to this recoil effect. A large volume (60µl) of transfer media and a large air interface have been shown to result in embryos which were expelled into the cervix, on the speculum, or adherent to the catheter.3 Removing the air column minimized such complications. We utilize a continuous fluid column with a volume of 30µl. The embryos are loaded preferentially toward the tip of the embryo column closest to the catheter opening. The concentration of protein in the transfer medium does not appear to affect results. In a study comparing 75%, 8%, and 2.25% concentrations of protein, no difference in outcome was noted. Replacing proteins with hyaluronan, however, was found to improve outcome in the mouse model.10 DIFFICULT EMBRYO TRANSFER-DOES IT MATTER? When one considers the term IVF-ET, there has been a plethora of research focusing on the IVF aspect of this discipline, yet amazingly little study and attention have been directed at embryo transfer. In the past, this may be due to the misconception on the part of clinicians that the ease or difficulty of the transfer really does not affect outcome. Evidence is to the contrary. Mansour found in a large prospective trial that difficult ETs were associated with a significantly lower pregnancy rate (4%) compared to easy transfers (20%). Englert also found a lower pregnancy rate with difficult transfers and with blood on the catheter or visible at the cervical opening.8 Difficult embryo transfers may also decrease pregnancy rates through the production of unwanted sub-endometrial myometrial contractions. Such contractions are more frequent after a traumatic embryo transfer. A

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study monitoring contraction activity by ultrasound found a lower pregnancy rate with excessive uterine contractions prior to transfer (greater than four per minute).9 The need for cervical dilatation because of difficulty with embryo transfer is also detrimental. Groutz found that among 41 women undergoing cervical dilation at the time of oocyte retrieval, only one achieved an intrauterine pregnancy.10 Touching the fundus with the catheter is another complication of difficult embryo transfers. Waterstone found that one clinician in their program who advanced the catheter until resistance was felt, then withdrew it 0.5cm prior to injection, had a pregnancy rate of 24%.11 The other clinician who routinely injected 5cm from the external os without touching the fundus achieved a 46% pregnancy rate. When difficulty is encountered with the insertion of the soft Wallace catheter during a trial transfer immediately before the actual embryo transfer, we have used a modification of the Wallace catheter with a malleable stylet. Using this system during difficult transfers, we have found a pregnancy rate (69%) comparable to that during routine transfers (63%), (Hesla, unpublished observations). ULTRASOUND GUIDANCE The use of transabdominal ultrasound guidance during ET has many advantages over blind catheter placement. The patient’s ovarian status can be reassessed to make sure that the risk of ovarian hyperstimulation syndrome is not great, and that embryo transfer therefore can be completed safely. The endometrium can be assessed, and the presence or absence of fluid in the cavity can be noted. The full bladder required to perform transabdominal ultrasound examinations is itself helpful in straightening the cervico-uterine axis and improving pregnancy rates.12 The patient is allowed to empty her bladder 15 minutes after the transfer and remains at bed rest for no more than one hour. Ultrasound guidance is especially helpful with the insertion of “soft” catheters. When placement is difficult, the problem may be visualized and modification of the angle between the cervix and uterus can be accomplished with manipulation of the speculum or the use of ring forceps. One can confirm that the catheter is not “digging in” to the endometrium, and placement of the tip 1.5cm proximal to the fundus can be assured. Blind catheter placement has been shown to result in the inadvertent location of the catheter tip outside the endometrial cavity in over 25% of cases.13 It also allows the physician to avoid hitting the fundus with the catheter, and enables the clinician to confirm that the catheter tip has past the internal os by at least 1cm prior to injection of the embryos. This is particularly important in cases where patients have an extremely lengthened endocervical canal. The end result is the atraumatic placement of the embryos 1–2cm from the fundus in the lumen of the

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endometrial cavity without trauma to the uterus or embryos and without inducing uterine contractions.

CONCLUSION Embryo transfer appears deceptively simple. However, as with all aspects of IVF, success depends on meticulous attention to the details of technique, catheter type and loading, and preparation of the cervix. The goal—atraumatic placement of the embryos near the fundus without pain, bleeding, trauma to the endometrium or embryos, and with the absence of uterine contractions. At the Colorado Center for Reproductive Medicine, we have found that the keys to this goal are the combination of the Wallace-Edwards catheter and transabdominal ultrasound guidance. The best measure of the efficiency of this embryo transfer technique is illustrated by oocyte donation cycles where presumably embryo quality is maximized. Our recent experience technique for embryo transfer in combination with blastocyst culture and transfer in oocyte donation cycles has demonstrated a greater than 60% implantation rate per embryo.14 Assuming that uterine receptivity, genetics, and unknown causes of implantation failure account for a portion of the 40% failure rate per embryo, the efficiency of embryo transfer with soft catheters and ultrasound guidance is quite high indeed.

APPENDIX

• • • • • •

PROTOCOL: ULTRASOUND-GUIDED EMBRYO TRANSFER Full bladder, transabdominal ultrasound guidance. Wash and lavage cervix with culture media. Trial transfer to the internal os. Wallace catheter, Airtite syringe, 30µl volume, embryos in last 10µl. Gentle insertion, manipulation of cervix with speculum and/or ring forceps as necessary to negotiate internal os. Examination of catheter following transfer for retained embryos, blood and mucus.

REFERENCES 1 Mansour R, Aboulghar M, Serour G, Amin Y. Dummy embryo transfer using methylene blue dye. Hum Reprod (1994); 9:1257–9. 2 Egbase PE, Al-Sharhan M, Al-Othman S, Al-Mutawa M, Udo EE, Grudzinskas JG. Incidence of microbial growth from the tip of the embryo transfer catheter after embryo transfer in relation to clinical

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pregnancy rate following in-vitro fertilization and embryo transfer. Hum Reprod (1996); 11:1687–9. 3 Poindexter A, Thompson D, Gibbons W, Findley W, Dodson M, Young R. Residual embryos in failed embryo transfer. Fertil Steril (1986); 46:262–7. 4 Visser D, Fourie S, Kruger H. Multiple attempts at embryo transfer: effects on pregnancy outcome in an in vitro fertilization and embryo transfer program. J Assist Reprod Gen (1993); 10:37–43. 5 Lavie O, Margalioth EJ, Geva-Elder T, Ben-Chetrit A. Ultrasonographic endometrial changes after intrauterine insemination: A comparison of two catheters. Fertil Steril (1997); 68:731–4. 6 Wisanto A, Janssens R, Deschacht J, Camus M, Devorey P, Van Steirteghem A. Performance of different embryo transfers in a human in vitro fertilization program. Fertil Steril (1989); 52:79–84. 7 Al-Shawaf T, Dave R, Harper J, Linehan D, Riley P, Craft I. Transfer of embryos into the uterus: How much do technical factors pregnancy rates? J Assist Reprod Gen (1993); 10:31–6. 8 Englert Y, Puissant F, Camus M, Van Houck J, Leroy F. Clinical study on embryo transfer after human in vitro fertilization. J In Vitro Fert Embryo Transfer (1986); 3:43–6. 9 Fanchin R, Righini C, Olivennes F, Taylor S, de Ziegler D, Frydman R. Uterine contractions at the time of embryo transfer alter pregnancy rates after in-vitro fertilization. Hum Reprod 1998; 13:1968–74. 10 Groutz A, Lessing JB, Wolf Y, Yovel I, Azem F, Amit A. Cervical dilation during ovum pick-up in patients with cervical stenosis: Effect on pregnancy outcome in an in vitro fertilization-embryo transfer program. Fertil Steril (1997); 67:909–11. 11 Waterstone J, Curson R, Parsons J. Embryo transfer to low uterine cavity. Lancet (1991); 337:1413. 12 Lewin A, Schenker JG, Avrech O, Shapria S, Safran AM, Friedler S. The role of uterine straightening by passive bladder distention before embryo transfer in IVF cycles, J Assist Reprod Gen (1987); 14:32–4. 13 Woolcott R, Stanger J. Potentially important variables identified by transvaginal ultrasound-guided embryo transfer. Hum Reprod (1997); 12:963–6. 14 Schoolcraft WB, Gardner DK. Blastocyst culture and transfer increases the efficiency of oocyte donation. Fertil Steril (2000); 74:482–6.

51 Endometriosis Mark I 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. Newly developed and 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. However, on their own, these treatments have largely failed to improve fertility in patients with endometriosis, which has led some to question the existence of a causal relation between the disease and impaired reproduction. Nevertheless, it does appear that assisted reproductive technology is becoming an indispensable asset in providing affected couples with viable pregnancies, although the nearly complete absence of adequate prospective trials has left many questions regarding protocol and efficacy unanswered.

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 either infertility or voluntary sterilization.2–4 The incidences of endometriosis ranged from 21–48% in the infertile women, while the disease 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

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women undergoing insemination with donor sperm because of 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 nonspecific polyclonal B-cell activation. In addition, some have reported increased production of cytokines and eicosanoids in the peritoneal fluid and sera, which may effect sperm motility and velocity, acrosome reactivity, sperm penetration, embryo implantation and early development.14,20 As with other factors, many conflicting reports have emerged. Further, it is not at all clear which is the cause and which the effect, or what part each abnormality actually plays in the pathogenesis of endometriosisassociated infertility. As stated, one argument, that has been posed against a causal relationship between endometriosis and infertility is in the outright failure of medical or surgical treatment to improve pregnancy success in these patients. The use of medical treatments, otherwise successful in alleviating the non-reproductive symptoms of endometriosis, has failed to demonstrate an improvement in fertility.21 Most studies investigating the surgical ablation of endometriotic lesions, by any one of a number of techniques, have failed to show an increased fecundity. One recent study, however, did show an improved rate of pregnancy for women treated with ablation of endometriotic lesions 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. Another recent randomized study, which looked at actual birth rates, failed to demonstrate a reproductive benefit for patients whose lesions were ablated.23

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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.24 However, it is not entirely clear that this approach is as effective for patients with endometriosis. Deaton et al25 demonstrated increased fecundity in patients treated with clomiphene citrate and IUI. However, Fedele and colleagues26 reported that the increased conception rate with COH and IUI did not follow with a significantly different pregnancy rate at six months. Further, 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.27 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.28 In a metaanalysis, Hughes29 reported that a diagnosis of endometriosis decreased the per cycle COH/IUI conception rate by half. Lastly, a recent prospective, randomized study reported live birth rates of 11% and 2% for endometriosis patients undergoing COH/IUI and no treatment, respectively.30 While this demonstrates a live birth odds ratio of 5.6 for the treatment group, the actual percentage of live births after treatment remains quite low.

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. 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, tubal disease, male factor, or a combination of etiologies, 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 infertility associated with endometriosis may help to answer some of the questions regarding this elusive association. It is thought that the use of IVF-embryo transfer (ET) in the infertile patient with endometriosis removes critical steps in reproduction, such as

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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 that of other infertility etiologies. Early studies, however, demonstrated that endometriosis patients, particularly those with moderate to severe disease, had lower pregnancy rates. Fortunately, with the development of GnRH agonists and transvaginal oocyte retrieval, most recent studies have demonstrated remarkable 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 wide range of reported pregnancy rates. Further, most studies are retrospective and observational and therefore of limited value in reaching definitive conclusions regarding therapy efficacy. With success in treating endometriosis patients using standard ART protocols, and an absence of evidence suggesting a deviation from normative IVF practices, most patients with the disease are treated as with other infertility etiologies. 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 controlled ovarian hyperstimulation 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 et al31 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 al32 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 owing to technical difficulties at the time of laparoscopic oocyte retrieval. Alternatively, other researchers have reported decreased folliculogenesis in patients with endometriosis.33–36 Further, Dlugi and colleagues37 reported a significantly lower number of preovulatory follicles in patients with endometriomas compared with patients with hydrosalpinges. In general, most contemporary studies utilizing GnRH agonists and transvaginal retrieval have not confirmed that endometriosis has a significant effect on oocyte yield. Dmowski et al38 retrospectively analyzed 237 IVF cycles and found no difference in either folliculogenesis or in the number of oocytes obtained for women with or without

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endometriosis. In a case-control study comparing 65 cycles of IVF for women with endometriosis, to 98 cycles of IVF in patients with tubal infertility, Bergendal et al39 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 compared with patients with more severe disease.40–42 The improvement in IVF outcomes, brought about by the development of GnRH agonists, is largely undisputed. Olivennes et al43 reported a significantly improved clinical prenancy rate for patients treated with GnRH agonists, when compared with standard, gonadotropin-only, ovarian stimulation protocols (Fig 51.1). Other investigations have reported similar results.44 Long term GnRH agonist suppression has been thought to further repress endometriotic lesions and improve IVF outcomes for patients with endometriosis. Dicker et al45 reported a significantly higher clinical pregnancy rate after six months of GnRH agonist therapy, compared with ovarian stimulation with

Fig 51.1 Comparison of clinical pregnancy rates for controlled ovarian hyperstimulation protocols including the use of no GnRH agonist, short GnRH administration, or long GnRH administration. gonadotropins alone. Chedid et al46 also investigated the use of a 3-month and a 3-week GnRH agonist down-regulation protocol and reported a significantly increased oocyte yield compared with controls receiving only gonadotropins. Although they noted an improved pregnancy rate, it did not reach statistical significance. Nakamura et al47 compared GnRH

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agonist suppression for 60 days with a shorter, mid-luteal downregulation, prior to ovulation induction. They reported pregnancy rates of 67% and 27%, for longer and shorter protocols, respectively. Marcus et al48 also reported a significantly higher pregnancy rate for patients treated with longer GnRH agonist protocols (Table 51.1), although they used different GnRH agonists for the two groups and assigned patients on the basis of their refusal to accept the longer regimen. Chedid et al46 found no difference between long and short GnRH agonist administrations. Clearly, prospective, randomized studies are needed to determine the optimum ovarian stimulation protocol for patients with endometriosis. In addition, the introduction of GnRH antagonists will likely add to the requirement that well designed studies are undertaken to generate solid evidence from which to draft future ART treatment plans for these patients. For now, endometriosis patients seem to respond to ovarian stimulation in a manner that is similar to other infertility etiologies. Although standard gonadotropin stimulation protocols work quite well, the addition of longer GnRH agonist down-regulation may increase IVF success and should be considered on a case by case basis. FERTILIZATION AND EARLY EMBRYO DEVELOPMENT It is unclear to what degree 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,49 while another reported a marked impairment in fertilization with the presence of an endometrioma.37 More recently, Bergendal et al reported fertilization rates of 60% and 78% for patients with endometriosis and tubal factor, respectively (P<0.00001).39 Other investigators have reported significantly lower fertilization success for stage III or IV endometriosis compared with stages I or II.42,43 With regard to early embryo development, researchers have reported fewer embryos reaching the 4 cell stage at 48 hours,50 a reduced number of blastomeres at 72 hours,51 and lower cleavage rates52 when endometriosis is compared with tubal factor or unexplained infertility. Further, Brizck et al53 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.

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Table 51.1. Comparison of IVF-ET outcomes for women with and without endometriosis. Study Group No. of Clinical Study Group No. of Clinical cycles pregnancies cycles pregnancies (%/cycle) (%/cycle) Mehadevan64 I-IV 14 14 I 111 40* Inoue41 (1983) (1992) Tubal 261 10 II 78 42 49 Wardle I-IV 17 6 III 51 47 (1985) Tubal 47 11 IV 69 42 Chillik31 Other 372 44 I/II 10 60 (1985) III/IV 14 7 Olivennes43 I-IV 360 29 33 (1995) I 24 13 Tubal 160 36 Matson (1986) II 37 14 Geber40 I/II 100 29 (1995) III 36 6 III/IV 29 52 IV 57 2 Tubal 1139 41 Tubal 40 18 Dmowski38 I/II 89 25 (1995) I/II 135 16 III/IV 30 30 Sharma65 (1988) III/IV 141 8 Other 118 21 Arici54 Tubal 994 13 I/II 43 12 32 (1996) Oehninger I/II 191 24 III/IV 46 15 (1988) Tubal 147 24 III/IV 35 20 I/II 61 13 Bergendal39 I-IV 65 28 Yovich35 (1990) (1998) III/IV 93 3 Tubal 98 30 Tubal 49 14 Pal42 I/II 45 44 (1998) III/IV 39 33 *Pregnancy rates in this study were reported per patient, not per cycle. Symbols I-IV represent endometriosis stage I through stage IV. Other: patients without a diagnosis of endometriosis. Conversely, there have been several large studies that have failed to detect an impairment in fertilization or early embryo development. Dmowski et al38 analyzed 237 cycles and found 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.54 In comparing the effect of progressive endometriosis stages on fertilization and embryo development, Inoue et al41 found no difference in either the fertilization rate or the embryo transfer rate for 309 patients with stage I through IV endometriosis. Further, Bergendal et al,39 although reporting impaired

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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. However, most 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. 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 may play in the fertilization of oocytes from women with endometriosis. Further, with a trend towards in vitro blastocyst development, embryos with poor developmental potential may be eliminated, allowing for only the fittest embryos to be transferred. 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 likely a result of the transfer of multiple embryos, a lower rate of implantation does not necessarily translate 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 51.1). Some early studies have shown a decrease in the implantation rate with a subsequent decrease in the pregnancy rate.33,34,49 In a small study, Chillik et al31 reported a significantly lower implantation and pregnancy rate for patients with stage III or IV endometriosis compared with patients with tubal factor or endometriosis of a lesser severity. Matson and Yovich33 demonstrated pregnancy rates of 18%, 13%, 14%, 6%, and 2%, for patients with tubal factor, and stage I through IV endometriosis, respectively. More recently, Arici et al54 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. While Simon et al55 also reported lower implantation and pregnancy rates for patients with endometriosis, versus 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

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endometriosis, who received oocytes from donors without the disease. However, patients who received oocytes from endometriotic ovaries had significantly lower implantation rates, irrespective of whether or not the recipient had the disease. 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.56 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.39–43 Geber et al40 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,43 while another reported 28% and 30%, respectively.39 Inoue et al,41 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.32,38,40,54 Pal et al42 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 endometriosis, clinical pregnancy rates did not differ significantly between the two groups. A few studies have associated endometriosis with increased pregnancy loss during IVF cycles. Oehninger et al32 noted a higher miscarriage rate after IVF among patients with stage III or IV endometriosis compared with those with less severe disease. Yanushpolsky et al50 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 either oocyte yield, embryo quality, pregnancy rate or miscarriage.57 Further, most studies have not reported a significant endometriosis-associated increase in pregnancy loss.39,40 ENDOMETRIOSIS AND GIFT There is limited data concerning the effect of endometriosis on gamete intrafallopian transfer. Guzick et al,58 in a retrospective, case-control 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

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patients with different stages of the disease or with other infertility etiologies.36 In an early observational study, Yovich and Matson59 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.60 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. Further, 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.61 Pregnancy rates were reported as 70% over two cycles of IVF compared with 24% for the 9 months after surgery. Until better data are available, however, no conclusions can be drawn regarding the role of surgery for endometriosis prior to ART. FUTURE DIRECTIONS Some researchers have suggested that endometriosis is associated with an impaired folliculogenesis and a decreased oocyte yield. Although the data is conflicting, it is possible that the introduction of GnRH antagonists may represent another large step forward in improving ovarian stimulation protocols and increasing IVF success. Further, 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 increasing number of women with endometriosis who have failed standard IVF will benefit from donation. There is evidence for and against an endometriosis-associated 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 by case basis. For patients with endometriosis who are experiencing fertilization difficulty, it is likely that intracytoplasmic sperm injection (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 al62 analyzed 980 cycles of ICSI for couples with male factor infertility, of which 101 cycles were also complicated by endometriosis.

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They found no significant difference in fertilization, implantation or pregnancy rates with coexisting endometriosis. Lastly, 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 of 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 where it will become routine to transfer no more than one or two embryos at a time, thereby significantly lowering the incidence of multiple pregnancies. Further, 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.

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

Fig 51.2 Live birth rates among non-donor ART cycles in the United States in 1996. (Adapted from ref. 63.)

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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. Perhaps the value of IVF in the treatment of endometriosis is best illustrated by data on a national scale, as reported in the United States ART registry.63 In 1996, the national live birth rate following ART for patients with endometriosis was 23.8% per cycle (Fig 51.2). This rate is quite impressive and comparable with other infertility etiologies. Although ART procedure alterations are site specific, the vast majority of endometriosis patients undergo the same treatment protocol as for those patients with tubal factor 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); 70:313–6.

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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 or mild endometriosis and otherwise unexplained infertility. Clin Endocrinol (1992); 36:177–81. 9 Matorras R, Rodriguez F, Perez C, Pijoan JI, Neyro JL, RodriguezEscudero FJ. 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. 10 Pittaway DE, Maxson W, Daniell J, Herbert C, Wentz AC. Luteal phase defects in infertility patients with endometriosis. Fertil Steril (1983); 39:712–3. 11 Hirschowitz JS, Soler NG, Wortsman J. The galactorrheaendometriosis syndrome. Lancet (1978); 1:896–8. 12 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. 13 Wheeler JM, Johnston BM, Malinak LR. The relationship of endometriosis to spontaneous abortion. Fertil Steril (1983); 39:656–60. 14 Burns WN, Schenken RS. Pathophysiology of endometriosisassociated infertility. Clin Obstet Gynecol (1999); 42:586–610. 15 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. 16 Kusuhara K. Luteal function in infertile patients with endometriosis. Am J Obstet Gynecol (1992); 167:274–7. 17 Matalliotakis I, Panidis D, Vlassis G, Vavilis D, Neonaki M, Koumantakis E. PRL, TSH and their response to the TRH test in patients with endometriosis before, during, and after treatment with danazol. Gynecol Obstet Invest (1966); 42:183–6. 18 Pittaway DE, Vernon C, Fayez JA. Spontaneous abortions in women with endometriosis. Fertil Steril (1988); 50:711–15. 19 Matorras R, Rodriguez F, Gutierrez de Teran G, Pijoan JI, Ramon O, Rodriguez-Escudero FJ. Endometriosis and spontaneous abortion rate: A cohort study in infertile women. Eur J Obstet Gynecol Reprod Biol (1998); 77:101–5. 20 Senturk LM, Arici A. Immunology of endometriosis. J Reprod Immunol (1999); 3:67–83. 21 Hughes E, Fedorkow DM, Collins J, Vendekerckhove P. Ovulation suppression versus placebo in the treatment of endometriosis. In: Lilford R, Hughes E, Vandekerckhove P, eds. Subfertility module of the Cochrane database of systematic reviews. The Cochrane Collection: Oxford, 1997.

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22 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. 23 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. 24 Guzick DS, Carson SA, Coutifaris C, et al. Efficacy of superovulation and intrauterine insemination in the treatment of infertility. N Eng J Med (1999); 340:177–83. 25 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. 26 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. 27 Nuojua-Huttunen S, Tomas C, Bloigu R, Tuomivaara L, Martikainen H. Intrauterine insemination treatment in subfertility: an analysis of factors affecting outcome. Hum Reprod (1999); 14:698–703. 28 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); 2602–5. 29 Hughes EG. The effectiveness of ovulation induction and intrauterine insemination in the treatment of persistent infertility: a meta-analysis. Hum Reprod (1997); 1965–72. 30 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. 31 Chillik CF, Acosta AA, Garcia JE, Perera S, Van Uem JF, Rosenwaks Z, Jones HW Jr. The role of in vitro fertilization in infertile patients with endometriosis. Fertil Steril (1985); 44:56–61. 32 Oehninger S, Acosta AA, Kreiner D, Muasher SJ, Jones HW Jr, Rosenwaks Z. In vitro fertilization and embryo transfer (IVF/ET): an established and successed therapy for endometriosis. J In Vitro Fert Embryo Transf (1988); 5:249–56. 33 Matson PL, Yovich JL. The treatment of infertility associated with endometriosis by in vitro fertilization. Fertil Steril (1986); 46:432–4. 34 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. 35 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.

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36 Chang MY, Chiang CH, Hsieh TT, Soong YK, Hsu KH. The influence of endometriosis on the success of gamete intrafallopian transfer (GIFT). J Assist Reprod Genet (1997); 14:76–82. 37 Dlugi AM, Loy RA, Dieterle S, Bayer SR, Seibel MM. The effect of endometriomas on in vitro fertilization outcome. J In Vitro Fert Embryo Transf (1989); 6:338–41. 38 Dmowski WP, Rana N, Michalowska J, Friberg J, Papierniak C, elRoeiy A. 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. 39 Bergendal A, Naffah S, Nagy C, et al. Outcome of IVF in patients with endometriosis in comparison with tubalfactor infertility. J Assist Reprod Genet (1998); 15:530–4. 40 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. 41 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. 42 Pal L, Shifren JL, Isaacson KB, Chang Y, Leykin L, Toth TL. Impact of varying stages of endometriosis on the outcome of in vitro fertilization-embryo transfer. J Assist Reprod Genet (1998); 15:27–31. 43 Olivennes F, Feldberg D, Liu HC, Cohen J, Moy F, Rosenwaks Z. Endometriosis: a stage by stage analysis—the role of in vitro fertilization. Fertil Steril (1995); 64:392–8. 44 Oehninger S, Brzyski RG, Muasher SJ, Acosta AA, Jones GS. In-vitro fertilization and embryo transfer in patients with endometriosis: impact of a gonadotrophin releasing hormone agonist. Hum Reprod (1989); 4:541–4. 45 Dicker D, Goldman JA, Levy T, Feldberg D, Ashkenazi J. 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. 46 Chedid S, Camus M, Smitz J, Van Steirteghem AC, Devroey P. Comparison among different ovarian stimulation regimens for assisted procreation procedures in patients with endometriosis. Hum Reprod (1995); 10:2406–11. 47 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); 9:113–7. 48 Marcus SF, Edwards RG. High rates of pregnancy after long-term down-regulation of women with severe endometriosis. Am J Obstet Gynecol (1994); 171:812–7. 49 Wardle PG, Mitchell JD, McLaughlin EA, Ray BD, McDermott A, Hull MG. Endometriosis and ovulatory disorder: reduced fertilisation

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in vitro compared with tubal and unexplained infertility. Lancet (1985); 2:236–9. 50 Yanushpolsky EH, Best CL, Jackson KV, Clarke RN, Barbieri RL, Hornstein MD. Effects of endometriomas on ooccyte quality, embryo quality, and pregnancy rates in in vitro fertilization cycles: a prospective, casecontrolled study. J Assist Reprod Genet (1998); 15:193–7. 51 Pellicer A, Oliveira N, Ruiz A, Remohi J, Simon C. 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. 52 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. 53 Brizek CL, Schlaff S, Pellegrini VA, Frank JB, Worrilow KC. Increased incidence of aberrant morphological phenotypes in human embryogenesis—an association with endometriosis. J Assist Reprod Genet (1995); 12:106–112. 54 Arici A, Oral E, Bukulmez O, Duleba A, Olive DL, Jones EF. The effect of endometriosis on implantation: results from the Yale University in vitro fertilization and embryo transfer program. Fertil Steril (1996); 65:603–7. 55 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. 56 Sung L, Mukherjee T, Takeshige T, Bustillo M, Copperman AB. Endometriosis is not detrimental to embryo implantation in oocyte recipients. J Assist Reprod Genet (1997); 14:152–6. 57 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. 58 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. 59 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. 60 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. 61 Pagidas K, Falcone T, Hemmings R, Miron P. Comparison of reoperation for moderate (stage III) and severe (stage IV)

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endometriosis-related infertility with in vitro fertilization-embryo transfer. Fertil Steril (1996); 65:791–5. 62 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. 63 Assisted reproductive technology in the United States: 1996 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril (1999); 71:798–807. 64 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. 65 Sharma V, Pampiglione J, Riddle A, et al. An analysis of factors influencing the establishment of a clinical pregnancy in an ultrasoundbased ambulatory in vitro fertilization program. Fertil Steril (1988); 49:468–78.

52 Polycystic ovaries and ART Howard S Jacobs, Adam H Balen, Jane MacDougall

INTRODUCTION Although in vitro fertilization (IVF) is not the first line treatment for polycystic ovary syndrome (PCOS), many patients with polycystic ovaries (PCO) may be referred for IVF either because there is another reason for their infertility or because they have not conceived after six or more ovulations (ie their infertility remains unexplained despite correction of anovulation). An understanding of the management of such patients is therefore important for specialists involved in IVF. In this chapter we consider four separate but related issues. First what are polycystic ovaries, and how are they diagnosed? Second, how prevalent is the problem in the context of IVF? Third, does a diagnosis of PCO matter, and do patients with polycystic ovaries respond differently at any of the stages of IVF? Finally, should such patients be managed differently, and if so, how? It is now clear that polycystic ovaries may be present in women who are not hirsute and who have regular menstrual cycles. A clinical spectrum exists from the typical SteinLeventhal picture of hirsutism, obesity, and oligomenorrhoea (PCOS) to the asymptomatic women with polycystic ovaries (PCO) as the sole finding.1 In patients with PCOS, metabolic and endocrine disturbances (elevated serum concentrations of luteinizing hormone (LH), testosterone, insulin, and prolactin) are common and may have implications for long term health. Polycystic ovary syndrome is a familial condition, and a number of candidate genes have been implicated.2 It appears to have its origins during adolescence, and its onset is thought to be associated with increased weight gain during puberty.3

DIAGNOSIS Diagnosis is most readily based on ovarian morphology. Ovaries are described as polycystic if there are 10 or more cysts, 2–8mm in diameter, arranged around a dense stroma or scattered throughout an increased amount of stroma.4 They should be distinguished from multicystic ovaries, which occur normally during puberty and are associated with recovering weight loss related amenorrhoea.5 Multicystic ovaries do not

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contain increased stroma, and the cysts are usually larger than in polycystic ovaries.4 Studies using computerized, three-dimensional reconstructions of ultrasound images of the polycystic ovary have shown that the major factor responsible for the increase in ovarian volume is the stroma, with little contribution from the cysts themselves.6 Using color and pulsed Doppler ultrasound, it has been shown that the stroma of the polycystic ovary has an increased rate of blood flow,7 consistent with the early histological studies that had shown increased stromal vascularity.8 Large amounts of vascular endothelial growth factor (VEGF) have been identified in the theca cells of the polycystic ovary and are reflected in such subjects by raised serum VEGF concentrations.9 The latter correlate with Doppler assessed ovarian blood flow.10 In general it is helpful to differentiate PCO from PCOS.1 The former specifically describes the morphological appearance of the ovary (in practice, the ultrasound features) whereas the latter term is only appropriate when PCO are found in association with a menstrual disturbance, most commonly oligomenorrhoea, the complications of hyperandrogenization (seborrhoea, acne and hirsutism) and obesity. On the other hand, PCOS may become apparent, may be “brought out,” with polycystic ovaries are “stressed,” either by overstimulation by insulin, as when obesity develops, or by gonadotropin stimulation, as occurs during infertility treatment. Serum LH concentrations are raised in about 40% of patients with PCOS.11 Moderate hyperprolactinemia (usually between 600–2000mU/l) is present in about 15%. In a retrospective study we found microprolactinomas in more than half of the 41 hyperprolactinemic patients with PCOS we had investigated by magnetic resonance imaging (MRI) scan (unpublished data). We therefore consider that hyperprolactinemic patients with PCOS should be offered endocrine evaluation before infertility treatment proceeds. Estradiol levels are usually similar to those found in normal women during the early follicular phase of the cycle. Estrone levels, however, may be raised because of extraovarian conversion of androstenedione in fat tissue. The significance of the hyperestrogenism is that these patients are subject to endometrial hyperplasia, an immediate problem in infertility treatment and, long term, a significant risk factor for endometrial carcinoma. Finally, of course, the polycystic ovary produces an excess of androgens. As with the clinical picture, endocrine changes are variable and patients with PCOS may have normal hormone concentrations. Thus their measurement is not as helpful as ultrasound in making the diagnosis. Turning to clinical features, body mass index correlates with hirsutism, cycle disturbance, infertility, and insulin resistance with its compensatory hypersecretion of insulin.12 In patients presenting for IVF it is seldom necessary to measure serum insulin concentrations, as the results do not determine the immediate management of the patient. Since the prevalence

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of diabetes in obese women with PCOS may be as high as 11%13 assessment of glucose tolerance is important in obese women.14 Weight loss improves the symptoms of the PCOS and improves the patient’s endocrine profile and response to treatment.15,16

PREVALENCE The prevalence of polycystic ovaries in women with ovulatory disorders is well documented. Thus 87% of patients with oligomenorrhoea and 26% with amenorrhoea were reported to have PCO17 while Polson et al, found a prevalence of 22% in a volunteer “normal” population.17 This initially surprising result has since been confirmed in several studies.18–20 The prevalence in patients referred for IVF is less well known. It would of course be unexpected for fewer than 20% to have PCO. Three of our own studies suggest that actually many more patients presenting for IVF have PCO by ultrasound criteria. The first involved a review of ultrasound scans performed in the early follicular phase of an IVF treatment cycle and noted that 50% of 42 patients had PCO.1 A more recent study identified 58 (33%) with PCO, compared with 117 with normal ovaries.21 In patients referred for natural cycle IVF, all of whom had regular ovulatory menstrual cycles, 43.5% had PCO.22 PCO with or without clinical symptoms are therefore common in patients referred for IVF. Infertility in patients with PCO is caused either by the PCOS (i.e. failure to ovulate at a normal rate, and/or hypersecretion of LH23) or by any of the other causes of infertility or a combination of the two. Ovulation induction is appropriate for the first group (PCOS). IVF may be necessary in the second group and in patients with PCOS who have not conceived despite at least six ovulatory cycles.

THE 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.24–26 Conventional IVF nowadays depends on inducing multifollicular recruitment.27 It is thus to be expected that the response of the polycystic ovary within the context of an IVF programme should also differ from the normal, but this has previously been assumed rather than documented. Jacobs et al, 1987 described an increase in follicle production in patients with PCO and others refer to the “explosive” nature of the ovarian response (Smitz et al, 1991)1,28 Dor et al, 1990, compared 16 patients with PCOS with a control group with normal ovaries, who

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were all undergoing IVF and noted an increase in follicle numbers, oocytes, and estrogen levels, associated with a decrease in fertilization rates.29 Some years ago we studied 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.30 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 hCG administration (5940±255 v. 4370±240 pmol/l, P<0.001), developed more follicles (14.9±0.7 v. 9.8±0.6, P<0.001) and produced more oocytes (9.3±0.6 v. 6.8±0.5, P=0.003). Fertilization rates were, however, reduced in PCO patients (52.8±3.4% v. 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. There were three high order multiple pregnancies in the PCO group but none 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 good quality embryos for transfer. The study indicated the importance of the diagnosis of polycystic ovarian morphology prior to “controlled” ovarian stimulation because it is less likely to be controlled in women with polycystic ovaries and these patients are more likely to develop OHSS and multiple pregnancy. Similar observations in women with polycystic ovaries undergoing IVF have been reported by others.31 There are several possible explanations for the excessive response of the PCO to ovarian stimulation. The polycystic ovary contains many partially developed follicles which are readily stimulated to give rise to the typical multifollicular response. Thecal hyperplasia (with in some cases raised levels of LH and/or insulin) provides large amounts of androstenedione and testosterone which act as substrates for estrogen production. Granulosa cell aromatase, although deficient in the “resting” polycystic ovary, is readily stimulated by follicle stimulating hormone (FSH). Therefore normal quantities of FSH act in the presence of large amounts of substrate (testosterone and androstenedione) to produce large amounts of intra-ovarian estrogen. Ovarian follicles are thus made increasingly sensitive to FSH (receptors for which are stimulated by high local concentrations of estrogen), and as a result there is multiple follicular development associated with very high concentrations of circulating estrogen. In some cases, this may result in the ovarian hyperstimulation syndrome (OHSS), to which patients with PCO are particularly prone.

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There are two additional factors to be considered. The first is that many women with PCOS, particularly those who are obese, have compensatory hypersecretion of insulin in response to the insulin resistance that is specifically related to the polycystic ovary syndrome32 and to that caused by obesity. Since the ovary is spared the insulin resistance, it 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 LH33 and granulosa cell production of estrogen in response to stimulation by FSH.34 The second factor to be considered relates to the already mentioned overexpression of VEGF in the PCO. VEGF is an endothelial cell mitogen which stimulates vascular permeability, hence its involvement in the pathophysiology of the OHSS.35 In the ovary VEGF is normally largely confined to blood vessels and after ovulation is responsible for invasion of the relatively avascular Graafian follicle by blood vessels which occurs as part of the formation of the corpus luteum. It is the increase of LH at mid cycle which leads to expression of VEGF, which is known to be an obligatory intermediate in the formation of the corpus luteum.36 In PCO, however, Kamat et al have shown widespread expression of VEGF in theca cells in the increased stroma.9 The studies of Agrawal et al have shown that, compared with women with normal ovaries, women with PCO and PCOS have increased serum VEGF, both before and during superactive LHRH analogue therapy and gonadotropin treatment.37 The above data serve to remind us of the close relation of PCO and OHSS. They also provide a possible explanation for the multifollicular response of the polycystic ovary to gonadotropin stimulation. Thus, one of the mechanisms which underpins the unifollicular response of the normal ovary is diversion of blood flow within the ovaries, first from the nondominant to the dominant ovary and, second, from cohort follicles to the dominant follicle. This results in diversion of FSH away from the cohort follicles and permits them to undergo atresia. We postulate that the widespread distribution of VEGF in the PCO prevents this diversion of blood flow, leaving a substantial number of small and intermediate sized follicles in “suspended animation” and ready to respond to gonadotropin stimulation. The distribution of VEGF in the polycystic ovary therefore may help to explain one of the fundamental features of the polycystic ovary, namely the loss of the intraovarian autoregulatory mechanism which permits unifollicular ovulation to occur.

SUPEROVULATION STRATEGIES FOR WOMEN WITH POLYCYSTIC OVARIES AND/OR THE POLYCYSTIC OVARY SYNDROME Pituitary desensitization with a gonadotropin releasing hormone agonist has become almost universal in assisted conception clinics. The reversible hypogonadotrophic hypogonadism so produced permits enhanced control

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of follicular development and improved pregnancy rates in IVF programs.38,39 Suppression of endogenous LH by gonadotrophin releasing hormone (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.1,40 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 the PCOS.41,42 There are few studies that specifically compare different treatment regimens for women with and without PCO, and those that do vary in their definition and diagnosis of the syndrome.1,43,44 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.45 With both long and short protocols, significantly more eggs are collected from women with polycystic than normal ovaries,1 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 levels43; 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 schedules,46 and there is every reason to suppose this conclusion will hold true for patients with PCO as well as for those with normal ovaries. Nonetheless it has to be accepted that a randomized controlled comparison of the various schedules has not been carried out specifically in PCO cases. The recent introduction of schedules of gonadotropin stimulation that incorporate treatment with GnRH antagonists holds promise for patients with PCO and PCOS although the results of specific trials in this condition are not yet available. The other debate in ovarian stimulation for women with the PCOS is whether using of FSH alone confers any benefit over HMG—is the hypersecretion of LH responsible for the exaggerated response to stimulation of the polycystic ovary? Does minimizing circulating LH levels by giving FSH alone improve outcome? Preparations of purified urinary FSH contain some LH activity, usually less than 1%, and preliminary work suggested that ovulation induction can be achieved without exogenous LH.47 In patients with hypogonadotrophic hypogonadism follicular maturation is, however, often incomplete and inconsistent because LH, by its action on thecal cells, is required for full ovarian steroidogenesis.48,49 Thus the presence of some LH in the

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hypogonadotrophic 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.1,50,51 So far as in vitro fertilization is concerned, we have recently reported the results of a meta-analysis of randomized controlled comparisons of urinary derived FSH and hMG.52 In contrast to the earlier study of Daya,53 we assessed the outcome in relation to the preceding schedule of treatment with GnRH analogue, 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. These results will of course need to be updated when data become available from randomized controlled trials using recombinant gonadotropin preparations, GnRH antagonists rather than superactive agonists and from trials that are focussed specifically on patients with PCO and PCOS rather than the usual melange of clinic patients. On the other hand, as described above, PCO occurs so commonly in patients presenting for IVF that the results from general clinic populations are likely to be sufficiently generalizable for them to be valid for PCO patients. At present therefore we recommend the long protocol of pituitary desensitization for women identified as having PCO. 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 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. When using recombinant gonadotropin preparations we recommend reducing the dose by at least 25 to 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 eight of stimulation with additional measurements of serum estradiol being helpful in some cases.

LUTEAL SUPPORT AND THE OVARIAN HYPERSTIMULATION SYNDROME It has been apparent for some time that patients with PCO undergoing straightforward ovulation induction are particularly at risk of developing OHSS.54,55 Recently this has been confirmed in IVF.28,56 In a total population of 1302 patients we identified 15 patients who had undergone ovarian stimulation for IVF or other assisted conception techniques at the Hallam Medical Centre, between July 1989 and July 1990, and who developed OHSS of sufficient severity to merit hospital admission (prevalence of 1.2% with 0.6% having severe OHSS). 53% of these

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patients had ultrasonically diagnosed PCO and 87% were undergoing their first attempt at IVF. All had received luteal support in the form of hCG. Although the pregnancy rate in this group was very high (93.3%) the multiple pregnancy rate was 57% with a miscarriage rate of 14.3%.56 As a result of this analysis, we recommend that patients undergoing IVF have a pelvic ultrasound scan performed either prior to or early in the treatment cycle. If PCO are identified the dose of gonadotropins should be minimized (see above). As mentioned earlier, recent studies have suggested that the increased propensity of polycystic ovaries to become overstimulated in caused by increased expression of VEGF in the stroma of the polycystic ovary, which itself has increased blood flow, as assessed by color Doppler.7 The association has been explored further by performing pulsed and color Doppler studies together with measurements of serum VEGF concentrations in 36 women with normal ovaries and 24 women with polycystic ovaries (10 of whom had the OHSS) undergoing IVF. Serum VEGF concentrations and blood flow were significantly higher in the women with PCO/PCOS than those with normal ovaries and this might explain the greater risk of OHSS in these patients.10 Careful monitoring of estrogen levels and numbers of follicles by ultrasound scan during stimulation for IVF can also help to identify those at risk. In patients thought to be at risk of OHSS (age less than 30 years, and/or PCO +/− estrogen concentrations <8000 pmols/l, and/or more than 20 follicles at oocyte collection) luteal support in the form of hCG should be withheld. It is now our practice to use progesterone pessaries instead of hCG as luteal support in all patients. Transfer of a maximum of two embryos reduces the multiple pregnancy rate with its attendant obstetric and neonatal problems. Additionally, embryos may be frozen for transfer at a later date, and, in the case of patients on LHRH analogues, the analogue continued until the onset of menstruation (by giving a long acting depot) and hormone replacement therapy for the frozen embryo replacement cycle started at that stage.

REFERENCES 1 Jacobs HS. Polycystic varies and polycystic ovary syndrome. Gynaecol Endocrinol (1987); 1:113–31. 2 Franks S. Molecular genetics of the polycystic ovary syndrome. In: Shoham Z, Howles CM, Jacobs HS, eds. Female Infertility Therapy: Current Practice. London: Martin Dunitz (1999); 35–44. 3 Balen AH, Dunger D. Pubertal maturation of the internal genitalia. Ultrasound Obstet Gynecol (1995); 6:164–5.

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4 Adams J, Polson DW, Abdulwahid N, et al. Multifollicular ovaries: clinical and endocrine features and response to pulsatile gonadotropin releasing hormone. Lancet (1985); ii:1375–78. 5 Brook CG, Jacobs HS, Stanhope R, Adams J, Hindmarsh P. Pulsatility of reproductive hormones: applications to the understanding of puberty and to the treatment of infertility. Bailliere’s Clin Endocrinol Metab (1987); 1:23–41. 6 Kyei-Mensah AA, LinTan S, Zaidi J, Jacobs HS. Relationship of ovarian stromal volume to serum androgen concentrations in patients with polycystic ovary syndrome. Hum Reprod (1998); 13:1437–41. 7 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. 8 Goldzieher JW, Green JA. The polycystic ovary. I Clinical and histological features. J Clin Endocrinol Metab (1962); 22:325–38. 9 Kamat BR, Brown LF, Manseau EJ. Expression of vascular endothelial growth factor vascular permeability factor by hugranulosa and theca lutein cells. Role in corpus luteum development. Am J Pathol (1995); 146:157–65. 10 Agrawal R, Sladkevicius P, Engman L, et al. Serum vascular endothelial growth factor concentrations and ovarian stromal blood flow are increased in women with polycystic ovaries. Hum Reprod (1998); 13:651–5. 11 Balen AH, Conway GS, Kaltsas G, et al. Polycystic ovary syndrome: the spectrum of the disorder in 1741 patients. Hum Reprod (1995); 10:2705–12. 12 Conway GS, Jacobs HS. Clinical implications of hyperinsulinemia in women. Clin Endocrinol (1993); 39:623–32. 13 Conway GS, Agrawal R, Betteridge DJ, Jacobs HS. Risk factors for coronary artery disease in lean and obese women with the polycystic ovary syndrome. Clin Endocrinol (1992); 37:119–25. 14 Conway GS. Insulin resistance and the polycystic ovary syndrome. Contemp Rev Obstet Gynaecol (1990); 2:34–9. 15 Kiddy DS, Hamilton-Fairley D, Bush A, Anyaoku V, Reed MJ, Franks S. Improvement in endocrine and ovarian function during dietary treatment of obese women with polycystic ovary syndrome. Clin Endocrinol (1992); 36:105–11. 16 Clark AM, Ledger W, Galletly C, et al. Weight loss results in significant improvement in pregnancy and ovulation rates in anovulatory obese women. Hum Reprod (1995); 10:2705–12. 17 Adams J, Polson DW, Franks S. Prevalence of polycystic ovaries in women with anovulation and idiopathic hirsutism. BMJ (1986); 293:355–8. 18 Polson DW, Wadsworth J, Adams J, Franks S. Polycystic ovaries: a common finding in normal women. Lancet (1988); ii:870–2.

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19 Clayton RN, Ogden V, Hodgekinson J, et al. How common are polycystic ovaries in normal women and what is their significance for the fertility of the population? Clin Endocrinol (1992); 37:127–34. 20 Farquhar CM, Birdsall M, Manning P, Mitchell JM. Transabdominal versus transvaginal ultrasound in the diagnosis of polycystic ovaries on ultrasound scanning in a population of randomly selected women. Ultrasound Obstet Gynecol (1994); 4:54–9. 21 Balen AH, Tan SL, MacDougall J, Jacobs HS. Miscarriage rates following in-vitro fertilization are increased in women with polycystic ovaries and reduced by pituitary desensitization with buserelin. Hum Reprod (1993); 8:959–64. 22 MacDougall JM, Tan SL, Hall V, Balen AH, Mason BA, Jacobs HS. Comparison of natural with clomiphene citratestimulated cycles in IVF: a prospective randomized trial. Fertil Steril (1994); 61:1052–7. 23 Balen AH, Tan SL, Jacobs HS. Hypersecretion of luteinising hormoneA significant cause of infertility and miscarriage. Br J Obstet Gynaecol (1993); 100:1082–9. 24 Balen AH, Braat DDM, West C, Patel A, Jacobs HS. Cumulative conception and live birth rates after the treatment of anovulatory infertility. An analysis of the safety and efficacy of ovulation induction in 200 patients. Hum Reprod (1994); 9:1563–70. 25 Balen AH. Effects of ovulation induction with gonadotropins on the ovary and uterus and implications for assisted reproduction. Hum Reprod (1995); 10:2233–7. 26 Shoham Z, Conway GS, Patel A, Jacobs. HS. Polycystic ovaries in patients with hypogonadotropic hypogonadism: similarity of ovarian response to gonadotropin stimulation in patients with polycystic ovarian syndrome. Fertil Steril (1992); 58:37–45. 27 Balen AH, Jacobs HS. Ovulation induction. In: Balen AH, Jacobs HS, eds. Infertility In Practice. Edinburgh: Churchill Livingsone (1997):131–80. 28 Smitz J, Camus M, Devroey P, Evard P, Wisanto A, Van Steirteghen AC. Incidence of severe ovarian hyperstimulation syndrome after gonadotropin releasing hormone agonist/HMG superovulation for invitro fertilization. Hum Reprod (1991); 6:933–7. 29 Dor J, Shulman A, Levran D, Ben-Rafael Z, Rudak E, Mashiach L. The treatment of patients with polycystic ovary syndrome by in-vitro fertilization: a comparison of results with those patients with tubal infertility. Hum Reprod (1990); 5:816–8. 30 MacDougall JM, Tans SL, Balen AH, Jacobs HS. A controlled study comparing patients with and without polycystic ovaries undergoing invitro fertilization and the ovarian hyperstimulation syndrome. Hum Reprod (1993); 8:233–7. 31 Homburg R, Berkowitz D, Levy T, Feldberg D, Ashkenazi J, BenRafael Z. In-vitro fertilization and embryo transfer for the treatment of

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infertility associated with polycystic ovary syndrome. Fertil Steril (1993); 60:858–63. 32 Dunaif A. Insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis. Endocr Rev (1997) 18:774–800. 33 Franks S. Polycystic ovary syndrome. New Engl J Med (1995); 333:853–61. 34 Adashi EY, Resnick CE, D’Ercole AJ, Svoboda ME, Van Wyk JJ. Insulin-like growth factors as intraovarian regulators of granulosa cell growth and function. Endocr Rev (1985); 6:400–20. 35 Jacobs HS, Agrawal R. Complications of ovarian stimulation. Bailliere’s Clin Obstet Gynaecol (1998); 12:565–79. 36 Ferrara N, Chen H, Davis-Smyth T, et al. Vascular endothelial growth factor is essential for corpus luteum angiogenesis. Nature Med (1998); 4:336–40. 37 Agrawal R, Conway G, Sladkevicius P, et al. Serum vascular endothelial growth factor and Doppler blood flow velocities in in vitro fertilization: relevance to ovarian hyperstimulation syndrome and polycystic ovaries . Fertil Steril (1998); 70:651–8. 38 Rutherford AJ, Subak-Sharpe RJ, Dawson KJ, Margara RA, Franks S, Winston RML. Improvement of in-vitro fertilization after treatment with buserelin, an agonist of luteinising hormone releasing hormone. BMJ (1988); 296:1765–8. 39 Frydman R, Fries N, Testart J, et al. Luteinising hormone releasing hormone agonists in in-vitro fertilization: different methods of utilization and comparison with previous ovulation stimulation treatments. Hum Reprod (1988); 3:559–61. 40 Fleming R, Coutts JRT. Luteinising hormone releasing hormone analogues for ovulation induction, with particular reference to polycystic ovary syndrome. In: Anti-Hormones in Clinical Gynaecology, Ed: Healy D, Bailliere’s Clin Obstet Gynaecol (1988); 2:677–88. 41 Fleming R, Jamieson ME, Hamilton MPR, Black WP, Macnaughton MC, Coutts JRT. The use of gonadotropin releasing hormone analogues in combination with exogenous gonadotropins in infertile women. Acta Endocrinol (1988); 119 (suppl 288):77–84. 42 Abdalla HI, Ahuja KK, Leonard T, Morris NN, Honour JW, Jacobs HS. Comparative trial of luteinising hormone releasing hormone analogue/HMG and clomiphene citrate/HMG in an assisted conception programme. Fertil Steril (1990); 53:473–8. 43 Salat-Baroux J, Alvarez S, Antoine JM, et al. Comparison between long and short protocols of luteinising hormone releasing hormone agonist in the treatment of PCOD by in-vitro fertilization. Hum Reprod (1988); 3:535–9.

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44 Tanbo T, Dale PO, Kjekshus E, Haug E, Abyholm T. Stimulation with HMG versus follicle stimulating hormone after pituitary suppression in polycystic ovary syndrome. Fertil Steril (1990); 53:798–803. 45 Salat-Baroux J, Antoine JM. Accidental hyperstimulation during ovulation induction. In: Ovulation Induction, Ed Crosignani PG, Bailliere’s Clin Obstet Gynaecol (1990); 4:627–37. 46 Tan SL, Kingsland C, Campbell S, et al. The long protocol of administration of gonadotropin releasing hormone agonist is superior to the short protocol for ovarian stimulation for in vitro fertilization. Fertil Steril (1992); 57:810–4. 47 Jones GS, Garcia JE, Rosenwaks Z. The role of pituitary gonadotropins in follicular stimulation and oocyte maturation in the human. J Clin Endocrinol Metab (1984); 59:178–83. 48 Couzinet B, Lestrat N, Brailly S, Forest M, Schaison G. Stimulation of ovarian follicular maturation with pure follicle stimulating hormone in women with gonadotropin deficiency. J Clin Endocrinol Metab (1988); 66:552–6. 49 Shoham Z, Balen A, Patel A, Jacobs HS. Results of ovulation induction ovary human menopausal gonadotrophin or purified folliclestimulating hormone in hypogonadotrophic hypogonadism patients. Fertil Steril (1991); 56:1048–53. 50 Homburg R, Eshel A, Kilborn J, Adams J, Jacobs HS. Combined luteinising hormone releasing hormone analogue and exogenous gonadotropins for the treatment of infertility associated with polycystic ovaries. Hum Reprod (1990); 5:32–5. 51 Sagle MA, Hamilton-Fairley D, Kiddy DS, Franks S. A comparative, randomized study of low dose human menopausal gonadotropin and follicle stimulating hormone in women with polycystic ovary syndrome. Fertil Steril (1991); 55:56–60. 52 Agrawal R, Holmes J, Jacobs HS. Follicle-stimulating hormone or human menopausal gonadotropin for ovarian stimulation in in vitro fertilization cycles: a meta-analysis. Fertil Steril (2000); 73:338–43. 53 Daya S. Follicle stimulating hormone versus human menopausal gonadotropin for in vitro fertilization: results of a meta-analysis. Horm Res (1995); 43:224–9. 54 Lunenfeld B, Insler V. Classification of amenorrhoeic states and their treatment by ovulation induction. Clin Endocrinol (1974); 3:223–37. 55 Schenker JG, Weinstein D. Ovarian hyperstimulation syndrome: A current survey. Fertil Steril (1978); 30:255. 56 MacDougall JM, Tan SL, Jacobs HS. In-vitro fertilization and the ovarian hyperstimulation syndrome. Human Reprod (1992); 7:597– 600.

53 Severe ovarian hyperstimulation syndrome 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 which need to be minimized or completely eliminated. Ovarian hyperstimulation syndrome consists of ovarian enlargement accompanied by overproduction of ovarian hormones and a host of other ovarian vasoactive substances (cytokins, angiotensin, vascular endothelial growth factor, and others) 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 classifications have been suggested to better categorize the condition and disseminate uniform guidelines for prevention and treatment. The original classification was suggested by Rabau et al and later expanded upon by other authors.2,3 These researchers have suggested a classification based on the severity of the syndrome (mild, moderate and severe), subdivided into six grades. While this classification seemed at the time to be comprehensive, it incorporated unnecessarily cumbersome subdivisions. The mild form of the syndrome denotes supraphysiologic levels of estradiol (E2) and progesterone (P4) accompanied by slight ovarian enlargement of <5cm and abdominal discomfort. Mild OHSS is routinely inflicted on a large proportion of women undergoing so called COH, thus mild OHSS is nothing more than an acknowledgement that COH indeed has been achieved. Moderate OHSS includes significant ovarian enlargement (5–12cm) and accompanying symptoms. Symptoms for moderate OHSS include 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. Likewise, moderate OHSS is of

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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, which is detectable ultrasonically, but not clinically. Additional features, which upgrade OHSS to the severe form include hepatic dysfunction and anasarca (Table 53.1). To the original classification of severe OHSS, Navot et al5 added a critical stage of OHSS, denoting a life threatening phase of the syndrome, implying severely contracted blood volume with hemoconcentration, organ failure, and/or thromboembolic phenomena (Table 53.1). Accordingly, severe OHSS is accompanied by a variety of symptoms and signs that include severe abdominal distention, dyspnea, tachypnea, lower abdominal pain, hypotension, oliguria, and hydrothorax in addition to a host of laboratory abnormalities. Although the exact etiological factor responsible for the pathogenesis of OHSS is unknown, the syndrome’s dependence on hCG provides one of the basic strategies for prevention.

Table 53.1. Novel criteria for severe and critical OHSS. SEVERE OHSS CRITICAL OHSS Variable enlarged ovary Variable enlarged ovary Ascites±hydrothorax Tense ascites±hydrothorax ±pericardial effusion Hematocrit >45% Hematocrit >55% WBC >15,000 WBC >25,000 Oliguria Oligoanuria Creatinine 1.0–1.5mg/dl Creatinine ≥1.6mg/dl Creatinine clearance ≥50ml/min Creatinine clearance ≥50ml/min Liver dysfunction Renal failure Anasarca Thromboembolic phenomena ARDS Table 53.2. Risk factors associated with OHSS. HIGH RISK LOW RISK Young (<35 years) Older (>36 years) PCOD-like Hypogonadotrophic Asthenic Habitus Heavy build High serum estradiol (ART >4000pg/ml; Low serum estradiol OI>1,700pg/ml) Multiple follicles (ART>20, OI>6) Few follicles Necklace sign Quiescent ovary Pregnancy Barren cycle hCG luteal supplementation Progesterone or no supplementation

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Clomiphene citrate and/or HMG protocol

PREVENTION OF SEVERE OHSS THE ROLE OF THE STIMULATORY AGENT AND PROTOCOL Ovarian hyperstimulation syndrome has intrigued clinicians for many years, because of 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 53.2 details the risk factors that should alert the clinician contemplating COH regarding the danger of severe OHSS. The criteria defining high versus low risk are different for assisted reproductive technologies (ART) and conventional ovulation induction (Ol). The single most important parameter to cause concern is the policystic appearance of the ovaries with the “necklace sign” or the “string of pearls” appearance on transvaginal ultrasound (Fig 53.1). In contrast, relatively quiescent ovaries with few antral follicles denote usually a slow response with little risk of OHSS. Early reports suggested a relation between the type of gonadotropin preparation utilized and the risk of OHSS. Recently, comparison between recombinant FSH (rFSH) and menotropins did not show significant difference between variable drug regimen. A large study by Bergh et al6 compared 119 cycles of rFSH (Gonal-F) to 114 cycles of uFSH-HP (Metrodin HP). Both groups were downregulated by a long GnRH-a protocol. All parameters studied, including E2 serum concentrations, ampoules utilized, days of stimulation, number of oocytes retrieved, and number of embryos obtained were significantly in favor of rFSH. Although clinical pregnancy rates and implantation rates were similar, significantly more embryos were frozen subsequent to rFSH stimulation.

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Fig 53.1 Transvaginal ultrasound depicting the ovary of a 31 year old woman with amenorrhea and PCOD. The picture illustrates the main features that constitute the high risk factors for the development of severe OHSS. • The string of pearls appearance of antral follicles • a dense stroma occupying the middle of the ovary • an enlarged ovary measuring 44.6×49.0mm • a total of 60 follicles on the right and 35 follicles on the left ovary. Ovulation was induced by a chronic low dose rFSH regimen. By day 21 two follicles have attained a mature size of >16mm and ovulation was triggered with 5000IU of hCG without complications. The respective rates of OHSS were 5.2% for rFSH and 1.7% for Metrodin HP. Another large study compared 585 patients receiving rFSH with Metrodin (396 patients).7 This report has shown similar advantages to rFSH in regard to length of treatment and ampoules utilized adding

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significantly higher ongoing pregnancy rate for rFSH when frozen thawed embryos were added to the equation. The rate of OHSS was 3.2% versus 2.0% for the rFSH and Metrodin respectively. This difference was not significant. The capacity of rFSH to enhance follicular recruitment and serum E2 concentrations may indeed carry a slightly increased risk of OHSS. However, the seemingly increased risk may also be due to lack of experience with rFSH. With increased awareness and understanding the unique features of rFSH, the actual rate of OHSS is bound to decrease. This seems to be borne out in more recent studies published comparing rFSH vs VFSH.8,9 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.10,11 Specifically, the so called chronic low dose regimen is more likely to result in mono or bifollicular response and significantly lower rate of OHSS. Similarly a step down protocol will allow more follicles to undergo atresia, rather than continuous rescue by a step up protocol. Thus the overall number of follicles capable of secretory activity is reduced 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 Sher12,13 and practiced widely later by several other researchers with variable results. Benadiva et al and Tortoriello et al have reported significant reduction in OHSS.14,15 While Shapiro et al and others have found no benefit in coasting.16,17 This discrepancy in the result of coasting is most probably due to the fact, that the latter researchers have discontinued gonadotropins for only two to three days while earlier studies continued coasting for up to six and even eight days. The use of GnRH-a in conjunction with COH, either as “long” or “short” protocol, has also a profound effect on the risk of OHSS. GnRH-a play a paradoxical role in OHSS by virtue of the control they afford, despite the 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 respective rise in serum E2 values increasing markedly the 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 with a concomitantly lesser risk of OHSS. Once the prerequisites for severe hyperstimulation are established, namely a multifollicular, high estrogenic milieu, the occurrence of OHSS is utterly hCG dependent. Thus either exogenously administered or pregnancy derived hCG are absolutely essential for the development of OHSS. In contrast, avoidance of hCG or substitution by a low affinity shorter acting compound are the mainstays for the prevention of OHSS.

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Indeed the acknowledgement 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 removed a major risk factor for OHSS in ART. However hCG as a surrogate for the midcycle LH surge is still universally used in ovulation induction as well as ART. The standard dosage of hCG used to trigger ovulation is 5000–10000IU. These dosages take 6–9 days respectively to clear the hCG from the circulation, thus exerting continuous ovarian stimulation up to the stage in which endogenous-pregnancy derived hCG is perceived. Since hCG has a very long half life and high affinity to the ovarian LH receptor it sustains the function of multiple corpora lutea up to the point of rescue by endogenous hCG. This ovarian effect of hCG exerts a stimulatory effect on the putative ovarian substance which directly promotes, or may even be the causal factor in ovarian hyperstimulation. The putative factor may be angiotensin II of the ovarian reninangiotensin cascade, vascular endothelial growth factor (VEGF), or interleukin 1, 2, or 6, all of which are either directly or indirectly enhanced by hCG.18,19 THE ROLE OF hCG AND ITS SUBSTITUTES The critical role of hCG in OHSS 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: it has a very short half life of about 20 minutes; and to create a surge of at least 24 hours either huge doses or repeated administration would be needed. Recombinant LH (rLH) on the other hand is still not commercially available. Recently a dose finding study, in which rLH was utilized to trigger ovulation was published.20 The recombinant LH utilized was however engineered with a high sialic acid content which very much extended its half-life. Any rLH with prolonged halflife will behave like hCG and may not substantially reduce the incidence of OHSS. A logical alternative is endogenous LH stimulation by either native GnRH or preferably GnRH-a. Early attempts to elicit an LH surge with synthetic GnRH in an hMGstimulated cycle yielded variable results.21,22 More recently, however, attempts to trigger ovulation with GnRH analogues have been more consistent in their results. Lanzone et al23 and Imoedemhe et al24 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. GnRH-a, through its prolonged half life and enhanced biological activity has become the agent of choice to stimulate the midcycle LH surge. Since then, there have been numerous reports of the use of GnRH-a to successfully induce follicular maturation in IVF cycles,25,26 as well as ovulation in non-ART cycles.27,28

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Several authors have addressed the efficacy of GnRH-a in preventing OHSS. Most reports support the hypothesis that GnRH-a allows ovulation, whilst avoiding OHSS. Emperaire and Ruffie studied 37 of 126 cycles in 48 patients undergoing ovulation induction with a regimen of either hMG or CC/hMG.29 All cycles were considered to be at high risk of OHSS and/or multiple pregnancy (E2 level>1000–1200pg/ml and >3 follicles of >17mm mean diameter). In these at risk cycles, the LH surge was provoked by intranasal buserelin 200µg three times at eight-hourly intervals. Ovulation was documented in all the cycles except one (97%). Eight pregnancies resulted (21.6%), and there were no cases of OHSS. Imoedemhe et al24 used two doses of GnRH-a (Suprefact 100µg) by nasal spray eight hours apart to induce follicular maturation 34–36 hours prior to oocyte recovery in 38 women considered at risk of OHSS (E2>4,000pg/ml) in an IVF program.24 Of the 707 oocytes recovered at egg retrieval, 93% were scored as mature, and 46% were successfully fertilized. Twenty six women had embryos replaced and 11 pregnancies occurred (28.9%), and there were no cases of OHSS. Itskovitz et al used buserelin acetate in dosages of either 250µg or 500µg injected subcutaneously in either a single or two divided doses 12 hours apart.26 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 al28 and Balasch et al30 used a single subcutaneous injection of triptorelin and leuprolide (0.5mg), respectively, to trigger ovulation in gonadotropin stimulated cycles which would otherwise have been canceled due to what was thought to represent high risk for OHSS. Fifty per cent and 17.4% conception rates were achieved respectively, while no patient developed OHSS. Kulikowski and colleagues used a single dose of 0.3mg 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>2500pg/ml).31 All patients had ovulation induced with a CC/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 ovulation induced with CC/hMG/GnRH-a, no OHSS was detected, while four patients became pregnant (25%). In summary, a number of investigators have used midcycle GnRH-a in cycles considered to be at high risk for development of OHSS, in varying dosages and time intervals. Pregnancy rates of 12.5–50% were achieved with a 0% incidence of OHSS. 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 have published a study in which three patients who used buserelin to induce a pre-ovulatory endogenous LH surge in lieu of hCG nevertheless developed moderate OHSS.32 Severe ascites, hypovolemia, or electrolyte imbalance did not occur, and no

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patients were hospitalized. These authors concluded that OHSS can occur independent of the ovulation triggering agent, and is due to a massive luteinization of the follicles after exaggerated follicular stimulation. Gerris et al also reported the occurrence of moderate OHSS in one patient following GnRH-a, administration,27 but in this case native GnRH was used, resulting in successful ovulation triggering, but without the critical gonadotropin suppression which is thought to be at least equally important in the prevention of OHSS.33 Casper cited a total of 163 cycles in which GnRH-a was used to trigger ovulation in the context of preventing OHSS.34 He stipulated that 900 cycles should have been randomized in order to find significant difference between GnRH-a and hCG. The problem with his assumption stems from assigning a 2% risk for severe OHSS. Although a 2% risk may be applicable to the average woman undergoing COH, most women in his survey might have had far greater risk, possibly in the 10–20% range. Thus there is a small possibility of moderate degree of OHSS developing despite the substitution of GnRH-a for midcycle hCG, especially in cycles with conception. Importantly, however, there have been no reports of severe or critical OHSS developing after using midcycle GnRH-a. The only two major drawbacks of GnRH-a triggered ovulation are the fact that it is frequently followed by extremely deficient luteal phase and it cannot be given in cycles in which GnRH-a was previously used. Both disadvantages may respectively be overcome by luteal supplementation of progesterone and use of GnRH antagonists (GnRH-ant) instead of long agonist protocols for the suppression of endogenous LH surge. GnRH-ant AND OHSS de Jong et al have used the GnRH-ant ganirelix to prevent OHSS by increasing the dose of the antagonist when target E2 values were inadvertently exceeded (16500pmol/l).35 Indeed E2 values have rapidly decreased with 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 would be 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 0.25mg of ganirelix daily seems to be sufficient to eliminate LH surge and result in favorable clinical outcome. Theoretically a larger dose of GnRH-a would be needed to induce LH surge in cycles suppressed by ganirelix, than in gonadotropin only induced ovulation. In addition progesterone supplementation will be mandatory throughout the luteal phase. RECOMBINANT LH AND OHSS

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The use of rLH as a midcycle substitute to natural LH surge will soon become feasible in all kinds of COH; whether gonadotropins alone are used, CC/hMG cycles or cycles cotreated with GnRH analogs are practiced. Loumaye et al have used doses of rLH between 5000 IU and 30000 IU to compare to 5000 IU of u-hCG.20 While all doses induced final follicular maturation, similar numbers of oocytes and equal fertilization rates, the incidence of ovarian enlargement and some ascites seemed to be directly dose dependent. There was no ovarian enlargement or ascites up to 10000 IU of rLH. One of 26 (3.8%) women on 30000 IU rLH and 13/121 (10.7%) on u-hCG had ovarian enlargement, ascites or accompanying symptomatology. One patient of 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 had a relatively long half life, compared to native LH. Alternately, it is possible that a single peak of LH is sufficient to induce final oocyte maturation as opposed to the 24 hour long naturally occurring LH surge. MISCELLANEOUS TECHNIQUES TO PREVENT OHSS Other modalities that were suggested for the prevention of OHSS include unilateral or bilateral follicular aspiration as a rescue for cycles not otherwise intended to undergo oocyte retrieval.36 Egbase advocated ovarian diathermy prior to initiation of COH.37 Ovarian diathermy should, however, be reserved to young patients with a severe condition of PCOS who tend to hyperstimulate even on the prolonged low dose FSH regimen. Recently metformin, an 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 follow. A very important safety net to guard against severe OHSS is the liberal application of embryo cryopreservation in cycles with early signs of hyperstimulation.38 With routine culture of embryos to the blastocyst stage it is possible to accurately assess the degree of OHSS prior to embryo transfer. Since blastocyst transfer takes place on the seventh day after hCG, absence of even a moderate degree of OHSS would be reassuring, and one may proceed with embryo transfer.5 Intravenous albumin, at the time of oocyte retrieval as a preventive measure is of questionable benefit in contrast to the significant value of albumin for the treatment of the full blown syndrome.19 Meta-analysis of randomized controlled trials conducted and published by the Cochrane Library39 has shown a clear benefit from administration of intravenous albumin at the time of oocyte retrieval in prevention of severe OHSS in highrisk cases. However, the results of this review cannot be regarded as

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conclusive as they are based on only three small trials. Further trials are urgently needed. In the near future GnRH-ant will be routinely used in clinical practice. Once we master the complexity of GnRH-ant for LH surge suppression and GnRH-a for triggering ovulation, ovarian stimulation is going to be controlled, and OHSS will be a forgotten entity. TREATMENT OF SEVERE OHSS MEDICAL APPROACH There are two possible approaches to the treatment of OHSS. Pathogenesis oriented treatment and empiric treatment. The former approach utilizes modalities which will specifically negate the putative causative factor(s) of OHSS. Indomethacin was such an agent, when prostaglandin was believed to play a role in OHSS. ACE inhibitors are another group of specific pharmacological agents which inhibit angiotensin II production, a probable pathogenic factor for the syndrome. Unfortunately, indomethacin did not benefit the syndrome, while ACE inhibitors are teratogenic and thus contraindicated whenever a pregnancy is contemplated. Although in the future cytokine inhibitors may by tried in OHSS, to date treatment of OHSS remains largely empiric in nature. Mild forms of OHSS require little more than reassurance. It is well established that in the absence of pregnancy, symptoms would resolve within two weeks after receiving hCG. If a pregnancy ensues, however, 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 by balanced salt solution. Gatorade, a popular drink among athletes, seems to be particularly suitable. The patient should be alerted to decreases in urine output, significant weight gain, or bloating, which may be self assessed by daily abdominal girth measurement. These symptoms may be the first warning signals of accumulation of ascitic fluid and accompanying hemoconcentration (hematocrit >45% or a 30% increment over baseline). The latter indicate that the condition has entered the category of severe OHSS and that hospitalization is required. Dramatic clinical deterioration is most likely to manifest eight to nine days after hCG administration, when endogenous, pregnancy derived hCG becomes perceptible. Severe OHSS has a wide range of systemic effects, which are secondary to depletion of intravascular plasma volume. Thus 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 RCV and total blood volume (RCA+plasma volume), the change in plasma volume must always be

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larger than the change reflected by the hematocrit.18 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% does not accurately reflect 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 small incremental rise in hematocrit between 40% and 45% is observed. Similarly, in the face of hemoconcentration, small drops in the 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 25000/mm3 may be seen. This massive neutrophilia may be attributed to hemoconcentration and generalized stress reaction. When oral fluid intake is insufficient to maintain plasma volume, intravenous fluid therapy becomes mandatory. Table 53.3 details the pros and cons of various therapies in the treatment of severe OHSS. Crystalloid alone, although seldom sufficient in restoring homeostasis because of massive protein loss, 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 1.51 to greater than 3.01. Although some authors advocate fluid restriction, to minimize the accumulation of ascites,40 one should rather deal with the discomfort of ascites than the consequences of hemoconcentration with the risk of thromboembolism and renal shutdown. In order to maintain fluid balance, urine output, oral and IV fluid intake, body weight, abdominal girth, hematocrit, and serum electrolytes have to be monitored. In addition coagulation parameters and liver enzymes are to be periodically assessed. Intravenous volume replacement should result in improved renal perfusion before fluid escapes into the peritoneal and/or pleural cavities. This transient haemodilution is achieved at the expense of increasing ascites, however. Whenever adequate fluid balance cannot be restored by crystalloid alone, plasma expanders should be utilized. Since albumin is the main protein lost in OHSS, low salt human albumin is the physiologic and thus volume expander of choice (Table 53.3).

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Table 53.3. Pros and cons of various therapies of OHSS. THERAPY PROS CONS IV crystalloids Alleviates hemoconcentration Lost immediately from Improves renal perfusion vascular tree Aggravates ascites Fluid restriction Controls ascites Reduces renal perfusion Promotes hemoconcentration Albumin Improves colloid-osmotic Human blood product pressure Improves renal perfusion Furosemide Reduces overall fluid excess Further reduces intravascular volume Indomethacin May block prostaglandinImplicated in renal failure induced hyperpermeability ACE inhibitors May block All-induced Teratogenic hyperpermeability Paracentesis Alleviates tense ascites Risk of hemorrhage, Improves renal perfusion infection, and leakage Heparin Decreases risk of Increases risk of hemorrhage thromboembolic phenomena Peritoneo-venous Replaces lost electrolytes and Risk of self toxicity shunt proteins Elaborate set-up and risk of infection Dopamine drip Improves renal blood flow Need for Intensive Care Unit Antiotensin II (All) Albumin at doses of 50–100 grams at 25% concentration should be administered intravenously and repeated every two to 12 hours until hematocrit falls below 45% and urine output increases. At a relatively advanced stage of OHSS, secondary to 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 often is a sudden, paradoxical onset of oliguria, increasing serum creatinine, and rapidly falling creatinine clearance.41 The sudden deterioration in fluid balance is probably the result of a significant rise in intra-abdominal pressure produced by tense ascites. Increased intraabdominal pressure may in turn impede renal venous outflow, causing congestion, renal edema, and decrease in renal function. A tense ascites is best treated through a surgical approach although diuretics may also be effective. When oliguria persists despite evidence of adequate

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hemodilution, intravenous furosemide (Lasix) at 10–20mg dose is often beneficial. In practice, an albumin-furosemide chase protocol seems to yield the best results. Two units of albumin (50g each), followed immediately by intravenous furosemide often will result in diuresis. In states of volume contraction, hemoconcentration and hypotension Lasix should be strictly avoided. In this precarious stage of OHSS, with impending renal failure, low dose dopamine drip should be used for renal rescue. PARACENTESIS The single most important treatment modality in life threatening 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 it has regained popularity (Table 53.3).3 Thaler,42 Borenstein,41 and Forman43 have all promoted paracentesis as safe and exceptionally beneficial. Dramatic improvements in the clinical symptoms of severe OHSS, with almost instantaneous diuresis, were reported.41 In a series of seven patients in whom paracentesis was performed, urine output rose from 780ml±407ml to 1670ml±208ml (P<0.05) and creatinine clearance from 75.4ml/min±16ml/min to 101ml/min±15ml/min (P<0.05), the hematocrit decreased from 46.3%±2.2% to 37.1%±2.5% (P<0.05), and a mean weight loss of 5.3kg was observed.44 In the study by Forman et al,43 37l of ascitic fluid with a protein content of 46g/l to 53g/l (1.85kg protein loss) 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 creatinine 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.44 Paracentesis is contraindicated in a patient who is hemodynamicaly unstable or if there is suspected hemoperitoneum. In other instances up to 41 of fluid may be removed by slow drainage to gravity.42 A new and innovative treatment for severe OHSS was suggested by Koike et al.40 The authors describe continuous peritoneovenous shunting in 18 patients with severe OHSS. This study group was compared to 36 control patients who had received intravenous albumin at a dose of 37.5g/day. Recirculation of ascites fluid rich in proteins is not a novel idea;45 however, the reliance on a continuous shunt from the peritoneal cavity into the antecubital vein is a logical way to replenish the vascular tree with the fluid volume, proteins and electrolytes that were lost from the vasculature. The study describes faster hemodilution, shorter hospital

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stay, and prompt improvement in symptoms thanks to diuresis and reduction in the amount of ascites. There are, however, some problems with the study in addition of the complexity of the setup. Firstly, 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.40 Most authors reserve treatment by heparin for special circumstances in which thromboembolic events have already occurred, or there is abnormal clotting, often owing to congenital coagulopathy. Although low dose, preventive treatment with heparin is of some theoretical value40 rapid alleviation of hemoconcentration is far more important. When the critical stage of OHSS is complicated by renal failure, thromboembolism, or ARDS, there is no choice but to surgically terminate the pregnancy.

REFERENCES 1 Abramov Y, Elchalal U, Schenker JG. 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, Soffer Y, Weinraub Z, Caspi E. 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 Bergh C, Howles CM, 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. Hum Reprod (1997); 12:2133–9. 7 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. 8 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 (Medtrodin HP) in women undergoing assisted reproductive techniques including

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intracytoplasmic sperm injection. On behalf of The French Multicentre Trialists. Hum Reprod (2000); 15:520–5. 9 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. 10 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., 11 Homburg R, Levy T, Ben-Rafael Z. A comparative prospective study of conventional regimen with chronic lowdose administration of follicle-stimulating hormone for anovulation associated with polycystic ovary syndrome. Fertil Steril (1995); 63:729–33. 12 Sher G, Salem R, Feinman M, Dodge S, Zouves C, Knutzen V. Eliminating the risk of life-endangering complications following overstimulation with menotropin fertility agents: a report on women undergoing in vitro fertilization and embryo transfer. Obstet Gynecol (1993); 81:1009–11. 13 Sher G, Zouves C, Feinman M, Maassarani G. ‘Prolonged coasting’: an effective method for preventing severe ovarian hyperstimulation syndrome in patients undergoing in-vitro fertilization. Hum Reprod (1995); 10:3107–9. 14 Benadiva CA, Davis O, Kligman I, Moomjy M, Liu HC, Rosenwaks Z. Withholding gonadotropin administration is an effective alternative for the prevention of ovarian hyperstimulation syndrome. Fertil Steril (1997); 67:724–7. 15 Tortoriello DV, McGovern PG, Colon JM, Skurnick JH, Lipetz K, Santoro N. “Coasting” does not adversely affect cycle outcome in a subset of highly responsive in vitro fertilization patients. Fertil Steril (1998); 69:454–60. 16 Shapiro D, Craven G, Mitchell-Leef J, et al. Allowing estradiol levels to fall in a controlled drift does not affect pregnancy rates and offers no protection against ovarian hyperstimulation syndrome. Fertil Steril (1997) (supplement):S169. 17 Lee C, Tummon I, Martin J, et al. Does withholding gonadotrophin administration prevent severe ovarian hyperstimulation syndrome? Hum Reprod (1998); 13:1157–8. 18 Bergh PA, Navot D. Ovarian hyperstimulation syndrome—a review of pathophysiology. J Assist Reprod Genet (1992); 9:429–38. 19 Orvieto R, Ben-Rafael Z. Ovarian hyperstimulation syndrome: a new insight into an old enigma. J Soc Gynecol Investig (1998); 5:110–3. 20 Loumaye E, Piazzi A, Engrand P. Results of a phase II, dose finding, clinical study comparing r-LH with hCG to induce final follicular maturation prior to IVF. Sixteenth World Congress on Fertility and

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Sterility, San Francisco October 4–9 1998 (Abst.0–236). San Francisco, 1998. 21 Breckwoldt M, Czygan PJ, Lehmann F. Synthetic LH-RH as a therapeutic agent. Acta Endocrinol (Copenh) (1974); 75:209–20. 22 Crosignani PG, Trojsi L, Attanasio A, Tonani E. Hormonal profiles in anovulatory patients treated with gonadotropins and synthetic luteinizing hormone releasing hormone. Obstet Gynecol (1975); 46:15– 22. 23 Lanzone A, Fulghesu AM, Apa R, Caruso A. LH surge induction by GnRH agonist at the time of ovulation. Gynecol Endocrinol (1989); 3:213–20. 24 Imoedemhe D, Chan R, Sigue A, Pacpaco E. A new approach to the management of patients at risk of ovarian hyperstimulation in an invitro fertilization programme. Hum Reprod (1991); 6:1088–91. 25 Gonen Y, Balakier H, Powell W. Use of gonadotropin-releasing hormone agonist to trigger follicular maturation for in vitro fertilization. J Clin Endocrinol Metab (1990); 71:918–22. 26 Itskovitz J, Boldes R, Levron J, Erlik Y, Kahana L. Induction of preovulatory luteinizing hormone surge and prevention of ovarian hyperstimulation syndrome by gonadotropin-releasing hormone agonist. Fertil Steril (1991); 56:213–20. 27 Gerris J, De Vits A, Joostens M. Triggering of ovulation in human menopausal gonadotrophin-stimulated cycles: comparison between intravenously administered gonadotrophin-releasing hormone (100 and 500ug), GnRH agonist (buserelin, 500ug) and human chorionic gonadotrophin (10000IU). Hum Reprod (1995); 10:56–62. 28 Shalev E, Geslevich Y. Induction of pre-ovulatory luteinizing hormone surge by gonadotrophin-releasing hormone agonist for women at risk for developing the ovarian hyperstimulation syndrome. Hum Reprod (1994); 9:417–9. 29 Emperaire J. Triggering ovulation with endogenous luteinizing hormone may prevent the ovarian hyperstimulation syndrome. Hum Reprod (1991); 6:506–10. 30 Balasch J, Tur R, Creus M, et al. Triggering of ovulation by a gonadotropin releasing hormone agonist in gonadotropin-stimulated cycles for prevention of ovarian hyperstimulation syndrome and multiple pregnancy. Gynecolog Endocrinol (1994); 8:7–12. 31 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. 32 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. 33 Kol S, Lewit N, Itskovitz-Eldor J. Ovarian hyperstimulation: effects of GnRH analogues. Ovarian hyperstimulation syndrome after using

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gonadotrophin-releasing hormone analogue as a trigger of ovulation: causes and implications. Hum Reprod (1996); 11:1143–4. 34 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. 35 de Jong D, Macklon NS, Mannaerts BM, Coelingh Bennink HJ, Fauser BC. High dose gonadotrophin-releasing hormone antagonist (ganirelix) may prevent ovarian hyperstimulation syndrome caused by ovarian stimulation for in-vitro fertilization. Hum Reprod (1998); 13:573–5. 36 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. 37 Egbase PE. Severe OHSS; How many cases are preventable? Hum Reprod (2000); 15:8–10. 38 Garrisi G, Navot D. Cryopreservation of semen, oocytes, and embryos. Curr Opin Obstet Gynecol (1992); 4:726–31. 39 Aboulghar M, Evers JH, IL-Anany H. Intra-venous albumin for preventing severe ovarian hyperstimulation syndrome (Cochrane Review). In: The Cochrane Library. Issue 1, 2000. Oxford: Update Software. 40 Koike T, Araki S, Minakami H, et al. Clinical efficacy of peritoneovenous shunting for the treatment of severe OHSS. Hum Reprod (2000); 15:113–7. 41 Borenstein R, Elhalal U, Lunenfeld B, Shoham Z. Severe OHSS; a reevaluated therapeutic approach. Fertil Steril (1989); 51:791–5. 42 Thaler I, Yoffe M, Kaftory JK, Brandes JM. Treatment of OHSS; the physiologic basis for a modified approach. Fertil Steril (1981); 36:110–3. 43 Forman RG, Frydman R, Egan D, Ross C, Barlow DH. Severe OHSS using agonists of gonadotropin-releasing hormone for in vitro ferilization; a European series and a proposal for prevention. Fertil Steril (1990); 53:502–9. 44 Rizk B, Aboulghar MA. Modern management of OHSS. Hum Reprod (1991); 6:1082–7. 45 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.

54 Bleeding, severe pelvic infection, and ectopic pregnancy Raoul Orvieto, Zion Ben-Rafael

Transvaginal ultrasonography guided aspiration of oocytes is a well accepted and a 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 done as a day care procedure under intravenous analgesia and sedation, 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, and spontaneous abortion and extrauterine pregnancy (EUP). The aim of the present review is to comprehensively discuss 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 the 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. The reported incidence of vaginal bleeding as a complication of ovum pick-up (OPU) varies from 0 to 1.3%;1,3 a recent study reported a rate of 0.09%.4 In most cases vaginal bleeding as a result of OPU stops spontaneously at the end of the procedure.3 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.

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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. Intraperitoneal bleeding tends to be severe with acute hemodynamic deterioration, whereas retroperitoneal bleeding usually has a later and more indolent presentation. 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 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 abdomen develops, diagnostic laparoscopy or exploratory laparotomy with subsequent hemostatis of the bleeding site(s) are required. The clinician must make sure to handle the fragile overhyperstimulated ovaries very cautiously. Our group has reported on three cases of severe intra-abdominal bleeding from ovarian puncture sites during OPU, leading to acute abdomen.1 In two of the patients, symptoms developed three hours after OPU (hemoglobin 9.0g/100mh and 8.1 g/100mh), and laparoscopic drainage and hemostatis were sufficient. The third patient became symptomatic after 4 hours (hemoglobin 7.3g%) and required exploratory laparotomy and hemostatis in addition to the transfusion of four units of blood as a life saving procedure. Description of the intraoperative measures needed to control intraabdominal hemorrhage is beyond the scope of this text, and the reader is referred elsewhere for a detailed review.5

PELVIC INFLAMMATORY DISEASE Pelvic inflammatory disease is an infrequent complication of ultrasound guided transvaginal aspiration of oocytes or embryo transfer, with a reported incidence of 0.2% to 0.5% per cycle.6–8 Signs or symptoms of pelvic infection, such as pyrexia, continuous low abdominal pain, dysuria, or offensive vaginal bleeding, are infrequent.6 However, this does not exclude occult, subclinical bacterial colonization, which may influence the success of the in vitro fertilization-embryo transfer (IVF-ET) treatment. Our group evaluated the outcome of all IVF-ET procedures performed in our unit between 1986 and 1992.8 Of the 4771 patients who underwent transvaginal OPU, 28 (0.58%) had symptoms of PID within 1–7 days. The

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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 >12000 cells/m3, and elevated erythrocyte sedimentation rate. All patients were admitted to hospital for treatment with intravenous antibiotics. 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.7 Three patients required laparotomy and adnexectomy, whereas in six patients culdocentesis was performed for adequate pelvic abscess drainage. 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 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,9,10 or rarely to inadvertent puncture of the bowel. Pelvic infection can occur as a direct consequence of transcervical ET. This is evidenced by cases of PID following ET in an agonadal donor egg recipient,11 or during cryopreserved ET12; 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. EFFECT OF ACUTE PELVIC INFECTION ON IVFET OUTCOME The first study of the impact of pelvic infection on IVF-ET outcome was reported by our group in 1994.8 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. 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 al13 reported on a case in which human oocytes were

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degenerated and fragmented, with no evidence of fertilization, in 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 al14 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.14 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.15,16 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,17 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.18 In vitro experiments have shown that temperature elevation leads to disintegration of the cytoskeleton19 and may affect the transport of organelles. In pregnancy, maternal heat exposure can cause intracellular embryonic damage20 and inhibit cell mitosis, proliferation and migration, resulting in cell death. In a study of guinea pig embryos, Edwards et al21 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.22

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TREATMENT 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 vaginal disinfection.23 Meldrum24 found no case of pelvic infection among 88 transvaginal retrievals, with the use of intravenous cefazolin and a vaginal preparation with povidoneiodine and saline irrigation; nor did Evers et al25 in 181 patients, using only 10% povidoneiodine. Borlum and Maiggard26 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 type of antibiotic, timing or duration of therapy, and efficacy of therapy have not yet been established.24,27 Indeed, some authors claim that these measures may not only further reduce the incidence of PID after oocyte retrieval, but can even increase the risks both of 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 al28 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.29,30 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 larger than 8cm or unresponsive to medication, transvaginal or percutaneous drainage is the treatment of choice,31 without or with ultrasound guided intracavitary instillation of a combination of antibiotics.32 Sometimes surgical laparoscopy or laparotomy is needed to evacuate the abscess or remove the infected tubes or adnexae. SUMMARY The appearance of PID at the critical time of implantation results in failure to conceive. This effect may be mediated by bacterial endotoxins, local inflammatory reaction against bacteria with the involvement of cytokines that affect implantation and early embryonic development, or temperature

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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, rigorous treatment of the bacterial infection and fever should be accomplished in an attempt 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.33 In 1992, the United States reported that almost 2% of all pregnancies were EUPs, and ectopic pregnancies accounted for 10% of all pregnancyrelated deaths.33,34 The rates of abortions, multiple pregnancies and EUPs are higher in pregnancies resulting from assisted reproduction techniques (ART) compared to spontaneous pregnancies. Many factors have been associated with the development of EUP. These include previous EUP, salpingitis, previous surgery to the fallopian tube, peritubal adhesions, pelvic lesions that distort the tube, developmental abnormalities of the tube, altered tubal motility, and ART. EUP AFTER ART The first IVF-ET pregnancy reported was an ectopic pregnancy.35 Today, the incidence of EUPs after IVF ranges from 2.1% to 9.4% of all clinical pregnancies.36,37 In 1996, the Society for Assisted Reproductive Technology (SART)38 reported a decrease in the incidence of EUP to 0.1% of transfers and 0.4% of intrauterine pregnancies, compared with 0.9% and 2.8%, respectively, in 1995. This finding might be 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. RISK FACTORS Data on risk factors for EUP after IVF are still unclear. Martinez and Trounson39 failed to identify any risk factors, whereas Karande et al40 pointed to a prior ectopic pregnancy. Verhulst et al41 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.37,42–44 Cohen et al45 showed that the

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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 those with one. In an analysis of the Bourn Hall Clinic data, Marcus and Brinsden46 found that the main risk factor was a history of PID. Though 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/E2 ratio on the day of ET had no associated history of EUP. Finally, Ankum et al47 in their meta-analysis of risk factors for EUP, concluded that the four strongest ones were previous EUP, documented tubal pathology, previous tubal surgery, and in utero exposure to diethylstilbestrol. These results were confirmed by Lesny et al,48 who also added one more, a difficult ET on day 2 rather than day 3. 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; the gravitational pull of the embryos to the hanging tubes, which are located lower than the uterine fundus; and the reflux expulsion of the embryo due to embryonic migration to the fallopian tubes, either spontaneously or secondary to uterine contractions.49 The technique of ET itself may also be a culprit in EUP, although this is controversial. For example, Yovich et al50 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 80µl of media are needed for the embryo to reach the fallopian tube,37 Knutzen et al,51 demonstrated easy passage of all or part of the material in 44% of patients in a mock intrauterine ET with 50µl of radiopaque dye. Lesny et al52 explained these findings by the propulsion of the embryo from the uterine fundus into the tubes by the junctional zone contractions. Therefore, being that 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,42 such as those derived from chromosomally abnormal gametes.53 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.54,55 However, this measure does not prevent the development of an interstitial pregnancy,44 although it certainly prevents the well known phenomenon of spontaneous pregnancies after IVF treatment, which occurs in 30% of the patients with patent tubes.56

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Another potential interfering factor in tubal function and embryo transfer is the different hormonal milieus resulting from ovulation induction protocols, particularly those including clomiphene citrate.41,57 This may result from the effect of the high E2 levels on tubal peristalsis through the control of tubal smooth muscle contractility and ciliary activity.50,57 Pygriotis et al,44 however, did not demonstrate a difference in E2 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 embryo transfers following natural cycles where the E2 levels were comparatively low. HETEROTOPIC PREGNANCY FOLLOWING ART The general incidence of combined intrauterine and extrauterine (heterotopic) pregnancy is 1:15000–30000, and it increases dramatically in pregnancies following ART or ovulation induction to 1:100.58–60 Although a distorted pelvic anatomy is responsible for the predisposition to both extrauterine and heterotopic pregnancy,61–63 heterotopic pregnancies are associated with a greater number of embryos transferred, whereas EUP is not: Tummon et al64 reported that when four or more embryos were transferred, the odds ratio for the development of a heterotopic pregnancy versus 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.65,66 A high index of suspicion and early intervention are mandatory to salvage the viable intrauterine pregnancy and prevent maternal mortality. 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.67,68

REFERENCES 1 Dicker D, Ashkenazi J, Feldberg D, Levy T, Dekel A, Ben-Rafael Z. Severe abdominal complications after transvaginal

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ultrasonographically guided retrieval of oocytes for in vitro fertilization and embryo transfer. Fertil Steril (1993); 59:1313–5. 2 Feldberg D, Goldman JA, Ashkenazi J, Shelef M, Dicker D, Samuel N. Transvaginal oocyte retrieval controlled by vaginal probe for in vitro fertilization: A comparative study. J Ultrasound Med (1988); 7:726–8. 3 Lenz S, Leeton J, Renou P. Transvaginal recovery of oocytes or in vitro fertilization using vaginal ultrasound. J In Vitro Fert Embryo Transfer (1987); 4:51–5. 4 Serour GI, Aboulghar M, Mansour R, Sattar MA, Amin Y, Aboulghar H. Complications of medically assisted conception in 3500 cycles. Fertil Steril (1998); 70:638–42. 5 Thompson JD, Rock WA. Control of pelvic hemorrhage. In: Rock JA, Thompson JD, eds. Te Linde’s Operative Gynecology. 8th edition. Lippincott-Raven: Philadelphia, PA (1997); 197–232. 6 Howe RS, Wheeler C, Mastroianni L Jr, Blasco L, Tureck R. Pelvic infection after transvaginal ultrasound-guided ovum retrieval. Fertil Steril (1988); 49:726–8. 7 Curtis P, Amso N, Keith E, Bernard A, Shaw RW. Evaluation of the risk of pelvic infection following transvaginal oocyte recovery. Hum Reprod (1992); 7:625–6. 8 Ashkenazi J, Farhi J, Dicker D, Feldberg D, Shalev J, Ben-Rafael Z. Acute pelvic inflammatory disease after oocyte retrieval: adverse effects on the results of implantation. Fertil Steril (1994); 61:526–8. 9 Yaron Y, Peyser MR, Samuel D, Amit A, Lessing JB. Infected endometriotic cysts secondary to oocyte aspiration for in vitro fertilization. Hum Reprod (1994); 9:1759–60. 10 Nargund G, Parsons J. Infected endometriotic cysts secondary to oocyte aspiration for in vitro fertilization. Hum Reprod (1995); 10:1555. 11 Sauer MV, Paulson RJ. Pelvic abscess complicating transcervical embryo transfer. Am J Obstet Gynecol (1992); 166:148–9. 12 Friedler S, Ben-Shachar I, Abramov Y, Schenker JG, Lewin A. Ruptured tubo-ovarian abscess complicating transcervical cryopreserved embryo transfer. Fertil Steril (1996); 65:1065–6. 13 Ng SC, Edirisinghe WR, Sathanathan AH, Ratnam SS. Bacterial infection of human oocytes during in vitro fertilization. Int J Fertil (1987); 32:298–301. 14 Giri SN, Emau P, Cullor JS, et al. Effect of endotoxin on circulating levels of eicosanoids, progesterone, cortisol, glucose and lactic acid, and abortion in pregnant cows. Vet Microbiol (1990); 21:211–31. 15 Ben-Rafael Z, Orvieto R. Cytokine involvement in reproduction. Fertil Steril (1992); 58:1093–9. 16 Tartakovski B, Ben-Yair E. Cytokines modulate preimplantation development and pregnancy. Develop Biol (1991); 146:345–52. 17 Saper CB, Breder CD. Endogenous pyrogens in the CNS: Role in the febrile response. Prog Brain Res (1992); 93:419–28.

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18 Hanson DF. Fever and the immune response. The effects of physiological temperatures on primary murine splenic T-cell responses in vitro. J Immunol (1993); 151:436–48. 19 Kitano Y, Okada N. Organization and disorganization of actin filament in human epidermal keratinocyte: Heat, shock treatment and recovery process. Cell Tissue Res (1990); 261:269–74. 20 Milunski A, Ulcickas M, Rothman KJ, Willett E, Jick SS, Jick H. Maternal heat exposure and neural tube defects. JAMA (1992); 268:882–5. 21 Edwards MJ, Mulley R, Ring S, Wanner RA. Mitotic cell death and delay of mitotic activity in guinea-pig embryos following brief maternal hyperthermia. J Embryol Exp Morphol (1974); 32:593–602. 22 Hendricks AG, Stone GW, Hendrickson RV, Matayoshi K. Teratogenic effects of hyperthermia in the bonnet monkey (Macaca radiata). Teratology (1979); 19:177–82. 23 Russell JB, DeCherney AH, Hobbins JC. A new transvaginal probe and biopsy guide for oocyte retrieval. Fertil Steril (1987); 47:350–2. 24 Meldrum DR. Antibiotics for vaginal oocyte aspiration. J In Vitro Fert Embryo Trans (1989); 6:1–2. 25 Evers JLH, Larsen JF, Gnany GG, Sieck UV. Complications and problems in transvaginal sector scan-guided follicle aspiration. Fertil Steril (1988); 49:278–82. 26 Borlum KG, Maiggard S. Transvaginal oocyte aspiration and pelvic infection. Lancet (1989); 2:53 (letter). 27 Van Os HC, Roozenburg BJ, Janssen-Caspers HAB, et al. Vaginal disinfection with Povidone iodine and the outcome of in vitro fertilization. Hum Reprod (1992); 7:349–50. 28 Peters AJ, Hecht B, Durinzi K, DeLeon F, Colston Wentz A. Salpingitis or oophoritis: What causes fever following oocyte aspiration and embryo transfer? Obstet Gynecol (1993); 81:876–7. 29 Wren M, Parson J. Ultrasound directed follicle aspiration in IVF. In: Chen C, Tan SL, Cheng WC, eds. Recent Advances in the Management of Infertility. McGraw-Hill, New York (1989); 105–81. 30 Ashkenazi J, Ben-David M, Feldberg D, Dicker D, Goldman JA. Abdominal complications following ultrasonically guided percutaneous transvesical collection of oocytes for in vitro fertilization. J In Vitro Fert Embryo Trans (1987); 4:316–8. 31 Russell JB, Decherney AH, Hobbins JC. A new transvaginal probe and biopsy guide for oocyte retrieval. Fertil Steril (1987); 47:350–2. 32 Caspi B, Zalel Y, Or Y, et al. Sonographically guided aspiration: an alternative therapy for tubo-ovarian abscess. Ultrasound Obstet Gynecol (1996); 7:439–42. 33 Centers for Disease Control and Prevention. Ectopic pregnancy— United States, 1990–1992. MMWR (1995); 1:46.

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34 Berg CJ, Atrash HR, Koonin LM, Tucker M. Pregnancy-related mortality in the United States, 1987–1990. Obstet Gynecol (1996); 88:161. 35 Steptoe P, Edwards R. Reimplantation of a human embryo with subsequent tubal pregnancy. Lancet (1976); 1:830–2. 36 Azem F, Yaron Y, Botchan A, et al. Ectopic pregnancy after in vitro fertilization-embryo transfer (IVF/ET): the possible role of the ET technique. J Assist Reprod Genet (1993); 10:302–4. 37 Zouves C, Erenus M, Gomel V. Tubal ectopic pregnancy after in vitro fertilization and embryo transfer: a role for proximal occlusion or salpingectomy after failed distal tubal surgery. Fertil Steril (1991); 56:691–5. 38 SART, ASRM. Assisted reproductive technology in the United States: 1996 results generated from the American Society for Reproductive Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril (1999); 71:798–807. 39 Martinez F, Trounson A. An analysis of factors associated with ectopic pregnancy in a human in vitro fertilization program. Fertil Steril (1986); 45:79–87. 40 Karande VC, Flood JT, Heard N, Veeck L, Muasher SJ. Analysis of ectopic pregnancies resulting from in vitro fertilization and embryo transfer. Human Reprod (1991); 6:446–9. 41 Verhulst G, Camus M, Bollen N, Van Steirteghem A, Devroy P. Analysis of the risk factors with regard to the occurrence of ectopic pregnancy after medically assisted procreation . Hum Reprod (1993); 8:1284–7. 42 Herman A, Ron-El R, Golan A, Weinraub Z, Bukovsky I, Caspi E. The role of tubal pathology and other parameters in ectopic pregnancies occurring in in vitro fertilization and embryo transfer. Fertil Steril (1990); 54:864–8. 43 Correy JF, Watkins RA, Bradfield GF, Garner S, Watson S, Gray G. Spontaneous pregnancies and pregnancies as a result of treatment on an in vitro fertilization program terminating in ectopic pregnancies or spontaneous abortions. Fertil Steril (1988); 50:85–8. 44 Pygriotis E, Sultan KM, Neal GS, Liu HC, Grifo JA, Rozenwaks Z. Ectopic pregnancies after in vitro fertilization and embryo transfer. J Assist Reprod Genet (1994); 11:80–3. 45 Cohen J, Mayaux MJ, Guihard-Moscato ML. Pregnancy outcomes after in vitro fertilization: A collaborative study on 2342 pregnancies. Ann NY Acad Sci (1988); 54:1–6. 46 Marcus SF, Brinsden PR. Analysis of the incidence and risk factors associated with ectopic pregnancy following in vitro fertilization and embryo transfer. Hum Reprod (1995); 10:199–203. 47 Ankum WM, Mol BW, Van der Veen, et al. Risk factors for ectopic pregnancy: a meta-analysis. Fertil Steril (1996); 66:513–6.

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48 Lesny P, Killick SR, Robinson J, Maguiness SD. Transcervical embryo transfer as a risk factor for ectopic pregnancy. Fertil Steril (1999); 72:305–9. 49 Russell JB. The etiology of ectopic pregnancy. Clin Obstet Gynecol (1987); 30:181–90. 50 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. 51 Knutzen UK, Sotto-Albors CE, Fuller D, Sher G, Shyrock K, Behr B. Mock embryo transfer (MET) in early luteal phase, the cycle prior to in vitro fertilization and embryo transfer (IVF/ET). Presented at the 45th Annual Meeting of the American Fertility Society, San Francisco, CA, Nov 13 to 16 1989. American Fertility Society, Program Supplement, pS152:299. 52 Lesny P, Killick SR, Tetlow RL, Robinson J, Maguiness SD. Embryo transfer—can we learn anything new from the observation of junctional zone contraction? Hum Reprod (1998); 13:1540–6. 53 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. 54 Svare J, Norup P, Grove Thomsen S, et al. Heterotopic pregnancies after in vitro fertilization and embryo transfer—A Danish survey. Hum Reprod (1993); 8:116–8. 55 Tucker M, Smith D, Pike I, Kemp J, Picker R, Saunders D. Ectopic pregnancy following in vitro fertilization and embryo transfer . Lancet (1981); 2:1278. 56 Ben-Rafael Z, Mashiach S, Dor J, Rudak E, Goldman B. Treatmentindependent pregnancy after in vitro fertilization and embryo transfer trial. Fertil Steril (1986); 45(4):564–7. 57 Fernandez H, Coste J, Job-Spira N. Controlled ovarian hyperstimulation as a risk factor for ectopic pregnancy. Obstet Gynecol (1991); 78:656–9. 58 Ben-Rafael Z, Carp HJ, Mashiach S, Blankstein J, Serr DM. The clinical features and incidence of concurrent intra and extra uterine pregnancies. Acta Eur Fertil (1985); 16(3):199–202. 59 Dimitry ES, Subak-Sharpe R, Mills M, Margara R, Winston R. Nine cases of heterotopic pregnancies in 4 years of in vitro fertilization. Fertil Steril (1990); 53:107–10. 60 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. 61 Goldman GA, Fisch B, Ovadia J, Tadpir Y. Heterotopic pregnancy after assisted reproductive technologies. Obstet Gynecol Surv (1992); 47:217–21. 62 Molloy D, Deambrosis W, Keeping D, Hynes J, Harrison K, Hennessey J. Multiple-sited (heterotopic) pregnancy after in vitro

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fertilization and gamete intrafallopian transfer. Fertil Steril (1990); 53:1068–71. 63 Li HP, Balmaceda JP, Zouves C et al. Heterotopic pregnancy associated with gamete intra-fallopian transfer. Hum Reprod (1992); 7:131–5. 64 Tummon IS, Whitmore NA, Daniel SAJ, Nisker JA, Tuzpe AA. Transferring more embryos increases risk of heterotopic pregnancy. Fertil Steril (1964); 61:1065–7. 65 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. 66 Rojanski N, Schenker JG. Heterotopic pregnancy and assisted reproduction—an update. J Assist Reprod Genet (1996) 13:594–601. 67 Rock JA, Damario MA. Ectopic pregnancy. In: Rock JA, Thompson JD, eds. Te Linde’s Operative Gynecology. 8th edition. LippincottRaven: Philadelphia, PA (1997); 501–27. 68 Yao M, Tulandi T. Current status of surgical and nonsurgical management of ectopic pregnancy. Fertil Steril (1997); 67:421–33.

55 Iatrogenic multiple pregnancy: the risk of ART Isaac Blickstein

INTRODUCTION The common denominator of most assisted reproduction techniques (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 physician-made—iatrogenic, multiple pregnancies (IMPs). There are two exceptions to this statement. First, single embryo transfer may still be associated with an increased risk of monozygotic (MZ) twins since ART augments the rate of zygotic splitting.1 Second, recent observations from the East Flanders Prospective Twin Survey may suggest that a genetic familial trait for spontaneous twins may be also involved in induced conceptions. Hence, women with “twins in the family” undergoing infertility treatment may be at increased risk of having multiples compared with women without that characteristic (Derom R, unpublished). 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 after transfer of three embryos.2 Roughly, these reference figures represent a 20 and 50 times increased frequency for iatrogenic twins and triplets, respectively, compared with naturally occurring multiples. Table 55.1 shows a simple model of an obstetrical service with 4000 live births/year, including 5% after iatrogenic pregnancies.3 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

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fecundity and increased need for fertility treatment. Thus, social trends act in concert with available ART to increase the risk of multiple pregnancy.

Table 55.1 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). Adapted from Blickstein.3 Singles Twins Triplets Births Neonates 100% Spontaneous 3948 48 4 4000 4056 5% Iatrogenic 140 50 10 200 270 95% Spontaneous 3750 46 4 3800 3854 Total 3890 96 14 4000 4124

Fig 55.1 Ratio of spontaneous to induced twins. Since the implementation of effective infertility treatment the ratio changed from one induced for every 40–50 spontaneous twin pregnancies, to one induced for every two to three spontaneous twin pregnancies. Adapted from the East Flanders Prospective Twin Survey.4 Fig 55.1 shows the ratio of spontaneous to induced twins in the 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

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one in every 2–3 twins.4 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.5 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.6,7 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.8 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 one of every 6–7 twin pregnancies following ART.9,10 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.11,12 Multiples are associated with higher frequencies of malformations of varied etiology. The yet unknown factor(s) that cause zygotic splitting has been implicated in causing structural malformations in MZs. In the subset with monochorionic (MC) placentas, also encountered in HOMPs, twintwin transfusion syndrome (TTTS) may affect as many as 5–10% of the pairs and result in major morbidity of one or both twins. Later in pregnancy, single fetal demise associated with MC placentas may result in severe end organ damage in the survivor. Finally, it has been shown that the risk of cerebral palsy (CP) is five- to six- and 23-fold increased in twins and triplets compared with singletons.13 A model based on British data related to transfer of two and three embryos2 and on British data related to CP in multiples14 suggested a significantly lower estimated CP rate (2.7/1000 neonates) after spontaneous pregnancies compared with transfer of three embryos (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)15 (Fig 55.2).

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Fig 55.2 Estimated risk of cerebral palsy per 1000 neonates following transfer of three and two embryos, and following MFPR of all triplets to twins. A threeto sixfold increased risk of cerebral palsy is expected. Adapted from Blickstein and Weissman.4 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 a major area of clinical importance, primarily because of the confirmed increased morbidity and mortality associated with MZ twinning. Firstly, 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. To overcome this problem we evaluated single embryo transfers following IVF with and without ICSI (Fig 55.3).1 The data indicated that splitting is expected in 4.9% after IVF without ICSI, 12-times higher than the 0.4% rate of MZs in spontaneous conceptions. 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 enfin successful, may have to consider reduction (=termination) of their “surplus” fetuses (=success). At the same time,

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there is little doubt that MFPR may be the only solution for a potentially successful outcome of a HOMP. MFPR, discussed elsewhere in this volume, is indeed associated with improved perinatal outcome, as expected from comparing HOMPs with twins or singletons. However, given the fact 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.16 Table 55.2 shows the minimal death rates associated with various MFPR procedures

Fig 55.3 Zygotic splitting. Frequency of twins following single embryo transfer in IVF cycles. The accepted 0.4% of spontaneous zygotic splitting was used as reference to show the 12-fold increased incidence of zygotic splitting in this series. Adapted from Blickstein et al.

Table 55.2. Minimal mortality rates in various MFPR combinations. MFPR 4→2 5→2 3→2 4→1 Minimal mortality 50% 60% 33% 75%

3→1 66%

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 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

2→1 50%

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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.17 Given the facts the 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 measurements for screening18 or on invasive cytogenetic procedures (amniocentesis or chorionic villus sampling (CVS)). Regrettably, the former has not been studied 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. However, there are two additional partners to the triangle. IMP following ART is usually achieved after longstanding 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. 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 treatment changes quite often to severe psychological morbidity. From the outset, couples are faced with dilemmas that they never faced before. For instance, couples initiating therapy were given questionnaires to determine attitudes regarding multiple pregnancy and MFPR.19 The results suggested declining ratings as the number of fetuses increased. IUI patients felt more favorable than the IVF group toward all gestational outcomes and less favorable toward MFPR. 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.20 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.21 The French group that followed couples during pregnancy and for 4 years postpartum provided some important clues to understanding this complex situation.22,23 Their first paper studied the effects of MFPR on the mothers’ emotional

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wellbeing 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 one 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 relationship 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 relationship 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.24 Twenty-one couples with complete sets of triplets aged 4–6 years 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 bed rest, 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 OHSS is considered before ART. Since maternal morbidity is undoubtedly increased in multiple gestations, it has been proposed that maternal mortality is also increased.25 However, since a multiple pregnancy is not registered as the direct cause of death, the risk is unknown. For example, eclampsia, tocolysis, and delivery related deaths were more common in twins.25 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.26 It is believed that IMP are not spared these risks. The epidemic of iatrogenic HOMPs enabled some insight into the increased maternal morbidity in these cases. The most significant morbidity found in triplets were pregnancy induced hypertension (27–

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33%), HELLP syndrome (9–10.5%), anemia (27–58.1%) and postpartum hemorrhage (9–12.3%).27,28 Since maternal morbidity clearly increases with plurality, it is expected that maternal morbidity will decrease following MFPR. Skupski et al29 found that severe pre-eclampsia was more common among IVF triplet pregnancies (26.3%) than among IVF triplets reduced to twins (7.9%). The prevalence of all pre-eclampsia cases also was 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 pre-eclampsia than is successful implantation alone. Maternal morbidity should also be considered in the context of maternal age. ART enabled pregnancies 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 Centers for Disease Control (NCHS/CDC Press release, 14 September, 1999) suggest that (1) between 1980–82 and 1995–97 the twin birth rate rose by 63% for women between the ages 40 and 44 and by nearly 1000% for women 45–49 years of age; (2) HOMP birth rate rose by nearly 400% for women in their 30s and by more than 1000% for women in their 40s. In 1997 more twins were born to women aged 45–49 than during the whole decade of the 1980s. 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, 7 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, pre-eclampsia, HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, and fetal growth retardation. Caesarean section was done in 64.8%.30 The age related risk for trisomy, depending on the source of the female gametes, is of primary importance when ART is performed in elderly women. 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 after 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 may be scheduled during the 30th-32nd week, with the possibility of fetocide at 33–35 weeks. This logical scheme eliminates the risk of losing

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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. Second, 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 pre-implantation diagnosis will 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 non-siblings who shared no common genes and, of course, shared nothing with the surrogate mother.31 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 be 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 a iatrogenic contra-evolutionary 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 is a direct relation between the number of transferred embryos and success rate of ART on 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 embryos are transferred and MFPR is not used (Fig 55.4). In the Reggio Emilia (Italy) center for reproductive medicine, 34.6% of the clinical pregnancies were multiples—20% twins and 14.6% HOMPs. Importantly, the number of gestational sacs diminished during pregnancy and the rate of sacs that spontaneously disintegrated was similar in singleton, twins, and triplets (26.2–28.7%) but significantly increased in quadruplets and quintuplets. Thus, expecting spontaneous reduction is not a practical solution for iatrogenic triplets and twins. Ethical, legal, religious, and technical (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) versus

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failure (IMP) rates using selected embryos. Genetical and biochemical markers would supplement morphological criteria as normal-appearing embryos may be genetically abnormal, and only a few have the glycolytic activity needed for implantation.33 The pre-implantation genetic studies will also replace invasive

Fig 55.4 Spontaneous loss in IMPs when all available embryos are transferred and MFPR is not used. Gray bars: % of clinical pregnancies at 35 days posttransfer, black bars: % of disintegrated gestational sacs. Data adapted from La Sala et al.32 procedures during pregnancy. For the time being, the first step has already been done. Recently, the British Human Fertilisation and Embryology Authority (HFEA) has stated that although the code of practice limits the number of embryos that may be transferred in a single cycle to three, HFEA welcomes the recommendation of the British Fertility Society that two embryo transfer should be the usual practice. This statement (Press release on 15 December 1998, www.hfea.gov.uk/) was based on a HFEA large, albeit retrospective, data base.2 Although some biases may have been introduced to the British study,33 it has been definitely shown that when there are four or more embryos available to a patient, the chances of live birth is no greater for three than for two transferred embryos, but the chance of a IMP is much increased with the former.2 The HFEA recommendations have been based on embryo transfer without specifying their quality and their implantation potential. At the meantime, it has become possible to culture embryos to the blastocyst stage, selecting the fittest embryos for transfer and synchronizing the

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embryonic with the endometrial stages. Blastocyst transfer has been associated with a much improved 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 days embryos and then, when blastocyst 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. In the first series of successful pregnancies following mixed embryo blastocyst transfers at the Kaplan Medical Center, we noticed some bizarre complex chorionicity arrangements, which have never been seen with usual IVF-ET protocols (Fig 55.5). It is therefore reasonable to conclude that demands from infertile couples and fertility clinics to maximize success rates conflict the need to reduce the number of IMPs. THE PREGNANCY PHASE Once pregnant, the woman is not infertile any more and there should be no difference in the management of spontaneous compared with iatrogenic pregnancies. However, the past reproductive history continues to follow the patient albeit her pregnancy may be absolutely normal. When a 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.

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Fig 55.5 Complex chorionicity. Sonographic image showing a 7 week quadruplet pregnancy after sequential transfer of two embryos and one blastocyst. This bichorionic quadruplet pregnancy comprises of monochorionic triamniotic triplets (upper sac) and a singleton (lower sac). Image courtesy of B Caspi, MD. 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. Then, the unprepared couples may consider MFPR or risky interventions as hostile suggestions. It follows that the dissociation between the reproductive and the MFM physicians 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 pre-eclamptic patient. This complicated pregnancy follow up is therefore never a one man show, and well orchestrated teamwork is encouraged. Indeed, it has been shown that special multiple pregnancy clinics do have better results.34 The extremely varied spectrum of IMPs is superimposed on the special doctor-patient relationship. It is beyond the scope of this chapter to

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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 they are expected to run different age related obstetrical risks. Likewise, 20 and 40 year old women may need similar ICSI techniques for severe oligospermia but differ in respect to anticipated age related pregnancy complications. The obligations for the fetus as a patient in multiple pregnancy are quite complicated.35 In addition to the relationship among physician, mother, and fetus, 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 BC triplet pregnancy (MC twins plus a singleton). Obviously, a three to two reduction will end with a 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, 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) in a premature delivery of multiples.25 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 of the utero-placental 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. Second, 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

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caesarean 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 1500g each, either route of delivery seems to be appropriate, irrespective of fetal presentation.36 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 were much faster than the preparation of sufficient cribs in the neonatal intensive care unit (NICU). As a result, over-production of preterm neonates overwhelms the capacity of many NICUs, leading to medical problems associated with overcrowded stations. A recent Canadian study compared the preterm birth rates in two 3 year periods, 1981–83 and 1992–94.37 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 attributed to 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 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 the average, 7 weeks shorter. For 1995, 92% of triplets 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, each of the multiples must be given equal opportunity to bond with his parents and, perhaps, according to psychological view, to continue its intra-uterine contacts with its siblings. For example, there is increasing evidence that co-bedding of twins in the NICU improves thermoregulation, feeding, and sleeping parameters.38 Indeed, the special

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and unique interaction between multiples during childhood and beyond, seems to reflect the unique relationship that exists between fetuses that grow together in utero. Fig 55.6 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 in multiple pregnancy.39 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, that professional help is required during infancy and childhood to the same extent that it has 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 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 affluent society.

EPILOGUE: RE-DEFINING SUCCESS Every day there are numerous healthy multiples delivered after 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.

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Fig 55.6 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.39 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 for 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 sixfold increased risk for a lifelong handicap such as cerebral palsy15 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 obstetrical, neonatal, and lifelong complications associated with IMPs. Second, 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. The apocalyptic views expressed in this chapter will remain pertinent as long as demands for better pregnancy rates by couples undergoing ART

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will be accepted by overzealous reproduction centers without a clear definition of what should be considered successful.

REFERENCES 1 Blickstein I, Verhoeven HC, Keith LG. Zygotic splitting after assisted reproduction. N Engl J Med (1999); 340:738–9. 2 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. 3 Blickstein I. Perinatal implications of iatrogenic multiple pregnancies. In: Voto LS, Margulies M, Cosmi EV, eds. 4th World Congress of Perinatal Medicine. Monduzzi Editore: Bologna, Italy, 1999; 167–72. 4 Leroy F. Les jumeaux dans tous leurs états. De Boeck-Wesmael: Bruxelles, Belgium, 1995; 87. 5 Blickstein I, Keith LG, eds. Iatrogenic multiple pregnancy: Clinical implications. Parthenon Publishing: Lancs, 2000; in press. 6 Blickstein I, Smith-Levitin M. Twinning and twins. In: Chervenak FA, Kurjak A, eds. Current perspectives on the fetus as a patient. Parthenon Publishing: Lancs, 1996; 507–25. 7 Blickstein I, Smith-Levitin M. Multifetal pregnancy. In: Petrikovsky BM, ed. Fetal disorders: diagnosis and management. John Wiley: New York, 1998; 223–47. 8 Blickstein I, Goldman RD, Smith-Levitin-M, Greenberg M, Sherman D, Rydhstroem H. The relation between inter-twin birth weight discordance and total twin birth weight. Obstet Gynecol (1999); 93:113–16. 9 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. 10 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. 11 Pharoah PO, Cooke RW. A hypothesis for the aetiology of spastic cerebral palsy—the vanishing twin. Dev Med Child Neurol (1997); 39:292–6. 12 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. 13 Blickstein I. Cerebral palsy in multifetal pregnancies: Facts and hypotheses. In: Chervenak FA, Kurjak A, eds. Fetal medicine: The clinical care of the fetus as a patient. Parthenon Publishing: Lancs, 1999; 368–73. 14 Pharoah PO, Cooke T. Cerebral palsy and multiple births. Arch Dis Child Fetal Neonatal Ed (1996); 75:F174-F177.

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15 Blickstein I, Weissman A. Estimating the risk of cerebral palsy after assisted reproduction. N Engl J Med (1999); 341:1313–4. 16 Blickstein I. Should the reduced embryos be considered in outcome calculations of multifetal pregnancy reduction? Am J Obstet Gynecol (1994); 171:866–7. 17 Meyers C, Adam R, Dungan J, Prenger V. Aneuploidy in twin gestations: when is maternal age advanced? Obstet Gynecol (1997); 89:248–51. 18 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. 19 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. 20 Laruelle C, Englert Y. Psychological study of in vitro fertilizationembryo transfer participants’ attitudes toward the destiny of their supernumerary embryos. Fertil Steril (1995); 63:1047–50. 21 Berkowitz RL, Lynch L, Stone J, Alvarez M. The current status of multifetal pregnancy reduction. Am J Obstet Gynecol (1996); 174:1265–72. 22 Garel M, Stark C, Blondel B, Lefebvre G, Vauthier-Brouzes D, Zorn JR. Psychological reactions after multifetal pregnancy reduction: a 2year follow-up study. Hum Reprod (1997); 12:617–22. 23 Garel M, Salobir C, Blondel B. Psychological consequences of having triplets: a 4-year follow-up study. Fertil Steril (1997); 67:1162–5. 24 Akerman BA, Hovmoller M, Thomassen PA. The challenges of expecting, delivering and rearing triplets. Acta Genet Med Gemellol Roma (1997); 46:81–6. 25 Blickstein I. Maternal mortality in twin gestations. J Reprod Med (1997); 42:680–4. 26 Conde-Agudelo A, Belizan J. Maternal mortality and morbidity associated with multiple pregnancy. Twin Research (1999); 2:S3. 27 Malone FD, Kaufman GE, Chelmow D, Athanassiou A, Nores JA, D’Alton ME. Maternal morbidity associated with triplet pregnancy. Am J Perinatol (1998); 15:73–7. 28 Albrecht JL, Tomich PG. The maternal and neonatal outcome of triplet gestations. Am J Obstet Gynecol (1996); 174:1551–6. 29 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. 30 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. 31 Simini B. Italian surrogate twins. Lancet (1997); 350:1307.

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32 La Sala GB, Montanari R, Cantarelli M, Valli B, Taricco F, Torelli MG. Iatrogenic multifetal pregnancies and SPIER. Twin Research (1999); 2:S6. 33 Meldrum DR, Gardner DK. Two-embryo transfer—The future looks bright. N Engl J Med (1998); 339:624. 34 Hartikainen-Sorri AL. Is routine hospitalization in twin pregnancy necessary? A follow-up study. Acta Genet Med Gemellol Roma (1985); 34:189–92. 35 Chervenak FA, McCullough LB. Ethics in Obstetrics and Gynecology. Oxford University Press: New York, 1994. 36 Blickstein I, Goldman RD, Kuperminc M. Delivery of breech-first twins: a multicenter retrospective study. Obstet Gynecol (2000); 95:37–43. 37 Joseph KS, Kramer MS, Marcoux S, Ohlsson A, Wen SW, Allen A, Platt R. Determinants of preterm birth rate in Canada from 1981 through 1983 and from 1992 through 1994. N Engl J Med (1998); 339:1434–9. 38 Mazela JL, Gadzinowski J. Co-bedding twins and multiples—is there strong clinical evidence? Twin Research (1999); 2:S17. 39 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.

56 Reducing the incidence of multiple gestation David R Meldrum

MULTIPLE PREGNANCY AS AN INDICATOR OF PROGRAM QUALITY The issue of multiple pregnancy is a predictable consequence of improvements in embryo laboratory quality (and hence embryo quality) and embryo transfer technique. If the implantation rate (IR) per embryo increases from 10% to 20%, the calculated expectation of twins with two embryos transferred would increase from 1% to 4%. Likewise, if 100% of the transfer medium is retained in the desired location the chance of multiple implantation would be higher than if 50% is expelled. Clearly, higher-quality programs will have more multiples, resulting in a greater need to restrict embryo number. Conversely, programs with less refined laboratory conditions and/or transfer techniques have fewer multiples and will have a marked reduction in their success rates if they restrict embryo number for transfer. Following this logic, adherence of all in vitro fertilization (IVF) programs to restrictive criteria for the number of embryos transferred can be expected to widen the variation in success rates among programs. It will allow some programs to more clearly identify suboptimal techniques since a low pregnancy rate demands more attention than a low implantation rate.

DOES INCREASING THE NUMBER OF EMBRYOS INCREASE THE PREGNANCY RATE? There has been a running controversy whether there is a continuing increase in successful pregnancy with an increase of embryo number beyond two or three. For example, Templeton and Morris reported on over 44000 cycles analyzed by the Human Fertilization and Embryology Authority in the United Kingdom.1 As expected, the risk of multiple births was lower with two compared with three embryo transfer. However, they also concluded that when more than four eggs were fertilized there was no higher pregnancy rate with three versus two embryos. We must assume that those programs with lower results would tend to transfer three embryos and that three rather than two embryos would be transferred

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when embryo quality was reduced. Without a prospective randomized trial, such data are fatally flawed. In the 1997 US Registry National Summary and Fertility Clinic Reports, the Centers for Disease Control and Prevention, Atlanta, Georgia found that the live birth rates were 9.1%, 20.3% and 35.8% with transfer of one, two, and three embryos in women younger than age 35.2 There was no further increase of the pregnancy rate with four or more embryos transferred, but again these data are flawed by the same biases discussed above. This is clearly shown by the lack of change of the multiple pregnancy rate above three. If the higher order embryo transfers were receiving additional good quality embryos, the multiple pregnancy rate would continue to steadily rise. It is a mathematical certainty that transfer of further good quality embryos will increase both the chance of pregnancy and multiple pregnancy.3 This is particularly true in women over age 40 for whom the transfer of four or more embryos has been associated with a higher pregnancy rate with both in vitro fertilization (IVF)4 and gamete intrafallopian transfer (GIFT).5 Of course, a compounding factor is that older women who produce more embryos likely have a better prognosis.

IDENTIFYING RISK FACTORS FOR MULTIPLE PREGNANCY The critical issue is how we can reduce the risk of multiples without compromising the pregnancy rate to any great degree. To do this we must identify those patients with a very good prognosis who are therefore at highest risk, for whom the transfer should be limited to two embryos. The two primary indicators of embryo quality are the patient’s history and characteristics and the quality of the embryos. The following patient factors have been statistically associated with pregnancy outcome. AGE Age is the single most important variable affecting both pregnancy outcome and the risk of multiple pregnancy. In spite of more embryos being transferred with increasing age, the delivery rate in the 1996 US Registry decreased from 32 to 25 to 12% in the <35, 35–39, and >39 year age groups.6 In the UK data, the multiple birth rate dropped from 39 to 33 to 27% at ages 30, 35 and 40 for women having at least five eggs fertilized and three embryos transferred.1 OVARIAN RESPONSE Women with a higher ovarian response and more fertilized eggs have a higher chance of pregnancy and multiple pregnancy. In the United

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Kingdom, the birth rate for a 35 year old having two embryos transferred increased from 9 to 17% with an increase from two to five or more fertilized eggs.1 Chenette et al reported that patients with high response had a significantly higher pregnancy rate than lower responders.7 Schoolcraft et al found that almost 90% of high order multiple pregnancy occurred in women with 10 or more follicles on the day of human chorionic gonadotropin (hCG).8 A high ovarian response may simply reflect a physiologically younger ovary and would correlate with low follicle stimulating hormone (FSH) levels. It may also allow more selection of the best embryos for transfer. The latter factor depends on timing of cryopreservation and transfer. If all embryos are allowed to develop until the day of transfer, the pregnancy rate and risk of multiples will be higher than if some are cryopreserved at the 2PN stage and therefore removed from the selection process since it is difficult to identify the best embryos at that stage. DAY 3 LEVELS OF FSH, ESTRADIOL The day 3 serum concentration of FSH is a better predictor of pregnancy outcome than age.9 One would presume, although this has not been examined, that the risk of multiple pregnancy would likewise be higher in women with low FSH levels. It is likely that FSH level or FSH dose would add some additional predictive value to the ovarian response. Increased day 3 FSH probably predicts a lower risk of multiple pregnancy since it correlates with reduced ovarian response. TREATMENT HISTORY/CAUSE OF INFERTILITY A prior success with IVF increases the chance of pregnancy but not multiple pregnancy, for a subsequent IVF cycle.1 This suggests that the increased outcome is more related to endometrial receptivity than to embryo quality. With each year of infertility there is a 2% reduction of both delivery and multiple birth.1 The rates of pregnancy and multiple pregnancy do not change over the first three IVF cycles but decrease by 40% for four or more prior failed attempts.1 Women with multiple failed IVF cycles (average of 4.8 cycles) have been found to have an improved prognosis with transfer of more embryos.10 Tubal infertility is associated with a 20–30% reduction of both pregnancy and multiple pregnancy.1 It is not clear whether the latter finding would be influenced by removing hydrosalpinges before IVF.11 EMBRYO QUALITY Morphologic quality of the embryos is the factor most predictive of successful implantation and the risk of multiple pregnancy12–15 next to age and ovarian response. As the time of transfer has progressed from day 2 to

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day 3 and recently to day 5, embryo quality becomes more predictive since embryos destined to be non-viable often progress more slowly and may have irregular morphology. Scott et al13 and Erenus et al14 reported that day 2 embryos, which had even blastomeres and no or minimal fragmentation, predicted higher pregnancy rates. Cohen et al studied embryo fragmentation in more detail, finding that <10% fragmentation had no apparent impact on implantation.12 They did find that increased variation of thickness of the zona pellucida was the most predictive criterion for implantation. Steer et al15 devised an embryo score by multiplying the morphologic score (grade 1 to 4) times the number of blastomeres (day 2 transfer). They found that the pregnancy rate continued to rise until a total score of 42. Above 42 there was only an increase of multiple pregnancy. However, all patients were under age 36. Older women have embryo morphology which is unchanged, but a high proportion of their embryos are genetically abnormal.16 Steer’s findings correlate well with those of others17 suggesting that only two or three good quality embryos should be replaced in women under age 35 (two 4 cell grade 4 embryos on day two=a score of 32). They should not be accepted as valid for women older than the population studied. Hu et al, using a similar scoring system, concluded that it would be necessary to limit transfer to two good quality embryos in women age 39 or younger to eliminate any risk of high-order multiple pregnancy, but that five embryos could be transferred in women over 40 regardless of morphologic quality.18 Van Royen et al19 found that the embryo criteria predicting implantation were absence of multinucleated blastomeres, four or more blastomeres on day 2 or 7 or more cells on day 3, and ≤20% anucleate fragments. A total of 106 transfers with two top quality embryos resulted in a 63% ongoing pregnancy rate and 57% were twins. With only one top grade embryo 58% were ongoing with 21% twins. With no top quality embryos 23% were ongoing with no twins. They concluded that single embryo transfer could be considered if a top quality embryo is available. Morphologic quality of blastocysts may be even more predictive of implantation. Using a scoring system to grade blastocyst expansion and trophoblast and inner cell mass quality, Schoolcraft et al8 found that transfer of a high quality blastocyst predicted a pregnancy rate of 85%. As experience accumulates with this technique, it may be possible to limit transfer to a single embryo in some women without affecting their chance of pregnancy.

WHAT IS THE OPTIMUM EMBRYO NUMBER? If we accept that it is a mathematical certainty that the pregnancy rate will increase with the number transferred, the question becomes: “What is a reasonable compromise between the chance of pregnancy and the risk of multiple pregnancy?” It also should be kept in mind that the rate of

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miscarriage is lower with three or four embryos compared with one or two20 since each implantation has a chance of miscarrying. When multiple sacs are detected, the “take home baby” rate improves.21 The best pregnancy rate with the lowest number of embryos transferred will be achieved by culturing all embryos to day 3 or 5 to maximize selection. At that point, the transfer of good quality day 3 embryos, on the basis of morphology and cleavage stage, should be limited according to age and ovarian response. The Society for Assisted Reproductive Technology has recently circulated suggested guidelines indicating that women under age 35 with additional embryos cryopreserved (indicating a good ovarian response), should have no more than two good quality embryos transferred (American Society for Reproductive Medicine, unpublished Practice Committee Report, November, 1999). In women age 35–39 they suggested no more than four, and in women over age 39 five good quality embryos. We have modified these criteria to limit transfer to three good quality embryos in women age 35–37 with a good ovarian response, since their success rate is better than that of women approaching age 40. This is supported by a study of such patients with only a slightly lower pregnancy rate with two fresh embryos and an equal cumulative fresh plus frozen rate to those women having three fresh embryos transferred.22 These guidelines should reduce the incidence of high order multiple pregnancy to an acceptably low level. In good responders, blastocyst transfer should be limited to two embryos that have achieved that stage8,23,24 except for some women over age 40 three may be acceptable. It is clear that in women under age 40, transfer of three good quality blastocysts is associated with an unacceptably high risk of triplets. Others have considered that the risk of twins and triplets must be reduced to an absolute minimum through the transfer of only one or two embryos.22,25 Staesson et al22 found an equal cumulative fresh plus frozen rate with two versus three fresh embryos replaced in women less than age 37 with good quality embryos. Gerris et al did a randomized study of one versus two good quality embryos in women less than age 34 having their first IVF cycle.25 The ongoing pregnancy rates were 39% versus 74%. The authors suggested that avoiding the 30% incidence of twins justified the lower fresh embryo transfer success rate. They did not determine cumulative (fresh plus frozen) pregnancy rates.

SUMMARY There has been an evolution of opinion over the past few years toward transfer of fewer embryos. This has resulted from improvements in embryo quality, which increases multiple implantation as well as giving an acceptable pregnancy rate with transfer of fewer embryos. Improvements in cryopreservation in turn have meant that any embryo

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frozen rather than transferred fresh will have a lesser reduction of its potential for implantation. On the other side is the desire from patients for the highest pregnancy rate with the least cost to them, and their common perception that twins are a positive outcome (two for the cost of one both regarding the IVF cycle and pregnancy). Even if the cumulative fresh plus frozen pregnancy rate may be similar in some circumstances, there is added cost and inconvenience for those women who would have conceived with more fresh embryos transferred. Third party payers understandably wish to reduce costs for multiples, since payment is usually their responsibility once pregnancy is established. Particularly in older women, transfer of more embryos not only increases the pregnancy rate and reduces the miscarriage rate but avoids the time lost during the frozen embryo cycle. Frozen embryo cycles also have much more limited outcome in women over 40 because of fewer extra high quality embryos being available to freeze. As with many controversies, the best answer lies between the extremes, transferring a limited number of embryos in younger women with good ovarian response and good embryo quality and more embryos when low prognosis factors are present such as advanced age, multiple failed cycles and poor embryo quality. Through these measures an acceptably low incidence of triplets can be expected, with our goal to refine the ability to identify embryos with high implantation potential and the continuing goal toward single embryo transfer for some couples.

REFERENCES 1 Templeton A, Morris JK. Reducing the risk of multiple births by transfer of two embryos after in vitro fertilization. New Engl J Med (1998); 339:573–7. 2 Centers for Disease Control, American Society for Reproductive Medicine, Society for Assisted Reproductive Technology, Resolve. 1997 Assisted Reproductive Technology success rates. Atlanta: US Department of Health and Human Resources, CDC, 1999. 3 Martin PM, Welch HG. Probabilities for singleton and multiple pregnancies after in vitro fertilization. Fertil Steril (1998); 70:478–81. 4 Widra EA, Gindoff PR, Smotrich DB, Stillman RJ. Achieving multipleorder embryo transfer identifies women over 40 years of age with improved in vitro fertilization outcome. Fertil Steril (1996); 65:103–8. 5 Qasim SM, Karacan M, Corson GH, Shelden R, Kemmann E. Highorder oocyte transfer in gamete intrafallopian transfer patients 40 or more years of age. Fertil Steril (1995); 64:107–10. 6 Society for Assisted Reproductive Technology, The American Fertility Society. Assisted Reproductive Technology in the United States: 1996 results generated from the American Society for Reproductive

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Medicine/Society for Assisted Reproductive Technology Registry. Fertil Steril (1999); 71:798–807. 7 Chenette PE, Sauer MV, Paulson RJ. Very high serum estradiol levels are not detrimental to clinical outcome of in vitro fertilization. Fertil Steril (1990); 54:858–63. 8 Schoolcraft WB, Gardner DK, Lane M, Schlenker T, Hamilton F, Meldrum DR. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril (1999); 72:604–9. 9 Toner JP, Philput CB, Jones GS, Muasher SJ. Basal follicle-stimulating hormone level is a better predictor of in vitro fertilization performance than age. Fertil Steril (1991); 55:784–91. 10 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. 11 Camus E, Poncelet C, Goffinet F, et al. Pregnancy rates after in-vitro fertilization in cases of tubal infertility with and without hydrosalpinx: a meta-analysis of published studies. Hum Reprod (1999); 14:1243–9. 12 Cohen J, Inge KH, Suzman M, Wiker SR, Wright G. Videocinematography of fresh and cryopreserved embryos: a retrospective analysis of embryonic morphology and implantation. Fertil Steril (1989); 51:820–7. 13 Scott RT, Hofmann GE, Veeck LL, Jones HW, Muasher SJ. Embryo quality and pregnancy rates in patients attempting pregnancy through in vitro fertilization. Fertil Steril (1991); 55:426–8. 14 Erenus M, Zouves C, Rajamahendren P, Leung S, Fluker M, Gomel V. The effect of embryo quality on subsequent pregnancy rates after in vitro fertilization. Fertil Steril (1991); 56:707–10. 15 Steer CV, 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); 7:117–9. 16 Munne S, Alikani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmental rates, and maternal age are correlated with chromosomal abnormalities. Fertil Steril (1995); 64:382–91. 17 Svendsen TO, Jones D, Butler L, Muasher, SJ. The incidence of multiple gestations after in vitro fertilization is dependent on the number of embryos transferred and maternal age. Fertil Steril (1996); 65:561–5. 18 Hu Y, Maxson WS, Hoffman DI, et al. Maximizing pregnancy rates and limiting high-order multiple conceptions by determining the optimal number of embryos to transfer based on quality. Fertil Steril (1998); 69:650–7.

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19 Van Royen E, Mongelschots K, De Newbourg D, et al. Characterization of a top quality embryo, a step towards single-embryo transfer. Hum Reprod (1999); 14:2345–9. 20 Balen AH, MacDougall J, Tan SL. The influence of the number of embryos transferred in 1060 in-vitro fertilization pregnancies on miscarriage rates and pregnancy outcome. Hum Reprod (1993); 8:1324–8. 21 Botchan A, Yaron Y, Lessing JB, Barak Y. When multiple gestational sacs are seen on ultrasound, “take-home baby” rate improves with invitro fertilization. Hum Reprod (1993); 8:710–3. 22 Staessen C, Janssenswillen C, Van Den Abbeel E, Devroey P, Van Steirteghem AC. Avoidance of triplet pregnancies by elective transfer of two good quality embryos. Hum Reprod (1993); 8:1650–3. 23 Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod (1998); 13:3434–40. 24 Milki AA, Fisch JD, Behr B. Two-blastocyst transfer has similar pregnancy rates and a decreased multiple gestation rate compared with three-blastocyst transfer. Fertil Steril (1999); 72:225–8. 25 Gerris J, De Neubourg D, Mangelschots K, Van Royen E, Van de Meerssche M, Valkenburg, M. Prevention of twin pregnancy after invitro fertilization or intracytoplasmic sperm injection based on strict embryo criteria: a prospective randomized trial. Hum Reprod (1999); 14:2581–7.

57 Multifetal pregnancy reduction and selective termination Shlomo Lipitz

INTRODUCTION The incidence of pregnancies with three or more fetuses has increased markedly over the past two decades. The change has been attributed to the introduction and widespread use of new techniques for ovulation induction and the replacement of multiple embryos during in vitro fertilization-embryo transfer (IVF-ET).1–3 These techniques have been a matter of concern, since triplet and higher order multiple pregnancies have long been associated with an increased risk of maternal complications, and a high prevalence of perinatal and neonatal morbidity and mortality.4– 8 Simultaneously, the developing experience with multifetal pregnancy reduction offers a new option for the management of such pregnancies. There is therefore a need to evaluate the management and outcome of high order multiple pregnancies. The incidence of triplets or higher order multiple pregnancies depends to a large extent on the availability and use of fertility therapies in a given population. Petrikovsky and Vintzeleos9 have stressed the influence of genetic and geographic factors. They reported an incidence of triplets that varied from 1:612 in the Yorube tribe in Western Nigeria to 1:7925 in the USA. In a recent series of 78 triplet pregnancies we found an incidence of 1:849 deliveries.10 This reflects the frequent use of fertility treatment, as only 12% of the triplet pregnancies were conceived spontaneously. Ovulation was induced by human menopausal gonadotropin (hMG) in 50% of the pregnancies, and by clomiphene citrate (CO in an additional 29%; the remaining 9% resulted from IVF-ET. The prevalence of spontaneous triplet pregnancies that reached 20 weeks gestation was 1:7358 deliveries.10 Our data are in agreement with a report from the British Association of Perinatal Medicine.3 This group found that among 156 high order multiple births in 1989 (143 triplets, 12 quadruplets and 1 quintuplet), 31% were conceived naturally, 34% had ovarian stimulation, usually with CC or hMG, and 35% had IVF-ET. All quadruplet and quintuplet pregnancies were established after assisted reproduction. The incidence of quadruplet pregnancies has varied from 1:5370 to 1:600,000, while the birth of quintuplets is a very rare event occurring

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once in 15–20 million deliveries. Using ovulation induction treatment with hMG, we found an incidence of 1:9190 (8 in 73,527 deliveries) for quadruplet and 1:24,500 for higher multiple gestations (two quintuplets and one sextuplet).11 Thus, it is evident that treatment of infertility has resulted in a dramatic increase in the number of triplets and high order multiple births in developed countries. In the United States, for example, a 113% increase was observed from 1972 to 1989.12 Ovulationstimulating drugs accounted for 50% of 1138 triplet pregnancies delivered in the United States.1 Similarly, in Britain there has been a marked increase in the number of triplets born since the late 1970s. This has been associated with a fourfold increase in the number of higher multiple births between 1971 and 1983, compared with the 15 year period 1956 to 1970.3 This marked increase in the numbers of high order multiple births seems to be continuing.2,3 PERINATAL MORTALITY Perinatal mortality rises progressively with the number of fetuses. Rates of 164, 200, 214 and 416 per 1000 births have been reported for triplets, quadruplets, quintuplets and sextuplets, respectively.13 We found a lower perinatal mortality rate in our study groups;10,11 93 and 119 per 1000 deliveries for triplets and higher multiple births, respectively. This may reflect advances in perinatal and neonatal care. Nevertheless, the mortality rates of extremely low birth weight infants remains high but could be reduced in the future. The mortality rates in our triplet series10 were similar to those reported for twin gestations by the Oxford National Perinatal Epidemiology Unit in England and Wales from 1975 to 1983, taking into account the exclusion of stillbirths before 28 weeks’ gestation.13 Preterm labor (<37 completed gestational weeks) is the major complication of multifetal pregnancies. The risk of preterm labor and delivery rises with the number of fetuses.14 Recent studies have reported the incidence of preterm labor in triplet pregnancies to be as high as 66%,15 80%16 and 86%.10 The risk is even greater for multiple pregnancies of higher order. In our series of high order multifetal pregnancies,11 only one pregnancy continued up to the 35th week of gestation. Similarly, in a review of eight sets of quintuplets, only one set completed 36 weeks of pregnancy.9 The average gestational age at delivery in triplet pregnancies has been reported as being 33.6±3.0 weeks (mean±SD),15 33.8±2.8 weeks,1 and 33.2±3.8 weeks,10 with a mean birth weight of 1871±555 g,15 and 1911±521 g.1 For quadruplets, the average gestational age at delivery has been reported as 31.1±6.8 weeks.9 In our series, 74% of the 39 live born infants from high order multifetal gestations weighed <1500g and 41% were below the tenth percentile for gestational age.11

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THE EFFECT OF FERTILITY DRUGS AND IVF-ET ON THE OUTCOME OF TRIPLET PREGNANCIES The outcome of induced compared to spontaneous triplet pregnancies has been the subject of an ongoing controversy. While some authors described a higher incidence of prematurity and low birth weight in babies born after induced conceptions,5,17 others have found these pregnancies to be associated with a more favorable outcome than spontaneous pregnancies.6,18,19 Ron-El et al5 were among the first to observe that spontaneous triplet gestations lasted 2 weeks longer than induced pregnancies. Furthermore, in a more recent study, Mordel et al17 found that 17 triplets, conceived following ovulation induction with menotropins, were delivered about 3–4 weeks earlier and weighed between 350–500g less than 7 who were delivered after spontaneous conception or 12 after ovulation induction with CC. In contrast, Holchberg et al6 compared 9 induced triplet pregnancies to 12 spontaneous pregnancies, and observed that the latter were born 2.5 weeks earlier. It should be noted, however, that most of the above triplet gestations were induced with CC rather than by menotropins. One explanation for the discrepancies between the various studies has been proposed by Newman et al.19 They have shown that the increase in birth weight and gestational age in their large series of spontaneous triplets, could be explained by difference in fetal gender and maternal parity. Once these confounding factors were controlled for using analysis of variance, the advantage disappeared. Although the incidence of spontaneous triplets increases with age,1 among women undergoing IVFET, multiple pregnancies are more common in younger patients.20 Maternal age should therefore be considered as an additional confounding factor. The outcome of triplet pregnancies following IVF-ET has been subject to particular concern.21 In a recent study, we directly compared the results of IVF triplet pregnancies with spontaneous and ovulation induced gestations.22 Our results were reassuring with regard to neonatal outcome. Despite the fact that a high rate of early pregnancy loss was observed in the IVF triplets, the mean birth weight of 1833±458g was comparable and the mean gestational age of 33.9±2.5 weeks was better than previously reported by Seoud et al (1666±443g and 31.8±2.7 weeks),20 and by Kingsland et al (1850g (range 720–2880) and 33.3 weeks (range 26–38).23 The stillbirth and prenatal mortality rates were also comparable to those previously reported for IVF triplets.24 We concluded that induction of ovulation and IVF-ET should not be considered a significant risk factor for the outcome of triplet pregnancies.

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MULTIFETAL PREGNANCY REDUCTION The procedure of multifetal pregnancy reduction has, in recent years, become both clinically and ethically accepted as a therapeutic option in pregnancies with four or more fetuses25–34 and in multifetal pregnancies in which one or more of the fetuses has congenital abnormalities.26,35 In cases of triplet gestations, however, this option remains controversial.25,31,32,34,36–38 Reports of the improving outcome of triplet pregnancies,10,15 the failure to demonstrate an improvement in the outcome of triplet pregnancies reduced to twins compared with those managed expectantly,36,37 and the procedure related risk of losing the entire pregnancy31,39 have complicated the clinical and ethical discussion surrounding this procedure in triplet gestations. SPONTANEOUS REDUCTION IN MULTIFETAL PREGNANCY Consideration of the clinical options and the ethical issues involved in the management of triplet or higher order gestations should include the probability of achieving a successful pregnancy outcome if an expectant management policy is undertaken. The occurrence of the disappearance of one or more of the fetuses in pregnancies that start as multiple gestation was reported by several authors. Dickey et al39 reported that when three viable embryos were diagnosed on first trimester ultrasound, the probability of delivering triplets and twins was 68.4% and 21%, respectively. This outcome was influenced by the chronological age of the mother. Blumenfeld et al40 followed by transvaginal ultrasound 88 women with multiple gestations (54 twin, 26 triplet, 5 quadruplet and 3 quintuplet) that were diagnosed at 5–6 weeks of gestation, in all of whom absorption of at least one gestation sac was detected at follow-up ultrasound. Of the 54 twin gestations, 51 ended in a birth of a singleton and 3 in miscarriage. Of the 26 pregnancies starting as triplets, 12 ended in singleton births, 12 in twins and two miscarried. The five quadruplet gestations resulted in one singleton birth, one set of twins, two triplets, and one ended in late miscarriage. Of the three quintuplet pregnancies, four fetuses vanished spontaneously. Overall, out of 221 fetuses identified, 107 (48%) vanished spontaneously. Manzur et al41 reported in 1995 on the outcome of triplet pregnancies after assisted reproductive techniques. Thirty-eight pregnancies were identified by transvaginal ultrasound between 24 and 28 days after transfer. The triplet delivery rate was 47.4%, whereas 31.6% delivered twins, 18.4% delivered singletons, and only one patient (2.6%) miscarried all three fetuses. The detection of three fetal heartbeats was associated

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with a triplet delivery rate of 69.2%, a twin delivery rate of 19.2% and 11.6% of singleton births. TIMING AND TECHNIQUES The first multifetal pregnancy reduction series was published in 1986 by Dumez and Oury,42 who reported fetal reduction in 15 women who carried triplet to sextuplet and reduced the number of fetuses to one or two. The procedures were performed by dilating the cervix and aspirating the gestational sacs transvaginally under ultrasound guidance. Some other authors have used transcervical aspiration of the gestational sac.43 This method, however, was thought to be associated with an increased incidence of fetal loss due to infection caused by introduction of bacteria from the cervix, or due to cervical incompetence brought about by cervical dilatation.30 Fetal reduction very early in gestation (6–8 weeks) by transvaginal puncture and embryo aspiration44 was also reported with fairly good pregnancy outcome. However, this method might have some theoretical limitations, such as (1) using general anesthesia, (2) the possibility of spontaneous fetal reduction at this stage of gestation, (3) the inability to perform early fetal screening, such as nuchal translucency test, or even fetal anomaly screening, which is done later on in pregnancy, and (4) the possibility of increasing infections. Multifetal pregnancy reduction using intrathoracic injection of potassium chloride, by both the transabdominal and the transvaginal approaches, has been extensively reported.25,26,28,31– 34,43–45 No method, however, has yet been proven to be superior to the others.32,34 Of the several techniques for MPR that have been reported, the most popular is the intrathoracic injection of potassium chloride by the transabdominal approach at 10–12 weeks, gestation. It has been reported that MPR performed at later weeks of pregnancy may be accompanied by increased risk of pregnancy loss. OUTCOME The pregnancy loss subsequent to fetal reduction has been reported as ranging from 0% to 40%. However, in three recent reports the reduction of triplets to twins resulted in a fairly consistent fetal loss of 8% to 10%.28,31,37,46 In our experience, the four pregnancies that were lost following reduction occurred among the first seven patients.47 The losses may have reflected a learning curve associated with the introduction of a new procedure. Multifetal pregnancy reduction appears to be a safe procedure in triplet pregnancies and imposes a relatively small risk of losing the entire pregnancy.33,48 Porreco et al37 showed no improvement in pregnancy outcome in 13 triplet pregnancies reduced to twins, compared with 11 triplet gestations managed expectantly. In contrast, Macones et

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al49 showed a mean gestational age at delivery for the reduced triplets of 35.6 weeks, compared with 31.2 weeks in the non-reduced triplets. The perinatal mortality rate was 30 per 1000 births in the reduction group and 210 per 1000 births in the non-reduced triplets. There were no statistically significant differences between the reduced and non-reduced twins. These results are comparable to those reported by others.28,36,38,46,48 In our prospective series, multifetal pregnancy reduction of triplets to twins was clearly associated with an improvement in the outcome of the pregnancies without an increased loss of the entire pregnancy.47 A spontaneous pregnancy loss of 20.7% in triplet gestations diagnosed soon after conception occurred prior to 25 weeks’ gestation, compared with a loss of 8.7% of the triplet pregnancies reduced to twins. Furthermore, the mean gestational age of the reduced pregnancies was increased by approximately 3 weeks and mean birth weight of the live born infants increased by 570g, compared to triplet gestations. The proportion of lower birth weight (<2500g) infants, very low birth weight (<1500g) infants and of premature infants (<37 completed gestational weeks) were all significantly decreased after the reduction procedure. Moreover, we recently compared the outcome of triplet pregnancies undergoing fetal reduction to twins who reached 24 weeks’ gestation with that of twin pregnancies managed in the same perinatal department.50 The outcome of triplet pregnancies reduced to twins did not significantly differ from the outcome of non-reduced twin pregnancies, and the results obtained were generally comparable to those reported in larger series of twin pregnancies.20,31,51,52 However, several findings suggested a tendency to a poorer outcome in the reduced twin group. A higher proportion of patients had premature rupture of membranes and there was a higher proportion of low and very low birth weight infants in the reduced twin group. These differences were not statistically significant possibly due to the small sample size. The transabdominal MPR approach is the most popular around the world. Based on the experience reported mainly by Evans et al in their multicenter report,34 it is clear that: the total pregnancy loss rate is correlated with the initial and final number of fetuses, gestational age at delivery is inversely correlated with the initial and final number of fetuses, significant maternal complications have not been reported, and the risk of congenital anomalies in the survivors is not increased. The pregnancy loss rate for transvaginal embryo aspiration44 or transvaginal intrathoracic potassium chloride (KCl) injection32 is probably higher mainly for triplet and quadruplet pregnancies. The corrected loss rate for transvaginal KCl injection was 11%.32 We now prefer to use the abdominal approach between 11–13 weeks of pregnancy. In doing so, cases that would have otherwise resulted in spontaneous abortions or spontaneous reduction in the number of fetuses by intrauterine demise are avoided. This approach and timing also allows early screening (nuchal

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translucency) and prenatal genetic and ultrasound diagnosis by ultrasound in order to perform selective reduction when warranted. Multifetal pregnancy reduction is beneficial mainly in pregnancies of four or more fetuses because of the risk of extreme prematurity. Reduction of triplets is more controversial mainly because of the improvement in neonatal survival. However, patients with triplets should receive all the information and offered the option of MPR. Although uncommon, occasionally, we still diagnose patients conceiving more than six fetuses. The highest number of fetuses we treated successfully was a pregnancy with 12 fetuses.53 Such pregnancies result from aggressive ovulation induction protocols mainly in PCO patients. In cases of more than six fetuses we performed the reduction in 2–3 steps, about 7 days apart. Evans et al34 reported that the overall loss rates were related to the starting number and the final number. However, they reported that the total pregnancy loss rates were lower when triplets were reduced to twins than when triplets were reduced to singletons. Also, elective reduction of twins to singletons differs substantially from MPR and no data suggest that this procedure significantly improves perinatal outcome. Bichorionic triplet pregnancy (1 singleton and 2 monochorionic twins) can result spontaneously as well as after ovulation induction treatment or IVF-ET. If the parents decide to undergo multifetal pregnancy reduction (after informed consent) there are three alternatives. (a) To reduce the singleton fetus—the risk of the procedure per se is similar to all other MPR. However, there is an increased risk later in the pregnancy because of the 15% chance of twin to twin transfusion syndrome (TTS) in the monochorionic biamniotic twins.

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Fig 57.1 Illustrative photo of a high-order multiple pregnancy. (b) To reduce one of the monochorionic twins—this procedure can not be performed by injecting potassium chloride because of the very high risk to the other co-twin (death or severe neurologic handicap). In case of structural abnormality in one of the monochorionic twins it may be considered to perform selective termination by performing coagulation of the cord or cord ligation. The latter procedures are much riskier as a method for MPR. (c) To reduce the two monochorionic twins—the risk of the procedure is similar to that of reducing triplets to singleton and is approximately 6–7% (not much different than MPR from triplets to twins). By choosing this option, the risk of TTS will be eliminated. From the clinical point of view the parents should choose mainly between options (a) and (c). In both cases they should give a written informed consent and have permission from the ethical committee in countries where it is necessary. PSYCHOLOGICAL IMPACT Women who undergo MPR are different from those having spontaneous or induced abortions. Most patients have conceived after longstanding infertility and are now presented with a situation that can endanger their

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pregnancy either by choosing MPR or by the decision of an expectant management with three or more fetuses. Requesting MPR is a paradoxical reaction for couples having conceived with assisted conception techniques. It is in any case a difficult decision to perform MPR. The role of the medical team is critical before and after the procedure. In a follow up study, Garel et al54 showed that 4 years after delivery, other than the obvious financial burden, mothers of triplets still report fatigue, emotional distress and difficult relationship with the children. Schreiner-Engel et al55 assessed the psychological impact of MPR. Most women investigated agreed that the reduction procedure was stressful, painful, emotionally disturbing, and frightening. Mourning for the lost fetuses was reported by approximately three quarters of the women, but for most it lasted only one month. Persistent depressive symptoms were mild, although moderate levels of sadness and guilt continued for many. Nevertheless, the majority of couples would make the same decision to have MPR in a future pregnancy.

SELECTIVE TERMINATION OF THE MALFORMED FETUS IN MULTIPLE PREGNANCY Selective termination of an affected twin (or in cases of multifetal pregnancies) during pregnancy is not “abortion” in the most simple sense. A woman elects to have an “abortion” because she has decided, for whatever reason, that she does not wish to have that child. A woman undergoes selective termination or multifetal pregnancy reduction precisely because she wishes to have a healthy child, either because the number of fetuses poses a clear and undisputed risk or because of a fetal abnormality that has been detected in one of the twins. Previously, the only options were to allow the pregnancy to continue and for the parents to be committed to the care of a potentially severely handicapped infant, or to abort the fetuses and terminate an otherwise wanted pregnancy. In the highly emotionally charged and ethically and medically different situation, selective termination provides couples with the possibility of having a presumably healthy infant, while sparing them the emotional and financial trauma associated with the birth of a severely handicapped infant. Application of modern techniques for clinical prenatal diagnosis, including amniocentesis, chorionic villus sampling, fetoscopy, ultrasonography, and fetal blood sampling, along with appropriate laboratory techniques, including cytogenetic, biochemical, and molecular genetic evaluations make it possible to detect an increasing number of fetal abnormalities in the antepartum period.56 After diagnosis of an abnormality in one fetus with an apparently normal co-twin, a couple should be given the choice of continuing the

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pregnancy with both the normal and abnormal fetuses, undergoing selective termination of the affected twin,57 or aborting both fetuses. The selective termination procedure is initiated by inserting the needle into the heart of the abnormal fetus. In cases with chromosomal aberrations, fetal blood is taken for rapid karyotyping to confirm that the abnormal twin is indeed the one being treated. In cases with structural abnormalities, an amniocentesis for the normal co-twin may be performed, according to the patient’s request. Feticide of the affected twin is performed by intracardiac injection of 2–3ml of 15% potassium chloride (KCl). This relatively small volume of KCl is harmless to the mother and is similar to the injected volumes used in multifetal pregnancy reduction of two or three fetuses in the first trimester. Cardiac puncture, air embolization, exsanguination, and cardiac tamponade are currently infrequently used alternatives. Pregnancies suspected of being monochorionic should not undergo the procedure with KCl injection because of the significant risk to the healthy fetus. Moreover, in cases where chorionicity is uncertain, careful ultrasonographic determination must be carried out before advising the parents that the procedure is relatively harmless to the healthy fetus. If ultrasonography is insufficient to determine chorionicity, amniocentesis and DNA fingerprinting for zygosity determination should also be considered. Surgical removal of the anomalous twin, occlusion of its umbilical cord, or selective removal of an acardiac fetus by hysterotomy58,59 have been reported in selected monochorionic pregnancies. Techniques of umbilical cord occlusion include embolization of the umbilical vessels, endoscopic or ultrasonographic cord ligation, endoscopic laser coagulation and diathermic coagulation.60–62 When the indication for reduction is a structural or chromosomal abnormality in a gender discordant pair, there is no difficulty in identifying the abnormal fetus immediately prior to the procedure. However, in cases of chromosomal abnormalities in a same-sex pair, cordocentesis or fluorescent in situ hybridization (FISH) analysis of amniotic fluid should be undertaken to confirm diagnosis according to the different positions of the fetuses. Termination is carried out only after the genetic results (approximately 36–72 hours) have been evaluated. No relation exists between the location of the affected sac and the risk for premature labor, or the procedure delivery interval, and this should not be considered in the decision of whether and when to perform the procedure.63 Follow up includes ultrasonographic examinations once every 2 weeks, frequent evaluation of fetal wellbeing with the use of the non-stress test and biophysical profile, and laboratory tests to detect possible disseminated intravascular coagulopathy (DIC). Assessment of the risk of preterm labor may be accomplished by one of several means, such as uterine contraction monitoring, determination of cervicovaginal

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fibronectin levels,64 or ultrasonographic measurements of cervical length. Tocolytic agents are not used routinely and are added only if uterine activity is recorded in association with cervical change. Glucocorticoids are routinely administered on a weekly basis when the procedure is performed from the 27th to the 32nd gestational week. In one of the cases reported by Evans et al,65 the healthy fetus was erroneously terminated after prenatal diagnosis of Down’s syndrome in the other twin by amniocentesis. This dramatic accident stressed the absolute need for precise identification of each twin during fetal tissue sampling procedures, as well as the risk of misleading reports on fetal positions at the time of amniocentesis, if 2–3 weeks elapse until the results became available. Documentation of which fetus has the anomaly at the time of prenatal diagnostic procedures is critical and must include an in utero mapping of both fetal and placental topography. Frequently, certain morphological abnormalities will be detectable on ultrasonography at the time of the selective termination procedure. If not, or if there is any doubt as to which is the affected fetus, then rapid determination of karyotype by direct preparation of chorionic villi, fetal blood sampling, or FISH would be appropriate. GESTATIONAL AGE CONSIDERATIONS In the late 1970s Alberg et al57 reported the first successful selective birth from a twin pregnancy discordant for Hurler syndrome. In 1978, Kerenyi and Chitkara66 reported selective birth in a twin pregnancy discordant for Down’s syndrome. Throughout the 1980s, several reports of second trimester selective termination appeared in the literature. None had sufficient data to reach reasonable conclusions concerning the safety and efficiency of the procedure.67–69 In a recent multicenter report on 183 such procedures, selective termination was technically successful in 100% of cases.65 No coagulopathy or ischemic damage was observed in survivors of dichorionic pregnancies and no maternal morbidity was noted. In experienced hands, selective termination of a dizygotic, abnormal twin was safe and effective when performed with KCL. A total of 83.8% viable deliveries occurred after 33 weeks, and only 4.3% between 25 and 28 weeks. Gestational age at the time of the procedure correlated positively with the rate of loss, and inversely with gestational age at delivery. However, this study included only five cases in which termination had been performed at >24 weeks’ gestation. Consequently, the appropriateness of selective termination has not yet been established for those patients presenting for treatment after pregnancy has become viable. This latter issue was addressed by a multicenter study conducted in Israel.63 Several states have laws with regard to the exact gestational week from which termination of pregnancy is prohibited under any conditions. Israeli law permits late termination in singleton as well as in multifetal

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pregnancies under special restriction and regulations. The opportunity exists to analyze the perinatal outcome of twin pregnancies after late selective termination of one malformed fetus. In the cited series, a total of 36 dichorionic twin pregnancies underwent selective fetal termination after 24 complete gestational weeks.63 Only five women (13.8%) delivered before 34 completed weeks. In only one case did perinatal death of the normal twin (2.8%) occur as a result of the termination of the malformed fetus. The international collaborative study of second trimester, selective terminations for fetal abnormalities in twin pregnancies65 reported first, a significantly higher miscarriage rate when the procedure was performed later in pregnancy (14.5%>16 weeks) compared with earlier procedures (9%<16 weeks), and second, that gestational age at delivery for those who reached viability was negatively correlated with gestational age at the time of selective termination. In that series, the rate of prematurity, namely <32 weeks, was 14.2%. Therefore, a higher rate of preterm labor and prematurity for late terminations (>24 weeks) may have been expected. However, the rate of prematurity in the Israeli series was relatively low (14.3% before 34 weeks), and the median gestational age at delivery in the study group (37 weeks) was similar to that of uninterrupted twin gestations in one American series.20 Consequently, there were no cases of significant morbidity associated with prematurity among the live born infants. Theoretically, when discordance for fetal anomaly is diagnosed late in pregnancy and selective termination is requested and approved, three options exist regarding the timing of the procedure. (1) As soon as possible, with the potential risks of prematurity associated with the procedure and its complications. (2) Following lung maturity acceleration and verification at about 32–34 weeks. (3) When labor starts (or after 36 weeks) to avoid possible prematurity of the unaffected fetus associated with the procedure. The two later options may prevent potential procedure related risks to the healthy fetus. However, live delivery of the malformed fetus may occur under these circumstances. Unfortunately, delaying the termination to a more advanced stage makes the ethical issue become even more problematic in many countries, including Israel, where late termination is permitted only under strict and specific conditions. In Israel, most committees that would approve selective termination because of a severe anomaly detected at 24–26 weeks’ gestation would not do so at 34–36 weeks, even if the law does not define an upper gestational limit. In view of the results in the Israeli multicenter study,63 we believe that when selective termination is a viable option, it should be effected shortly after the diagnosis.

REFERENCES

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1 Elster AD, Bleyl JL, Craven TE. Birth weight standards for triplets under modern obstetric care in the United States, 1984–1989. Obstet Gynecol (1991); 77:387–93. 2 Rein MS, Babieri RL, Greene MR The causes of high-order multiple gestation. Int J Fertil (1990); 35:154–6. 3 Levene MI, Wild J, Steer P. Higher multiple births and the modern management of infertility in Britain. Br J Obstet Gynaecol (1992); 99:607–13. 4 Gonen R, Heyman E, Asztalos EV, Ohlsson A, Pitson LC, Shennan AT. The outcome of triplet, quadruplet and quintuplet pregnancies managed in a perinatal unit: obstetric neonatal and follow-up data. Am J Obstet Gynecol (1990); 162:454–9. 5 Ron-El R, Caspi E, Schreyer P, Weintraub Z, Arieli S, Goldberg MD et al. Triplet and quadruplet pregnancies and management. Obstet Gynecol (1981); 57:458–63. 6 Holchberg G, Biale Y, Lewenthal H, Insler V. Outcome of pregnancy in 31 triplet gestations. Obstet Gynecol (1982); 59:472–6. 7 Loucopoulos A, Jewelewicz R. Management of multifetal pregnancies: sixteen years experience at the Sloane Hospital for Women. Am J Obstet Gynecol (1982); 143:902–5. 8 Deale CJC, Cronje HS. A review of 367 triplet pregnancies. S Afr Med J (1984); 66:92–4. 9 Petrikovsky BM, Vintzeleos AM. Management and outcome of multiple pregnancy of high fetal order: Literature review. Obstet Gynecol (1989); 44:578–84. 10 Lipitz S, Reichman B, Paret G, et al. The improving outcome of triplet pregnancies. Am J Obstet Gynecol (1989); 161:1279–84. 11 Lipitz S, Frenkel Y, Watts C, et al. High order multifetal gestation— management and outcome. Obstet Gynecol (1990); 76:215–8. 12 Kiely JL, Kleinman JC, Keily M. Triplets and higher-order multiple births. Am J Dis Child (1992); 146:862. 13 Botting BH, Mcdonald-Davies I, McFarlane AJ. Recent trends in the incidence of multiple births and associated mortality. Arch Dis Child (1987); 62:941–50. 14 Neilson JP, Crowther CA. Preterm labour in multiple pregnancies. Fetal Matern Med Rev (1993); 5:105–19. 15 Newman RB, Horner C, Miller MC. Outpatient triplet management: a contemporary review. Am J Obstet Gynecol (1989); 161:547–55. 16 Sassoon DA, Castro LC, Davis JL, Bear M, Hobel CJ. Perinatal outcome in triplet versus twin gestations. Obstet Gynecol (1990); 75:817–20. 17 Mordel N, Laufer N, Zajicek G, et al. Menotropis are a possible risk factor for premature deliveries in triplet pregnancies. Gynecol Endocrinol (1991); 5:197–201.

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18 Pons JC, Mayenga JM, Plu G, Forman RG, Papiernik E. Management of triplet pregnancy. Acta Genet Med Gemellol (Roma) (1988); 37:99– 103. 19 Newman RB, Jones JS, Miller MC. Influence of clinical variables on triplet birth weight. Acta Genet Med Gemellol (Roma) (1991); 40:173– 9. 20 Seoud MAF, Toner JP, Kruithoff C, Muasher S. Outcome of twin, triplet and quadruplet in vitro pregnancies: The Norfolk experience. Fertil Steril (1992); 57:825–34. 21 Botting B, MacFarlane A, Price AM. Triplet and higher order multiple births. The Sixth Report of the Interim Licensing Authority for Human In Vitro Fertilization and Embryology. (1991); 32–7. 22 Lipitz S, Seidman DS, Alcalay M, Achiron R, Mashiach S, Reichman B. The effect of fertility drugs and in vitro methods on the outcome of 106 triplet pregnancies. Fertil Steril (1993); 60:1031–4. 23 Kingsland CR, Steer CV, Pampliglione JS, Mason BA, Edwards RG, Campbell S. Outcome of triplet pregnancies resulting from IVF at Bourn Hallam 1984–1987. Eur J Obstet Gynecol Rep Biol (1990); 34:197–203. 24 Rizk B, Doyle P, Tan SL, et al. Perinatal outcome and congenital malformations in in vitro fertilization from babies from the Bourn Hallam Group. Hum Reprod (1991); 6:1259–64. 25 Berkowitz RL, Lynch L, Chitkara U, et al. Selective reduction of multifetal pregnancies in the first trimester. N Engl J Med (1988); 318:1043–7. 26 Wapner RJ, Davis G, Johnson A, et al. Selective reduction of multifetal pregnancies. Lancet (1990); 335:90–3. 27 Evans JI, Fletcher JC, Zador IE, et al. Selective first trimester termination in octuplet and quadruplet pregnancies: clinical and ethical issues. Obstet Gynecol (1988); 71:289–96. 28 Lynch L, Berkowitz RL, Chitkara U, et al. First trimester transabdominal multifetal pregnancy reduction: a report of 85 cases. Obstet Gynecol (1990); 75:735–8. 29 Evans MI, May M, Drugan A, et al. Selective termination: clinical experience and residual risks. Am J Obstet Gynecol (1990); 162:1568– 75. 30 Dommergues M, Nisand I, Mandelbrot L, et al. Embryo reduction in multifetal pregnancies after infertility therapy: obstetrical risks and perinatal benefits are related to operative strategy. Fertil Steril (1991); 55:805–11. 31 Berkowitz RL, Lynch L, Lapinski R, Bergh P. First trimester transabdominal multifetal pregnancy reduction: a report of two hundred completed cases. Am J Obstet Gynecol (1993); 169:17–21. 32 Timor-Tritsch IE, Peisner DB, Monteagudo A, et al. Multifetal pregnancy reduction by transvaginal puncture; evaluation of the

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technique used in 134 cases. Am J Obstet Gynecol (1993); 168:799– 804. 33 Evans MI, Dommergues M, Wagner RJ, et al. Efficacy of transabdominal multifetal pregnancy reduction: collaborative experience among the world’s largest centers . Obstet Gynecol (1993); 82:61–2. 34 Evans MI, Dommergues M, Timor-Tritsch I, et al. Transabdominal versus transcervical and transvaginal multifetal pregnancy reduction: international collaborative experience of more than one thousand cases. Am J Obstet Gynecol (1994); 170:902–9. 35 American College of Obstetrics & Gynecology. Multifetal pregnancy reduction and selective fetal termination. ACOG Committee Opinion 94, 1991 36 Melgar CA, Rosenfeld DL, Rawlinson K, Greenberg M. Perinatal outcome after multifetal reduction to twins compared with non-reduced multiple gestations. Obstet Gynecol (1991); 78:763–7. 37 Porreco RP, Burjke MS, Hendrix ML. Multiple reduction of triplets and pregnancy outcome. Obstet Gynecol (1991); 78:335–9. 38 Vauthier-Bouzes D, Lefebvre G. Selective reduction in multifetal pregnancies: technical and psychological aspects. Fertil Steril (1992); 57:1012–6. 39 Dickey RP, Olar TT, Curole DN, et al. The probability of multiple births when multiple gestational sacs or viable embryos are diagnosed at first trimester ultrasound. Hum Reprod (1990); 5:880–2. 40 Blumenfeld Z, Dirnfeld M, Abramovici H, Amit A, Bronshtein M, Brandes JM. Spontaneous fetal reduction in multiple gestations assessed by transvaginal ultrasound. Br J Obstet Gynaecol (1992); 99:333–7. 41 Manzur A, Goldsman MP, Stone SC, Frederick JL, Balmaceda JP, Asch RH. Outcome of triplet pregnancies after assisted reproductive techniques: how frequent are the vanishing embryos? Fertil Steril (1995); 63:252–7. 42 Dumez T, Oury JR Method for first trimester selective abortion in multiple pregnancy. Contrib Gynecol Obstet (1986); 15:50–3. 43 Salat-Baroux J, Aknin J, Antoine JM, et al. The management of multiple pregnancies after induction for superovulation. Hum Reprod (1988); 3:399–401. 44 Itskovitz J, Boldes R, Thaler I, et al. First trimester selective reduction in multiple pregnancy guided by transvaginal sonography. J Clin Ultrasound (1990); 18:323–7. 45 Shalev J, Frenkel Y, Goldenberg M, et al. Selective reduction in multiple gestations: pregnancy outcome after transvaginal and transabdominal needle-guided procedures. Fertil Steril (1989); 52:416– 20.

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46 Boutlot P, Hedon B, Pelliccia G, et al. Obstetrical results after embryonic reductions performed on 34 multiple pregnancies. Hum Reprod (1990); 5:1009–13. 47 Lipitz S, Reichman B, Uval J, et al. A prospective comparison of the outcome of triplet pregnancies managed expectantly or by multifetal reduction to twins. Am J Obstet Gynecol (1994); 170:874–9. 48 Tabsh KM. Transabdominal multifetal pregnancy reduction: report of 40cases. Obstet Gynecol (1990); 75:739–41. 49 Macones GA, Schemmer G, Pritts E, Weinblatt V, Wapner RJ. Multifetal reduction of triplets to twins improves perinatal outcome. Am J Obstet Gynecol (1993); 169:982–6. 50 Lipitz S, Yuval Y, Achiron R, Schiff E, Lusky A, Reichman B. Comparison of the outcome of twin pregnancies reduced from triplets with non-reduced twin gestations. Obstet Gynecol (1996); 87:511–4. 51 Lowry MP, Stafford J. Northern region twin survey. J Obstet Gynecol (1988); 8:228–34. 52 Taffel SM. Health and demographic characteristics of twin birth: United States, 1988. National Center for Health Statistics. Vital Health Stat (1992); 21 50: 53 Lipitz S, Frenkel Y, Seidman DS, Zolti M, Meizner I, Mashiach S. Successful outcome of multifetal reduction in a pregnancy with 12 live fetuses. Hum Reprod (1994); 9:1190–1. 54 Garel M, Salobiz C, Blondel B. Psychological consequences of having triplets: a 4-year follow-up study. Fertil Steril (1997); 67:1162–5. 55 Schriener-Engel P, Walther UN, Mindes J, Lynch L, Berkowitz RL. First trimester multifetal pregnancy reduction: acute and persistent psychological reaction. Am J Obstet Gynecol (1995); 172:541–7. 56 Drugan A, Dvorin E, Koppitch FC, et al. Counseling for low maternal serum AFP should emphasize all chromosomal anomalies, not just Down syndrome! Obstet Gynecol (1989); 73:271. 57 Alberg A, Metelman F, Cantz M, Gehler J. Cardiac puncture of fetus with Hurler’s disease avoiding abortion of unaffected co-twin. Lancet (1978); 2:990–9. 58 Robie GF, Payne G, Morgan MA. Selective delivery of an acardiac twin. N Engl J Med (1989); 320:12–3. 59 Fries MH, Goldberg JD, Bolbus MS. Treatment of an acardiac acephalus twin gestation by hysterotomy and selective delivery. Obstet Gynecol (1992); 79:601–4. 60 Porreco RP, Barton SM, Haverkamp AD. Occlusion of umbilical artery in an acardiac acephalic twin. Lancet (1991); 337:326–7. 61 Deprest J, Evrard VA, Van Schouebrooeck D, Vandenbergh K. Endoscopic cord ligation in selective feticide. Lancet (1996); 348:890– 1. 62 Ville Y, Hyett J, Vandenbussche FPA, Nicolaides KH. Endoscopic laser coagulation of umbilical cord vessels in twin reversed arterial perfusion sequence. Ultrasound Obstet Gynecol (1994); 4:396–8.

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63 Lipitz S, Shalev E, Meizner I, et al. Late selective termination of abnormalities in twin pregnancies: a multicenter report. Br J Obstet Gynaecol (1996); 103:1212–6. 64 Lockweed CJ, Senyei AF, Dische MR, Casal D, Shah ND, Thung SN. Fetal fibronectin in cervical and vaginal secretions as a predictor of preterm delivery. N Engl J Med (1991); 325:669–72. 65 Evans MI, Goldberg JD, Dommergues M, et al. Efficiency of secondtrimester selective termination for fetal abnormalities: international collaborative experience among the world’s largest centers. Am J Obstet Gynecol (1994); 171:90–4. 66 Kerenyi T, Chitkara U. Selective birth in twin pregnancy with discordancy for Down syndrome. N Engl J Med (1978); 304:1525–7. 67 Rodek C, Mibashan R, Abramowicz J, Campbell S. Selective feticide of the affected twin by fetoscopic air embolization. Prenat Diagn (1982); 2:189–94. 68 Golbus MS, Cunningham N, Goldberg JD, Anderson R, Filly R, Callen P. Selective termination of multiple gestations. Am J Med Genet (1988); 31:339–48. 69 Westendorp AK, Miny P, Holzbreve W, DeWilde R, Aydinili K. Selective fetocide by direct injection of isotonic potassium chloride. Arch Gynecol Obstet (1988); 244:59–62.

58 Egg donation Mark V Sauer, Matthew A Cohen

INTRODUCTION Human oocyte donation, pioneered in 1983, has evolved in a relatively short time into a common procedure that addresses a variety of problems. It has provided key insights into the physiology and pathophysiology of reproduction and, like other assisted reproductive technologies, has engendered controversy. Not surprisingly, the first report of successful oocyte 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 The greatest impact of mammalian embryo transfer occurred in the mid to late 1970s in the cattle industry. By 1990 almost 19,000 calves were born annually in the United States from embryo transfer.2 The vast majority of mammalian oocyte donations resulted from embryos fertilized in vivo, recovered by uterine lavage, and then transferred to the recipient uterus. Using this technique, in 1983 researchers at the University of California, Los Angeles, fertilized an oocyte by in vivo artificial insemination in a donor and then transferred the recovered embryo into a synchronized recipient.3 A total of 14 insemination cycles led to two ongoing pregnancies.4 In 1984 the first delivery was reported.5 Simultaneously, researchers at the Monash University in Melbourne began transferring embryos fertilized in vitro from oocytes obtained laparoscopically from infertile donors with excess oocytes.6 In 1984 they reported the first live birth following oocyte donation and in vitro fertilization.7 Synchronization of the recipient and donor was achieved using oral estradiol valerate and intravaginal progesterone pessaries prescribed to the agonadal recipient. Donor uterine lavage was popularized since it was far less invasive than laparoscopy, but by 1987 uterine lavage was discontinued in humans because of the fear of HIV transmission. Furthermore, around this time the introduction of transvaginal oocyte aspiration under ultrasound guidance enabled oocyte donation to become an office procedure, greatly reducing its inconvenience while improving its safety.

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The popularity of oocyte donation is evidenced by the rapidly increasing demand for services. In the US, 6,643 oocyte donation cycles were reported to the Centers for Disease Control in 1997, up from 5,162 in 1996.8 This increase is due in part to the rising percentage of women who remain childless past the age of 30 years, a number that has sharply increased over the past generation.9 Many women are marrying later, or are pursuing a vocation and are not ready to begin a family.10 Unfortunately, there is a natural decline in fecundability with increasing age, and many healthy women are experiencing infertility primarily as a result of normal ageing.

INDICATIONS FOR OOCYTE DONATION The indications for oocyte donation have grown since its inception. Originally envisioned as a treatment option for women with either primary or secondary premature ovarian failure (POF),11 the list now includes many other reasons (Table 58.1). Non-iatrogenic POF, defined as women less than 40 years old with persistent amenorrhea and elevated gonadotropins, is estimated to affect 1% of the female population.12 The

Table 58.1. Indications for oocyte donation. Premature ovarian failure Gonadal dysgenesis Repetitive IVF failure Natural menopause Inheritable disorders majority of cases are idiopathic, but about 20% are suspected to be autoimmune in nature or due to concomitant glandular autoimmune disease.13 Thus it is important to ensure that subclinical failure of the thyroid, parathyroid, and adrenal glands has been ruled out as well as diabetes mellitus and myasthenia gravis. Any of these conditions may adversely affect pregnancy outcome as well as affect the general health and wellbeing of the patient. If POF occurs before the age of 30 a karyotype should be requested to ascertain the presence of Ychromosome mosaicism. Patients discovered to be mosaic are at risk of gonadal tumors and require extirpation of the abnormal gonad.14 A bone density evaluation is also 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 (for example, DiGeorge syndrome),16 galactosemia,17 and ataxiatelangiectasia18 and require special evaluation.

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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 as treatment of malignancies, but surgical castration more often results from non-cancerous conditions including infection, torsion, or overly aggressive removal of intraovarian lesions (for example, dermoids, endometriomas). IVF failure is common when a poor ovarian response to gonadotropins occurs. Many of these patients can be identified prior to the expense and psychological distress of multiple failed cycles. The first consideration is the age of the patient. It has long been known that fecundability decreases with age, and this is true with IVF as well (Fig 58.1).8 Many IVF centers have a maximum age limit beyond which they will not perform IVF without oocyte donation (45 years of age at Columbia University). Women of advanced reproductive age have far greater success with donated oocytes.21 Ovarian reserve is evaluated with serum follicle stimulation hormone (FSH) levels on day 2 or 3 of the menstrual cycle.22 Values greater than 15mIU/ml, and certainly greater than 20mIU/ml, are prognostic for a greatly

Fig. 58.1 Live births per transfer for fresh embryos from own and donor eggs, by age of recipient, 1997. Reproduced with permission from ref. 8.

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reduced IVF success rate. Another useful serum marker is day 2 or 3 estradiol.23 Values greater than 45pg/ml are predictive of lower pregnancy rates and, if greater than 75pg/ml usually attempts end in failure. It is important each laboratory determines 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, LH, and estradiol at baseline and again after 5 days (days 5–9) of 100 mg clomiphene citrate.24 Serum FSH values greater than 25mIU/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 for physiological menopause is very effective.21 The Ethics Committee of the American Society for Reproductive Medicine 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, 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 assisted reproductive technologies. Furthermore, denying healthy older women donated oocytes while allowing older men complete access to reproductive care is both prejudicial and sexist.29 Less controversial is the use of oocyte donation for inheritable conditions such as X-linked or autosomal traits and chromosomal translations.30 However, with progress in preimplantation diagnosis, this need may decrease.31

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RECIPIENT SCREENING In addition to a complete medical history and physical exam, suggested medical screening for oocyte recipients is shown in Table 58.2. Most of the tests are standard for expectant mothers and IVF candidates. Patients of advanced maternal age are at higher risk for certain medical conditions such as diabetes mellitus and heart disease and require additional testing. Other indications may warrant further evaluation, 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 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 The role of the psychologist 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 resolve their differences. The presence of endometriosis does not affect the pregnancy rate in 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.

Table 58.2. Suggested medical screening of oocyte recipient(s). Oocyte recipient Male partner Complete blood count with platelets Blood Rh and type Blood Rh and type Hepatitis screen Serum electrolytes, liver and kidney function VDRL Sensitive TSH (thyroid stimulating hormone) HIV-1, HTLV-1 Rubella and hepatitis screen Semen analysis and culture VDRL HIV-1, HTLV-1 Urinalysis and culture Cervical cultures for gonorrhea and chlamydia Pap smear

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Transvaginal ultrasound Uterine cavity evaluation (sonohysterogram or hysterosalpingogram) Electrocardiogram * Chest x-ray* Mammogram* Glucose tolerance test* Cholesterol and lipid profile* *If over 39 years of age A mock endometrial preparation cycle and timed endometrial biopsy is performed in many programs to ensure that a luteal phase defect or an inadequate response to endometrial priming is not present. Glandular/stroma dysynchrony is 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 less than 6 or 7mm is associated with poor outcome. Another study showed that all endometrial biopsies were in phase if the thickness was greater than 7 mm.41 The great majority of women will have adequate responses to hormone replacement and 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 or mock cycle, a trial of low dose aspirin (81mg daily) during the transfer cycle may increase pregnancy rates. Weckstein et al found that in women with a previous endometrial thickness of less than 8mm, 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.

OOCYTE DONOR RECRUITMENT Perhaps the greatest obstacle to 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 etiologies underlying their own infertility making them imperfect donors. Furthermore, with the advent of increasingly successful embryo

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cryopreservation, “extra oocytes” became quite scarce. An obvious source 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 greater than 35 years of age.50 Known designated donors are another option, typically a family member (for example, sister, niece). The final source of donors are women recruited from the general population, most often through advertisement. There exists a longstanding debate as to whether it is morally correct 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 bodily risk and no direct medical benefit to the donor. For this reason, many countries do not permit oocyte donation (for example, Germany, Norway, and Sweden).51 Others allow only IVF patients with excess oocytes to donate, such as Israel. Great Britain and Canada allow anonymous oocyte donation, but strongly discourage payment to the donor, except for verified expenses. The US 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 acquire sufficient donors to meet demand.53 How much these payments should entail remains hotly debated.54 Another area of controversy is the issue of anonymity. Most donors express a strong desire to not be identified by the children and to have no legal obligations as parents. There is, however, an opposing view that similar to adopted children, offspring of oocyte donation should have the same right to ultimately identify their genetic mother.55 Should such legislation be enacted, a deleterious effect on donor recruitment can be expected.

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 those of standard IVF. Even less risk of severe ovarian hyperstimulation syndrome occurs in donors compared with patients undergoing IVF, since pregnancy does not occur and moderate cases are not exacerbated.56 In addition to a complete medical history and physical exam, the suggested medical screening of oocyte donors is shown in Table 58.3. Of utmost importance is the screening for infectious diseases. Unlike sperm, which are amenable to cryopreservation, oocytes cannot be frozen for subsequent use. In sperm donation, cryopreservation allows a quarantine period and follow up screening for infectious diseases. Obviously, this is not possible for oocyte donors. Transvaginal ultrasound examination screens for pelvic pathology and ovarian morphology.

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It is preferable oocyte donors be under 30 years of age as younger donors appear to have higher pregnancy rates.46,57,58 Pregnancy rates of donors over 30 years old are still acceptable, however, and other traits and characteristics (for example, a close physical

Table 58.3. 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 match to the recipient) may make a particular donor who is older desirable to a recipient. The prior fertility history of the donor does not appear to affect pregnancy outcomes.57,58 Psychological evaluation by a licensed mental health practitioner is recommended for anonymous donors and is mandatory for known donors. Evaluation should define the motivation to donate, as well as the financial status of the donor to ensure the donation is not because of excessive inducement or coercion. 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 58.4.59 The presence of any of the disorders listed, because of their genetic ties, should exclude her from participating. Donors should be under 35 years of age to reduce the risk of aneuploidy. Exceptions can be made in circumstances such as sister to sister donation where the benefits of shared genetic background may balance the known risks (which can be largely discovered by

Table 58.4. 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

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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 Age if living Age at death Cause of death Grandfather (pat) Grandmother (pat) Grandfather (mat) Grandmother (mat) Father Mother Brothers Sisters Family Genetic History Familial Conditions Self Mother Father Siblings Comments High blood pressure Heart disease Deafness Blindness Severe arthritis Juvenile diabetes Alcoholism Schizophrenia Depression or mania Epilepsy Alzheimer’s disease Other (specify) Malformations Cleft lip or palate Heart defect Clubfoot Spina bifida Other (specify) Mendelian disorders Color blindness Cystic fibrosis Hemophilia Muscular dystrophy Sickle cell anemia

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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. amniocentesis). Donors should also be tested for disorders common to their ethnic background. These include cystic fibrosis in whites, a sickle cell test for blacks and people of Mediterranean ancestry, 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 alpha-thalassemia. Jews of eastern European ancestry should be screened for Tay-Sachs, Gaucher, and Canavan diseases. It is important to inform the recipient couple that even with appropriate 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.60

ENDOMETRIAL STIMULATION AND SYNCHRONIZATION Endometrial preparation of the recipient is modeled on the natural menstrual cycle, using estrogen and progesterone (Fig 58.2).21 The initial estrogenic phase is most often maintained using either daily oral estradiol 4–8mg or transdermal estrogen 0.2–0.4mg. The initial results of oocyte donation cycles were significantly better than typically seen after standard IVF. The apparent detriment of standard IVF to embryo implantation was felt to be secondary to the supraphysiological concentrations of estrogen attained after controlled ovarian hyperstimulation.61,62 Transdermal estrogen can adequately prepare 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 is 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.63 Others, however, have shown no detriment with high levels of estrogen.64 The length of estrogenic exposure can vary widely with little apparent clinical effect, again mimicking the variable follicular phase found in natural menstrual cycles. Anywhere

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Fig. 58.2 A representative scheme for cycle synchronization. Modified and reproduced with permission from ref. 21. from 6–38 days of prescribed estrogen prior to progesterone appears adequate.36,65,66 Most programs prescribe at least 14 days of estrogen prior to progesterone, but these 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 simple to achieve. The recipient begins estrogen several days prior to initiating 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 (for example, 1 mg leuprolide acetate daily until suppressed, then 0.5mg daily thereafter) in order to render them functionally agonadal. Alternatively, ovulating

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recipients can be 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.65 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.67 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 19.68 The dose of progesterone is typically 100mg IM daily or 100–600mg transvaginally daily. Some 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 levels are high probably because of the absence of the hepatic first pass effect on clearance. Like 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.69 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,70 while others have advanced this to the 5th week.71 Clinically, we begin weekly monitoring of serum progesterone concentrations 10 weeks after embryo transfer when a serum level of ≥30ng/ml typically is attained. At that point exogenous steroids are superfluous.

OBSTETRICAL OUTCOME Several groups have evaluated the obstetrical outcome of pregnancies following oocyte donation and concluded that results are favorable.28,72–75 Common to all reports, however, were increases in the incidence of pregnancy induced hypertension (PIH) and delivery by caesarian section. Soderstrom et al compared 51 oocyte donation deliveries to 97 IVF deliveries and noted a higher rate of PIH (31% v 14%) and caesarian section (57% v 37%) with oocyte donation.74 PIH was evaluated by the study of 72 pregnancies from donated gametes with age and parity matched controls.76 Pre-eclampsia was noted to be much higher in the donated gamete group (18.1% v 1.4%) suggesting an autoimmune component to the disorder. Two studies evaluated older oocyte donation patients and found most complications such as gestational diabetes and preterm labor were associated with multiple pregnancies.28,73 Another study showed that 59 children of oocyte donation aged 6 months to 4

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years had growth and development comparable to children from IVF and the general population.77 In summary, while oocyte donation pregnancies should be considered high risk, the complications are manageable and parents can reasonably expect healthy children.

FUTURE DIRECTIONS The next frontier in oocyte donation may include the use of anucleate 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.78 Improvements in oocyte freezing may soon permit “egg banks” to be set up, reducing the need to synchronize patients while allowing for quarantine.79,80 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. Non-surgical transfer of an in-vivo fertilised donated ovum to an infertility patient. Lancet (1983); 1:816–7. 4 Buster JE, Bustillo M, Thorneycroft IH, et al. Non-surgical 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, Findlay J, Wood C, Renou P. 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 1997 Assisted reproductive technology success rates. Atlanta, Georgia: US Department of Health and Human Services, CDC, 1999. 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.

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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 1998. San Francisco. California (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, Oehninger S, Robinson S, Rosenwaks Z. Follicle-stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome. Fertil Steril (1989); 51:651–4. 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, Gardiner AC, Blazar AS, Berk CA. 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.

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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 post-menopausal 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 Munne 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, Scott RT, Kreiner D, Rosenwaks Z. 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, Kirkland A, Thomas A, Studd JW. 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 maturation in women undergoing hormone replacement for ovum donation. Fertil Steril (1996); 66:380– 3. 42 Li TC, Dockery P, Ramsewak SS, Klentzeris L, Lenton EA, Cooke ID. 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.

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43 Weckstein LN, Jacobson A, Galen D, Hampton K, Hammel J. Lowdose aspirin for oocyte donation recipients with a thin endometrium: prospective, randomized study. Fertil Steril (1997); 68:927–30. 44 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. 45 Meldrum D. Female reproductive aging—ovarian and uterine factors. Fertil Steril (1993); 59:1–5. 46 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. 47 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. 48 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. 49 Marina S, Exposito R, Marina F, Nadal J, Masramon M, Verges A. Oocyte donor selection from 554 candidates. Hum Reprod (1999); 14:2770–6. 50 Feinman M, Barad D, Szigetvari I, Kaali SG. Availability of donated oocytes from an ambulatory sterilization program . J Reprod Med (1989); 34:441–3. 51 Gunning JH. Oocyte donation: the legislative framework in Western Europe. Hum Reprod (1998); 13(Suppl 2):98–104. 52 Sauer MV. Reproductive prohibition: restricting donor payment will lead to medical tourism. Hum Reprod (1997); 12:1844–5. 53 McEaughlin EA, Day J, Harrison S, Mitchell J, Prosser C, Hull M. Recruitment of gamete donors and payment of expenses. Hum Reprod (1998); 13:1130–2. 54 Sauer MV. Indecent proposal: $5,000 is not “reasonable compensation” for oocyte donors. Fertil Steril (1999); 71:7–10. (edit) 55 Annas GJ. The shadowlands—secrets, lies, and assisted reproduction. N Engl J Med (1998); 339:935–9. 56 Sauer MV, Paulson RJ, Lobo RA. Rare occurrence of ovarian hyperstimulation syndrome in oocyte donors. Int J Gynaecol Obstet (1996); 52:259–62. 57 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. 58 Cohen MA, Lindheim SR, Sauer MV. Donor age is paramount to success in oocyte donation [in process citation]. Hum Reprod (1999); 14:2755–8.

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59 Brown S. Genetic aspects of donor selection. In: Sauer MV, ed. Principles of oocyte and embryo donation, (SpringerVerlag: New York, 1998); 53–63. 60 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. 61 Edwards RG. Why are agonadal and post-amenorrhoeic women so fertile after oocyte donation? Hum Reprod (1992); 7:733–4. (edit) 62 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. 63 Krasnow JS, Lessey BA, Naus G, Hall LL, Guzick DS, Berga SL. Comparison of transdermal versus oral estradiol on endometrial receptivity. Fertil Steril (1996); 65:332–6. 64 de Ziegler D. Hormonal control of endometrial receptivity. Hum Reprod (1995); 10:4–7. 65 Serhal PF, Craft IL. Ovum donation—a simplified approach. Fertil Steril (1987); 48:265–9. 66 Younis JS, Mordel N, Ligovetzky G, Lewin A, Schenker JG, Laufer N. 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. 67 Navot D, Scott RT, Droesch K, Veeck LL, Liu HC, Rosenwaks Z. The window of embryo transfer and the efficiency of human conception in vitro. Fertil Steril (1991); 55:114–8. 68 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. 69 Lewin A, Benshushan A, Mezker E, Yanai N, Schenker JG, Goshen R. 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. 70 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. 71 Scott R, Navot D, Liu HC, Rosenwaks Z. A human in vivo model for the luteoplacental shift. Fertil Steril (1991); 56:481–4. 72 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. 73 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.

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74 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. 75 Abdalla HI, Billett A, Kan AK, et al. Obstetric outcome in 232 ovum donation pregnancies. Br J Obstet Gynaecol (1998); 105:332–7. 76 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. 77 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. 78 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. 79 Tucker MJ, Morton PC, Wright G, Sweitzer CL, Massey JB. Clinical application of human egg cryopreservation. Hum Reprod (1998); 13:3156–9. 80 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.

59 Gestational surrogacy Peter R Brinsden

OVERVIEW Surrogacy has been practised 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 practised 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”. The “genetic couple” in an IVF surrogacy arrangement therefore became the full genetic parents of the resulting child. “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 United States in 1985.2 IVF surrogacy is now accepted in the United Kingdom as a treatment option for infertile women, provided there are clearly defined medical indications. A report, commissioned by the British Medical Association (BMA) in 1990,3 provided the first evidence that surrogacy was formally accepted as a legitimate treatment option in the United Kingdom. Most other countries in Europe do not allow surrogacy. In 1984 the Warnock Committee4 recommended to the UK government that surrogacy should be prohibited. Opinions 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 19876 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

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further rejected,7 in spite of the 1985 resolution. The 1987 report made it clear that doctors “should not participate in any surrogacy arrangements.”7 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 counselling, and, very much as a last resort option, should IVF surrogacy be used as a treatment to overcome a couples infertility problem. In the same year, the Human Fertilisation and Embryology Act (1990)8 was passed through the UK Parliament and did not ban surrogacy. The most recent report of the BMA9 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 United Kingdom, 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 United States, where commercial surrogacy arrangements are allowed. There are still relatively few publications in the literature of experience with gestational surrogacy, and, in particular, there have been very few long term follow up studies of the babies or of the couples involved in surrogacy arrangements10–13 in spite of strong recommendations to do so.14,15 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. 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 formalised in 1990. The outcome of the treatment of 49 genetic couples treated at Bourn Hall since then is detailed later in this chapter, together with a review of the results from other clinics in the United States.

METHODS DEFINITIONS OF TERMS There has always been confusion among patients, practitioners and between different countries on the definition of the different forms of surrogacy. It is common practice to use the term “surrogate mother” or

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“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 and these embryos 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 59.1. Absence of an uterus after hysterectomy for uterine or cervical carcinoma, or after haemorrhage, 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 embryo transfer cycles. There are certain medical conditions that 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 who request it purely for career or social reasons are not considered for treatment. Other groups, primarily from the United States, which have reported series of patients treated by gestational surrogacy, have given similar indications.10,16

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Table 59.1. Indications for treatment by IVF surrogacy. After hysterectomy for cancer Congenital absence of the uterus Hysterectomy for post-partum hemorrhage Repeated failure of IVF treatment Recurrent abortion Hysterectomy for menorrhagia 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 practise, treatment by surrogacy accounts for less than 1% of the total annual throughput of cases, out of a total of about 1200 IVF and frozen embryo replacement cycles. It is practiced in only a limited number of the major IVF centers both in the United Kingdom and in the United States. SELECTION OF PATIENTS FOR TREATMENT All “genetic couples” are referred by general practitioners or gynaecologists 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 counselling 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 committee17 (Appendix 1), 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, a close friend or that they may be able to find a suitable host through one of the major patient infertility support groups in the United Kingdom, or through Childlessness Overcome Through Surrogacy (COTS), a support group set up to help couples seeking surrogate hosts and potential hosts seeking couples to help. Other groups have also reported using sisters,20 mothers,21 and support groups or other agencies.10,16 All groups practicing IVF 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.16,22 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

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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 counselled in depth. If this process is satisfactory and there are no obvious reasons why the arrangement should not be allowed to proceed from a medical and counselling 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, whose guidelines are given in Appendix 1 to this chapter. 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 Human Fertilisation and Embryology Authority18 drawn up by the HFEA 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 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; that they 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.” There are very many issues that must be discussed with both the genetic couple and the proposed host surrogate—these include the following.

• • • • • • • • • • •

FOR THE GENETIC COUPLES9: 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 control that the host should have over the child of the genetic couple both during the pregnancy and after 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

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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 HOST9: The full implications of undergoing treatment by IVF surrogacy The possibility of multiple pregnancy 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 caesarian 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 United Kingdom are expected to only claim “reasonable expenses”—commonly ranging between £7000 and £10000 ($10000–$15000) 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 it up. Another issue that is often raised in counseling is whether the genetic mother may be able to breast feed 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 breast feeding 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 breast feed then it is worth an attempt, but there is a strong possibility of disappointment.

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PATIENT MANAGEMENT MANAGEMENT OF THE GENETIC MOTHER Most “genetic mothers” treated in this clinic are fully assessed by their gynaecologist before referral. The workup 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 pre-menstrual symptoms or symptoms of ovulation. This can be confirmed by one or more estimations of serum follicle stimulating 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 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.23–25 In all treatment cycles, the embryos obtained from the genetic couple must be frozen for a six-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 six or more 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 six months before it can be used. MANAGEMENT OF THE SURROGATE HOST In the UK, the recruitment of a host surrogate must be carried out by the genetic couple themselves. Only normal fit women who, in our own practice, are 38 years of age or less and have had at least one child, are considered. The relationships between the surrogate hosts and the genetic mothers in our own series are shown in Table 59.2. The ethics committee

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has recommended (Appendix 1) 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.

Table 59.2. Relationship of genetic mothers to surrogate hosts. • Relations – Sister to sister – Sister to sister in law – Step daughter to stepmother • Friend to friend • Through an organisation (COTS) 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, 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. 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 later 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 LH-RH analogue regimen combined with 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.26,27 RESULTS 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.25,28 A more detailed breakdown of the outcome of our own series is given in Table 59.3. 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.29

35% 20% 5% 15% 25%

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In the original series reported by Utian et al10 they achieved a clinical pregnancy rate of 18% [7/59] per cycle initiated and 23% clinical pregnancy rate per embryo transfer.

Table 59.3. Summary of results of treatment by IVF surrogacy at Bourn Hall Clinic, 1990–1998. Treatment of genetic couples No. of patients who started treatment 49 Range=22–40 Mean age at start (years) 32.9 Total stimulated cycles 80 Range =1–5 Mean No. of oocytes recovered 10 Range=2–24 Mean No. of embryos frozen 5.4 Range=0–13 Treatment of host surrogates No. of hosts started treatment 53 No. of cycles to embryo transfer 87 Mean No. of embryos transferred 2.1 Final outcomes Mean no. of host transfers 1.6 Clinical pregnancies/surrogate host 31/53 58.5% Delivered/ongoing pregnancies/host transfer cycle 18/87 21% Delivered/ongoing pregnancies/surrogate host 18/53 34% Clinical pregnancies/genetic couple 31/49 63% Delivered/ongoing pregnancies/genetic couple 18/49 37% Other more recently reported series from the United States have shown ongoing or delivered pregnancy rates of 36% (172 of 484 surrogate hosts)30 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 less than 40 years of age.31 What has recently become apparent is that very little investigation of the immediate and long term outcome of the babies born as a result of gestational surrogacy has been carried out. However, Parkinson et al32 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 new borns 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 five times lower in the surrogate hosts than in the standard IVF patient controls. Apart from birth weights and

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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.12,13,33

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.3,14,15,34–37 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 is very few. In the past 10 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 following. 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. The question of whether it is ethical to pay hosts (and if so how much) has always caused concern. In the United States, payment is “up front” and revealed, whereas in the United Kingdom altruistic surrogacy is what everyone aspires to, but it is in effect impractical and payment is often hidden as “reasonable expenses”. Many will also consider it unethical not to pay hosts for the sacrifices that they make to help other couples. The long term effects on the children born as a result of gestational surrogacy are not known. The ASRM14 and others9 strongly recommend long term follow up. Bourn Hall is currently associated with two research projects aiming to follow up these children. 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

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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,29 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 post-hysterectomy cases, this reduced follicular response may be as a result of reduced vascular supply to the ovaries. • The follicular responses of women with the Rokitansky-Kuster-Hauser (RKH) syndrome were remarkably good. Four women with RKH syndrome underwent ten stimulation cycles in the program of Ben Raphael et al38 with HMG two to three ampoules 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 et al39 sent questionnaires to all treatment centres performing surrogacy procedures and asked them to follow up the frequency of congenital abnormalities among the progeny born to RKH syndrome women. Results of 162 IVF cycles produced 34 live born children, half of whom were female. No congenital anomalies were found among these women. 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 United Kingdom40 29 of the total of 113 licensed clinics perform or have performed surrogacy. In general, very few problems were reported, the most significant of which were as follows. There was one report of a surrogate who failed to surrender the baby after the birth but she 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.

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FUTURE DIRECTIONS AND CONTROVERSIES In the United Kingdom and the United States, 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 that 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.9 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.41 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 United States there is much greater acceptance of “gestational surrogacy”, especially now that it is superceding “natural surrogacy” as the treatment option of choice for most couples. In the surrogacy programme 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 19908 and also followed by the independent ethics committee to Bourn Hall (Appendix 1). 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 example, 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 be answered 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”42 and also the case of Smith vs Jones.43,44 In the “baby M case,” the final decision was that the genetic couple would have precedence for

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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.45 In the United States, 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 summarised by Schuster.36,37 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.44,46 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 the adequacy of existing law in this difficult area”.47 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; and • 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 1990.8 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”.47 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 team35 was presented to the UK Parliament and published in October 1998. The following is a summary of their recommendations. (1) 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. (2) Agencies involved in surrogacy arrangements should be registered by the UK Department of Health 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. (3) 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

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arrangements in the UK: To continue the current provision relating to non-enforceability of surrogacy contracts The continuation of current provisions prohibiting commercial agencies from assisting in the creation of surrogacy arrangements and prohibiting advertisements in relation to surrogacy New statutory provisions defining and limiting lawful payments to surrogate mothers Provision for promulgation of a code of practice governing surrogacy arrangements generally 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 To make new provisions for the granting of parental orders to commissioning couples. Parental orders should only be obtained in the High Court and judges should be able to order DNA tests and guardians ad litem 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 United Kingdom, the Channel Islands, or the Isle of Man for a period of 12 months immediately preceding the application for a parental order. At the time of writing, it seems likely that these recommendations by the surrogacy review team will be implemented in full, but there will be further debate. In our response to the original discussion document, we stated that we believed that the majority of clinicians and scientists providing treatment by IVF surrogacy were comfortable with the concept that surrogate hosts should not be paid, but that they should be fully reimbursed for all reasonable expenses incurred as the result of acting as a surrogate host. However, we said that we fully appreciated that pure altruism, although ideal, was unlikely to attract sufficient numbers of women who would be willing to act as surrogate hosts. If all payments were to be made illegal, it would almost certainly mean that treatment by natural surrogacy would cease in the United Kingdom, other perhaps than in sister to sister arrangements, or the treatment would “go underground”—a most undesirable outcome. Most couples and professionals involved in surrogacy believe that payment of a “reasonable fee”, in addition to expenses, and recompense for loss of earnings would be reasonable. However, what constitutes a “reasonable fee” is difficult to determine; it should recognize the very real commitment that a host makes when carrying and bearing a child for another woman. It would be impossible to prevent the covert payment of fees and it may therefore be better to acknowledge that, in an attempt to prevent exorbitant fees being demanded, payment of a fixed reasonable amount should be accepted. Treatment by “gestational surrogacy” is already fully regulated in the United Kingdom, since it can only be practised in centres licensed by the HFEA. This should be sufficient to ensure that proper clinical and

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scientific services, counseling and legal advice are provided to both commissioning couples and host surrogates. If “gestational surrogacy” is to be allowed to continue, which there is a general consensus that it should be,41 then the existing regulations are probably sufficient. “Natural surrogacy” is completely unregulated in the UK, and we believe should be brought under the control of a regulatory body. The major problems that have arisen with surrogacy arrangements have nearly all involved “natural surrogacy”. We believe therefore that all surrogacy arrangements should be regulated by a committee, probably under the control of the HFEA. RELIGIOUS ISSUES The attitudes of different religious denominations towards surrogacy are summarised in Table 59.4. Although the Catholic Church is strongly against all forms of assisted conception, the Anglican Church has become less rigid. The Jewish religion does not forbid the practice of surrogate motherhood in the case of full or gestational surrogacy. From the religious point of view, the child will belong to the father who gave the sperm and to the woman who gave birth.48 The Islamic view appears absolute and, in the same way that donor gametes are not permitted, so surrogacy is not allowed.

CONCLUSION In the 10 years that treatment by gestational surrogacy has been practised at Bourn Hall Clinic, we have shown that the treatment of young women without a uterus or for other clear indications, is successful, and relatively free of the complications associated with natural surrogacy. The practise of gestational surrogacy is almost entirely confined to the United Kingdom, where it can only be carried out in clinics licensed by the HFEA and to the United States, where there is less regulation, but where certain states have enacted legislation on the parentage of children born as the result of surrogacy arrangements. 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. 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 six 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

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required, both in the short and the long term, on all aspects of the treatment. We believe 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,

Table 59.4. Religious attitudes to surrogacy. • Christian view Not acceptable—Catholic or Anglican “Contrary to unity of marriage and dignity of the creation of the person…” • Jewish view Not forbidden “The child belongs to the father who gave the sperm” • Islamic view Not acceptable “Pregnancy should be the fruit of a legitimate marriage” “If a host did deliver, the child would be hers” • Buddhist view Not prohibited, but generally against, because of family ties and legal and moral reasons 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 10 years of our series 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 three years. Another more minor problem which we have 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 and approximately 40% of the pregnancies have aborted spontaneously,25,28 an outcome which obviously causes severe stress to both parties. The host feels guilt that she has lost the genetic couples 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.

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At Bourn Hall we believe that a limited IVF surrogacy service should be part of a comprehensive infertility treatment program that most larger centres 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 our policy of careful selection and screening of both genetic and host couples, together with independent counseling, we have shown that 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. ACKNOWLEDGEMENTS I would like to thank Reverend Dr Tim Appleton for his help and support as an independent counselor with the surrogacy programme at Bourn Hall over the past 10 years. 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.

APPENDIX 1 BOURN HALL ETHICS COMMITTEE GUIDELINES FOR SURROGACY INTRODUCTION Bourn Hall Ethics Committee is prepared to consider IVF surrogacy in cases where an embryo or embryos from the commissioning couple are transferred to the uterus of the host. The use of donor eggs or donor sperm and natural surrogacy may be considered in exceptional circumstances. They consider that surrogacy should only be undertaken as a last resort. The need to safeguard the welfare of any children born as a result of surrogacy arrangements will be a guiding principle. The Committee considers that every case must be looked at by the Ethics Committee on its own merit, based on information provided by the Clinic. PROCEDURES Following examination by a clinician, the prospective genetic parents and host and partner must be counselled by a professional counsellor. If the clinician and counsellor, who are not members of the Ethics Committee, are satisfied they will prepare a report, a copy of which must be submitted to each member of the Ethics Committee. The case will then be considered by the Ethics Committee in consultation with the clinician and counsellor. If they are satisfied that the case falls within the Guidelines

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and is acceptable, the Ethics Committee will make their recommendations to the Clinic. The genetic parents and host and her partner will be asked to take independent legal advice and encouraged to take out insurance. Cases will not be considered if there is any doubt that the genetic couple will comply with the requirements for a parental order under section 30 of Human Fertilisation and Embryology Act 1990 or subsequent legislation. CATEGORIES ACCEPTABLE FOR TREATMENT 1. Total or partial absence of the uterus either of congenital origin or after surgery. 2. Repeated miscarriage. 3. Multiple failure of infertility treatment. The clinicians must be satisfied that, there is no reasonable prospect of success in the future. MOTIVES CONSIDERED UNACCEPTABLE 1. Social reasons. 2. Prospective genetic parents with severe health problems. Clinicians and the Committee will need to be satisfied that the strain of bringing up a child might not damage the mother’s health so seriously as to jeopardise the welfare of that child and the family.

1. 2. 3. 4. 5.

6. 7. 8.

CONSIDERATIONS WHICH APPLY TO ALL CASES The Clinic must not be involved in initiating or making arrangements between genetic and host couples. The relationship between genetic couple and host must be carefully considered and avoid creating conflicting family relationships. Independent counselling must be available to both genetic and host couples. HIV, hepatitis B, and hepatitis C antibody tests are required of both genetic and host couples. The age of the genetic mother and of the host is important. In view of the HFEA Code of Practice, the Committee considers that 35 should be the maximum age of the genetic mother unless there are exceptional circumstances; however, the Committee will consider genetic mothers up to and including 38. The host should generally be below 40. The principal motive of a prospective host should always be to help an infertile couple. A prospective host should have had at least one child before becoming a surrogate. The commissioning couple in a surrogacy arrangement should be married. The host should preferably be in a stable relationship. If the host is single then she should be adequately supported.

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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. BMA Publications, 1990. 4 Report of the Committee of Inquiry into Human Fertilisation and Embryology. London. Her Majesty’s Stationery Office: London, 1984. 5 British Medical Association. Annual Representative Meeting Report, 1985. 6 British Medical Association. Surrogate motherhood. Report of the Board of Science and Education. BMA Publications, 1987. 7 British Medical Association. Annual Representative Meeting Report, 1987. 8 Human Fertilisation and Embryology Act 1990. Her Majesty’s Stationery Office, London, 1990. 9 British Medical Association Report. Changing Conceptions of Motherhood. The Practice of Surrogacy in Britain. British Medical Association Publications: London, 1996. 10 Utian WF, Goldfarb JM, Kiwi R, et al. Preliminary experience with in vitro fertilization-surrogate gestational pregnancy. Fertil Steril (1989); 52:633–8. 11 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. 12 Fisher S and Gillman I. Surrogate motherhood: attachment, attitudes, and social support. Psychiatry (1991); 54:13–20. 13 Blyth E. Interviews with surrogate mothers in Britain. J Reprod Infert Psychology (1994); 12:189–98. 14 Ethics Committee of the American Fertility Society. Ethical considerations in the new reproductive technologies. Fertil Steril (1986); 46 (suppl.1):62–8. 15 American College of Obstetricians and Gynecologists. Committee on Ethics: Ethical issues in surrogate motherhood. Washington DC: American College of Obstetricians and Gynecologists, 1990. 16 Sheean LA, Goldfarb JM, Kiwi R, Utian WH. In vitro fertilisation (IVF)—surrogacy: Application of IVF to women without functional uteri. J Invitro Fert Embryo Trans (1989); 6:134–7. 17 Ethics Committee to Bourn Hall Clinic. Guidelines for treatment by in vitro fertilisation surrogacy at Bourn Hall Clinic. Personal Communication, 1999. (Appendix 1).

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18 Code of Practice for Clinics Licensed by the Human Fertilisation and Embryology Authority. Human Fertilisation and Embryology Authority: London, 1996. 19 Surrogacy Arrangements Act 1985. Her Majesty’s Stationery Office, London. 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 Trans (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 Trans (1988); 5:31–4. 22 Ethics Committee of the American Fertility Society. Surrogate gestational mothers: women who gestate a genetically unrelated embryo. Fertil Steril (1990); 53:64S-7S. 23 Marcus SF, Brinsden PR, Macnamee MC, Rainsbury PA, Elder KT, Edwards RG. Comparative trial between an ultrashort and long protocol of luteinising hormone-releasing hormone agonist for ovarian stimulation in invitro fertilization. Hum Reprod (1993); 8:238–43. 24 Macnamee MC and Brinsden PR. Superovulation Strategies in Assisted Conception. In: Brinsden PR, ed. A Textbook of In Vitro Fertilization and Assisted Reproduction. Carnforth and New York: Parthenon Publishing, 1999; 91–101. 25 Brinsden PR. IVF Surrogacy. In: Brinsden PR, ed. A Textbook of in vitro Fertilization and Assisted Reproduction. Carnforth and New York: Parthenon Publishing, 1999; 361–8. 26 Sathanandan M, Macnamee M, Rainsbury P, Wick K, Brinsden P, Edwards R. Frozen-thawed embryo replacement in artificial and natural cycles; a prospective study. Hum Reprod (1991); 5:1025–8. 27 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. 28 Brinsden PR, Appleton TC, Murray E, Hussein M, Akagbosu F, Marcus SF. Treatment by in vitro fertilisation with surrogacy—the experience of a single centre in the United Kingdom. Br Med J (2000); 320:924–8. 29 Meniru GI, Craft IL. Experience with gestational surrogacy as a treatment for sterility resulting from hysterectomy. Hum Reprod (1997); 12(1):51–4. 30 Batzofin J, Nelson J , Wilcox J, Potter D, Rogoff R, Norbryhn G, Hatkoff C, Feinman M. Gestational surrogacy: Is it time to include it as part of ART? ASRM 1999 Programme Supplement; P-017 (Abst). 31 Corson SL, Kelly M, Braverman A, English ME. Gestational carrier pregnancy, Fertil Steril (1998); 69:670–4 32 Parkinson J, Tran C, Tan T, Nelson J, Batzofin J, Serafini P. Peri-natal outcome after in vitro fertilizationsurrogacy. Hum Reprod (1999); 14(3):671–6.

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33 Van den Akker OBA. Organisational selection and assessment of women entering a surrogacy agreement in the UK. Hum Reprod (1999); 14(1):262–6. 34 Cohen B, Friend TL, Legal and ethical implications of surrogacy mother contracts. Clin Perinatal (1987); 14:281–92. 35 Brazier M, Golombok S and Campbell A. Surrogacy: Review for the UK health minister of current arrangements for payments and regulation. Report of the review team. London: Department of Health, 1998. 36 Shuster E. Non-genetic surrogacy: No cure but problems for infertility? Hum Reprod (1991); 6(8):1176–80. 37 Shuster E. When genes determine motherhood: Problems in gestational surrogacy. Hum Reprod (1992); 7(7):1029–33. 38 Ben-Raphael Z, Barr-Hava I, Levy T, Orvieto R. Simplifying ovulation induction for surrogacy in women with Mayer-Rokitansky-KusterHauser syndrome. Hum Reprod (1998); 13(6):1470–1. 39 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. 40 Balen AH, Hadyn CA. British Fertility Society Survey of all Licensed Clinics that perform surrogacy in the UK. Hum Fert (1998); 1:6–9. 41 Bromham DR. Surrogacy: the evolution of opinion. Br J Hosp Med (1992); 47(10):767–72. 42 Rothenberg KH, Baby M. The surrogacy contract, and the healthcare professional: unanswered questions. Law Med Healthcare (1988); 16:113–20. 43 Andrews LB. The stork market: the law of the new reproductive technologies. Am Bar Assoc J (1984); 78:50–6. 44 Annas G. Using genes to define motherhood: the California solution. N Engl J Med (1992); 326:417–20. 45 Smith v Jones. Los Angeles Superior Court, Los Angeles County. 9 June 1987. No CF 025653. 46 Oxman RB. California’s experiment in surrogacy. Lancet (1993); i:341:1468–9. 47 Surrogacy. Review for the UK health ministers of current arrangements for payments and regulation. Consultation document. Department of Health, London, 1997. 48 Schenker JG. Infertility evaluation and treatment according to Jewish Law. Em J Obstet Gynaecol Reprod Biol (1997); 71(2):113–21.

60 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 welldocumented in the literature. It is considered an emotionally difficult experience impacting all aspects of a couple or 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 well-being, 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 etiological 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 and 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 resiliency, the impact of reproductive medical treatments, and the efficacy of therapeutic psychological interventions.

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STRESS AND ART Assisted reproductive technology, while opening up expanded opportunities for the treatment of infertility, has generated its own psychological challenges for patients. For most couples, assisted reproductive technology (ART) is the last, best option for having a child and occurs 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, dependant, and anxious. Although emotionally depleted, couples are attracted to a technology which offers hope where, a few years ago, none existed. They find themselves drawn into new emotional turbulence of contrasting feelings of hope and despair which seems 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 success5,6,7 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 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 to 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 GENDER DIFFERENCES AND ART STRESS The majority of studies on stress during ART are of women and, overall, women react more intensely to infertility and ART than men.24 Prior to IVF, women report more anxiety, 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,

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invasive procedures for oocyte retrieval, and embryo transfer—is frequently given by woman as a cause of psychological distress.9 If treatment fails, depression persists longer for women than their partners, lasting up to six 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 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 early studies on 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 such informal activities as talking to spouse, family and friends. In terms of the effects of coping on post IVF treatment, Hynes et al found that women who used problem focused coping had a higher level of wellbeing 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 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

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contrasting approach describes stress as neither an event nor a response, but rather a combination of factors: the perceived meaning of the event and a self appraisal of the adequacy of coping resources.29 Thus, it is not the stress itself but the perception of the stress which determines how ART patients experience and handle it. The aspects of ART that are perceived stressful to patients are multifaceted and affect all parts of their life: marital, social, physical, emotional, financial, 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 the marital relationship with an emotionally laden experience and, by removing the conjugal act for procreation, sexual intimacy is lost. Couples, also, 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, 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 discovering that the intervention had not been successful, with the last cycle being as stressful as the first.12 Within a treatment cycle, patients view IVF/ART as a series of stages which must be successfully completed before moving onto the next phase of treatment: monitoring, oocyte retrieval, fertilization, embryo transfer, waiting period, and pregnancy test stages. The level of stress, anxiety, and anticipation raises 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 had 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.

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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 technological advances in ART and the recognition of the psychosocial issues and demands facing infertile patients, mental health professionals have become an increasingly important member 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 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, influences a patient’s perception of care and, in turn, his or her stress level. Sensitivity, warmth, patience, and responsiveness create an environment of support. Also, general clinic routine and ambience reflect support and respect of patients when it is provided in an efficient, organized, clean, uncrowded, and aesthetically 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.”

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TYPES OF ART SUPPORT SERVICES

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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 will be described, a detailed explanation of methodology will not be addressed.4 Moving from specific to general, the method of providing patient support services can be categorized as: psychological assessment and evaluation; therapeutic counseling; supportive counseling; information and education; and clinic administration. PSYCHOLOGICAL ASSESSMENT AND EVALUATION Psychological screening of participants using ART varies from program to program, as there are currently no laws or regulations in any country requiring psychological evaluation prior to 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 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 Not withstanding 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 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.

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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: requiring all anonymous recipients of donor eggs, sperm, 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 by a consulting psychologist. 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 both the donor/carrier and infertile patient. (If a gestational carrier is being used by genetic recipient parents, psychological testing of the genetic parents is not required.) A clinical interview is held with the donor 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. required of IVF participants when the physician is concerned about psychological vulnerability, marital instability or if a situation is presented to our internal ethics committee which warrants psychological assessment before a decision about treatment can be made. Our mental health professional staff follows 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 health care providers “to do no harm”.

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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 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 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.38 Guidelines for when to refer patients for psychological assessment and intervention are displayed in Table 60.1.

Table 60.1. When to refer to an infertility counselor. Situations The following situations serve as guidelines for referral for psychological assessment and 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

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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 SUPPORTIVE COUNSELING

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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: 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, general infertility patients (non-ART), and pregnancy after infertility. These groups are open ended, no cost to patients, and run by a staff infertility counselor and, if needed, a nurse. 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 requests for contact are situations where

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patients have undergone selective reduction or patients have carried multiple pregnancies. • giving each patient current information about local and national infertility support groups (such as RESOLVE, Inc.), such as monthly updates on meetings, support groups, living room sessions, telephone counseling, newsletters, and articles. INFORMATION AND EDUCATION

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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 which 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: monthly IVF 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. This class is held in the evening, a light dinner is provided, written materials on the medical and emotional aspects of IVF 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 members, and internet websites about clinic and community support services on infertility, endometriosis, premature ovarian failure, polycystic ovarian disease, adoption, pregnancy, 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

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shown that patients only retain 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 on adoption to medical diagnosis and treatment of infertility. CLINIC ADMINISTRATION

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•

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 aesthetically pleasing, clean, well maintained office staffed by friendly, professionally dressed, well trained 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 included: 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 gynaecology department, sensitivity needs to be considered and reasonable efforts made to separate pregnant patients and small children from infertility patients by adjusting appointment/schedules and/or seating arrangements.) private rooms where nurses or other clinical staff can instruct or consult with patients. private sections where billing and scheduling issues can be discussed by administrative staff with patients in a confidential manner. a quiet, secure “donor room” for men to give semen samples, with pornographic magazines, video player, and a comfortable chair or bed. 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. soothing, calming background music piped throughout the office. an annual or biannual “baby party” for patients to come back with their children and celebrate with staff. miscarriage/pregnancy loss cards sent by the clinical staff when it is learnt that, after a patient has been discharged from care, an ART pregnancy has been lost. primary care nursing, where a patient is assigned to one nurse, facilitating better continuity and coordination of treatment. 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. patient surveys, suggestion boxes, and written feedback, which encourage open communication regarding satisfaction, thoughts on improving care or services, and constructive criticism. 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. staff support offering confidential assistance, direction, and referral for personal

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problems and professional burnout, by the staff mental health professional or through an employee assistance program (EAP). After all, happy staff members are productive workers, and 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, if it were offered during treatment.19,42,43 While a minority of patients will 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 on patient satisfaction suggest that many patients are dissatisfied with support services offered with their IVF centers.43–46 This information, coupled with the high dropout rates in ART programs most likely due to psychological reasons,47 suggest that IVF programs need to provide better and more comprehensive psychosocial support services. 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. However, the more holistically a patient is handled—supported medically and emotionally—the more likely she/he is to be treatment compliant and satisfied for 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 60.2 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 who have

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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 who undergo it need as much support psychologically as

Table 60.2. Strategies for 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

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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 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.

REFERENCES 1 Menning BE. The emotional needs of infertile couples. Fertil Steril (1980); 34:313–9. 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, Dunkel-Schetter C, eds. Infertility: perspectives from stress and coping research. New York: Plenum Press (1991): 3–16. 4 Burns LH and Covington SN. Psychology of infertility. In: Burns LH, Covington SN, eds. Infertility counseling: a comprehensive handbook for clinician, New York: Parthenon Publishing (1999); 3–25. 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.

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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 women presenting for infertility evaluation. Fertil Steril (1989); 52:425–32. 17 Edelmann RJ, Connolly KJ, Cooke ID, Robson J. Psychogenetic infertility: some findings. J Psychosom Obstet Gynecol (1991); 12:163–8. 18 Stanton AL, Danoff-Burg S. Selected issues in women’s reproductive health: Psychological perspectives. In: Stanton AL, Gallant SJ, eds. The psychology of women’s health: progress and challenges in research and application. Washington DC: American Psychological Association (1996): 261–305. 19 Boivin J. Is there too much emphasis on psycholosocial counselling for infertile patients? J Assist Reprod Genet (1997); 14:184–6. 20 Freeman EW, Rickels K, Tausig J, et al. Emotional and psychosocial factors in follow-up of women after IVF-ET treatment. Acta Obstet Gynecol Scand (1987); 66:517–21. 21 Newton CR, Hearn MT, Yuzpe AA. Psychological assessment and follow-up after in vitro fertilization: assessing the impact of failure. Fertil Steril (1990); 54:879–86. 22 Berg BJ, Wilson JF. Psychological functioning across stages of treatment for infertility. J Behav Med (1991); 14:11–26. 23 Lalos A, Lalos O, von Schoultz B. The psychosocial impact of infertility two years after completed surgical treatment. Acta Obstet Gynecol Scand (1985); 65:599–604. 24 Newton CR, Sherrard W, Glavac I. The Fertility Problem Inventory: measuring perceived infertility-related stress. Fertil Steril (1999); 72:54–62.

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25 Boivin J, Shoog-Svanberg A, Andersson L, et al. Distress level in men undergoing intractoplasmic sperm injection versus in-vitro fertilization. Hum Reprod (1998); 13:1403–6. 26 Nachtigall RD, Becker G, Wozny M. The effects of gender-specific diagnosis on men’s and women’s response to infertility. Fertil Steril (1992); 57:113–21. 27 Hynes GJ, Callan VJ, Terry DJ, et al. The psychological well-being of infertile women after a failed IVF attempt: the effects of coping. Br J Med Psychol (1992); 65:269–78. 28 Stanton AL, Burns LH. Behavioral medicine approaches to infertility counseling. In: Burns LH, Covington SN, eds. Infertility counseling: a comprehensive handbook for clinicians. New York: Parthenon Publishing (1999): 129–47. 29 Cohen SJ, Kessler RC, Underwood GL. Strategies for measuring stress in studies of psychiatric and physical disorders. In: Cohen SJ, Kessler RC, Underwood GL, eds. Measuring stress: a guide for health and social scientist. New York: Oxford University Press (1995): 3–25. 30 Boivin J, Takefman J. Stress level across stages of in vitro fertilization in subsequently pregnant and nonpregnant women. Fertil Steril, (1995); 64:802–10. 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 ed. 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. Guidelines for gamete and embryo donation. Fertil Steril (1988); S3,70, 1S-13S. 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. Integrating infertility counseling into clinical practice. In: Burns LH, Covington SN, eds. Infertility counseling: a comprehensive handbook for clinicians, New York: Parthenon Publishing (1999): 475–89.

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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 Vitro Fert Embryo Transfer (1989); 6:242–56. 44 Sabourin S, Wright J, Duchesne C, Belisle S. Are consumers of modern fertility treatments satisfied? Fertil Steril (1991); 56:1984– 1090. 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.

61 Worldwide legislation Jean Cohen, Howard W Jones, Jr

INTRODUCTION The development of methods of assisted reproductive technology (ART) has been a major breakthrough in the treatment of infertile couples. This area of assisted reproductive technology interfaces with fundamental issues of life for many people. One of the basic human rights is that of a woman to be able to decide when and how to conceive. Do scientists or doctors have to be corralled by legislations or regulations? One would imagine that as for infertility surgery or medical treatment, ART decisions would be the result of a particular colloquy between women or couples and doctors. But in many countries, ART has produced the desire for control and regulation of this form of infertility treatment. These regulations induce important implications for patients. Such regulations remain open to questioning or discussion since there are not even two countries where regulations are similar. During the first 10 years after Louise Brown was born, there was no legislation in any country. This made possible much progress, such as embryo freezing, coculture, hatching, and so forth. Nowadays, there are several countries which operate under voluntary guideline systems without problems. The fear to see Man act as God in a field concerning the creation of life has led a certain number of societies to wish to control and regulate this form of treatment of infertility. In addition the newspapers’ headlines concerning clonage, selection of sex, posthumous use of human sperm, surrogacy, and errors in laboratories, prompted authorities to take action. In many countries, as we shall see, the legislator has defined what is allowed and what is forbidden, and has as well adjusted the structures of control in order to guarantee a quality of care and a protection for couples and children to be. Many doctors and biologists have called for these regulations in order to protect themselves from contingent suing. Many couples—having associations of patients acting for them—have also collaborated to the elaboration of those laws which protect them as well. However, everybody knows that no law or regulation can stop unscrupulous people from doing unscrupulous things, whether it is in the

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field of medicine, journalism, business, or education. But everyone feels reassured by regulations. The result of this evolution on the international level is the existence of multiple attitudes. Certain countries have no regulation. Others have voluntary guidelines that doctors follow. Others have laws voted by their legislative authority. Legislative regulations are nevertheless often unrealistic and are different from one country to another. No two countries have adopted the same regulations. In fact, as far as ART is concerned, the decisions and the choices depend on the country in which one lives and practices; and we know of paradoxical situations which have generated “procreative tourism”. DIVERGENT LAWS AND PRACTICES Many authors1–5 have tried to restate the elements of assisted reproduction practice in different countries whether it concerns laws (statutes or judge mode), guidelines decided by ethical committees, scientific societies, or even voluntary guidelines accepted by all the ART centers. All of these are extremely diverging, and we shall try to describe them looking for who decides. It is impossible in such a short chapter to study the situation of each country. We shall quote a few of them. The International Federation of Fertility Societies (IFFS) has elaborated an international statement, which is the most recent and the most comprehensive on the subject to this day.6 The tables and the information contained in this chapter come from the document IFFS Surveillance 98, of which we are general editors. We wish to thank all the participants quoted in the document as well as the ASRM for having given us the authorization to reproduce the contents. We have updated or corrected a few data of this original document, but we cannot assume that we have eliminated every mistake or reported fully on all the new developments.

LEGISLATION AND GUIDELINES The techniques of surveillance fall into three categories. (1) Those sovereign nations or political entities with legislative (government) regulations that are mandatory and (or) statutory; (2) Those sovereign nations or political at entities with guidelines intended to be followed voluntarily by those practicing ART; and (3) Those sovereign nations or political at entities without either guidelines or regulations, although some of them are considering/implementing legislation.

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(1) NATIONS OR STATES WITH REGULATIONS There are at least 22 such bodies (Table 61.1). Not even two of them have similar regulations. No country can be cited as a regulatory model, but the UK Human Fertilisation and Embryology Act of 1990 may be considered as remarkably effective. In France, for example, the law was voted in in 1994 and is still not completely applicable. In Australia about half of the states are subject to legislative control and half are free of it. In countries where laws exist, doctors know exactly what they are allowed to perform and in what conditions. Theoretically drifts are evaded but at the price of formalities and rules which limit the possibilities of treatment and research. Patients are protected from unethical acts but lose confidentiality. (2) VOLUNTARY GUIDELINES There are at least 12 such entities. The guidelines are provided by a medical society, or by the ministry of health, for example. It is difficult to document the degree to which guidelines are followed. There is abundant anecdotal evidence to suggest that there may be widespread violation in many countries. In this case, patients are not protected by the law: they must sue the doctors or the centers if they consider that they have got a prejudice. The advantage of guidelines is that they may be modified easily according to the evolution of technics. (3) COUNTRIES WITHOUT GUIDELINES OR REGULATIONS There are at least six such nations. However, some of them have some kind of medical accreditation for the ART laboratories. The conclusion of the IFFS participants in Surveillance 98 was as follows: Surveillance of ART is best accomplished by the adoption of rules and procedures by legislative action, namely, the adoption of regulations. The creation of a licensing agency and the issuance of a license for various procedures of ART, both clinical and laboratory, after fulfillment of requirements seems to work well. Because of the inertia of the

Table 61.1. Legislation and guidelines. Country Legislation Argentina Australia (West) +

Guidelines +

Neither

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Australia (South) Australia (Victoria) Australia (Remainder) Austria Belgium Brazil Canada* Czech Republic Denmark Egypt* Finland France Germany Greece Hong Kong Hungary India Ireland Israel Italy Japan Jordan Korea Mexico the Netherlands Norway Poland Portugal Saudi Arabia Singapore South Africa Spain Sweden Switzerland Taiwan Turkey United Kingdom United States of America

1185

+ + + + + + + + + +

+ +††

+ + + + + + + + + + + + + + + + + + + + + + + + + + +

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*See text. †Not official. ‡Some guidelines exist from the national ethics committee. legislative process, it is recommended that any bill to adopt regulations in the clinical or laboratory area or to revise them contain a strong provision for informed medical and scientific input to assure medical and scientific accuracy of the regulations and that the bill also provide a simple mechanism for the update of the regulations in order to conform to medical and scientific developments and to social changes. States now operating under regulations might well consider legislation to facilitate prompt modifications of regulations in order to accommodate medical, scientific, and social needs. The licensing body should be truly independent of governments and should be publicly accountable. The regulatory framework proposed should allow the maximum responsible expression of creativity for doctors and scientists and of responsible choice and self determination for patients. Inherent in the licensing concept is the possibility to suspend or withdraw a license to an individual or a program for violation of any rule or procedure promulgated by the licensing authority.

MARITAL STATUS IN ART Most legislations or guidelines in the various countries have recommended that ART should be restricted to heterosexual couples, legally married or at least leaving in a stable relationship (Table 61.2). In France a cohabitation of 2 years is required. Some countries like Israel, Finland, Spain and the UK permit its applications to single women.

MICROMANIPULATION In most of the countries, micro-insemination is allowed under the guidelines or laws or is used without any special regulation. In no country is the technique clearly not allowed

Table 61.2. Marital status in ART. Country Legislation Guidelines Couple restrictions Argentina + No requirement (heterosexual couples) Australia (West) + Marriage, stable relationship Australia (South) + No requirement

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Australia (Victoria) Australia (Remainder) Austria Belgium Brazil Canada Czech Republic Denmark Egypt Finland France Germany Greece Hong Kong Hungary India Ireland Israel Italy Japan Jordan Korea Mexico the Netherlands Norway Poland Portugal Saudi Arabia Singapore South Africa Spain Sweden Switzerland Taiwan Turkey United Kingdom

+ + + + + + +

+

+ +

+ + + + + + + + + + + + + + + + + + + + +

1187

Stable relationship Stable relationship Stable relationship No requirement Marriage, stable relationship No requirement Stable relationship No requirement, excludes lesbians Marriage Stable relationship, single woman Marriage, stable relationship (heterosexual couples) Stable relationship Marriage Marriage Marriage, stable relationship Marriage Marriage Marriage, stable relationship No requirement Marriage Marriage Marriage Marriage, stable relationship No requirement Stable relationship Stable relationship Stable relationship Marriage Marriage No requirement No requirement, women >18 yrs Stable relationship Stable relationship Marriage Marriage No requirement

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United States of America

+

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Stable relationship

except in the Netherlands, where Microsurgical Epididymal Sperm Aspiration (MESA) and Testicular Sperm Extraction (TESE) are not allowed. It is the same situation concerning assisted hatching. Nuclear or cytoplasmic transfers are usually considered as pre-embryo research and follow the rules concerning embryo research in the country. In most of the countries, there are guidelines, statutes or practices designed to prevent transmission of diseases in assisted reproductive technology. The list of disease differ in different countries. It includes: hepatitis B and C, sexually transmitted diseases such as chlamydia, gonorrhea, syphilis, infectious diseases such as cytomegalovirus, toxoplasma, rubella, herpes, hereditary and genetically transmitted diseases such as cystic fibrosis, Tay-Sachs disease, and hemoglobinopathies. In three of these countries, there are guidelines to prevent transmission of diseases, but no list is given. In 10 countries, there are no guidelines or statutes to prevent transmission of diseases. However, some of the centers use their own list for prevention of transmission of diseases (Table 61.3).

Table 61.3. Are there guidelines, statutes, or practices designed to prevent transmission of infectious disease in any aspect of assisted reproductive technology? Country Yes Argentina + Australia (West) + Australia (South) + Australia (Victoria) + Australia (Remainder) + Austria Belgium + Brazil + Canada + Czech Republic + Denmark + Egypt + Finland + France + Germany + Greece +

No

+

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Hong Kong Hungary India Ireland Israel Italy Japan Jordan Korea Mexico the Netherlands Norway Country Poland Portugal Saudi Arabia Singapore South Africa Spain Sweden Switzerland Taiwan Turkey UK USA

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+ + + + + + + + + + + + Yes

No +

+ + + + + + + + + + + THE NUMBER OF EMBRYOS TO TRANSFER

The prevention of multiple pregnancies is the aim of limiting the number of embryos to transfer. There turns out to be a great variety of legislative and guideline attempts to achieve this goal. A comprehensive comparative study of the results of these various attempts would be very helpful, but does not seem to exist. The situation has developed very quickly in many countries over a few years. For example, Swedish centers have adopted the transfer of two embryos (even one) without any legislation on the subject. In the United States, the Americal Society of Reproductive Medicine has recently modified its recommendations in favor of fewer embryos transferred. In 1998, IFFS Surveillance indicated that among the 23 entities with legislation, there are nine entities which limit the number of embryos to be

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transferred, the number varying from two to a maximum of four. Regulations are often rather specific and allow no exceptions. The penalties for a violation are not trivial. For instance, a clinic’s license to practice might be withdrawn (UK), and imprisonment and a fine of a least SF 100,000 (Switzerland), loss of the license probably (South Australia), imprisonment and a fine (Germany), and loss of license (Sweden). Interestingly enough, 14 of the nations operating with legislation do no indicate a limit to the number to be transferred. Six of the 10 guideline countries do not have any specification with their number to transfer. None of the seven sovereign states operating without legislation or guidelines, of course, has a limit in the nature of things. Nevertheless, it is interesting that the respondents of all seven countries indicated that it was customary in their countries to transfer two or three, although in the case of one country (Canada), it was stated that in certain circumstances up to six were transferred (Table 61.4). In ART, the most efficient solution is the elective transfer of two embryos or the elective transfer of two (even one) blastocysts. An efficient cryopreservation program is essential for supernumerary embryos in order to obtain the same pregnancy rate by adding transfers of fresh and frozen embryos to avoid high multiples. Such a policy has shown its efficiency in Sweden, for example. The transfer of two embryos would avoid the economic cost of multiple gestation which is by itself superior to the cost of treatment cycles. International surveys show that these rules are not spontaneously accepted, mainly because comparisons between centres are based on the bottom line pregnancy rate. Perhaps a better indicator of programme success could be the implantation rate, or total pregnancy rate including fresh and frozen transfers a projection that can be made according to the probability of pregnancy with frozen embryos in the clinic.

Table 61.4. ART—The number to transfer. Country Legislation Guidelines Neither Transfer limit Unlimited Argentina + + Australia (West) + + Australia (South) + 3 Australia (Victoria) + + Australia (Remainder) + 2(3) Austria + + Belgium + +(2–4+) Brazil + 4 Canada + +(2–4) Czech Republic + +(4) Denmark + 2–3

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Egypt + + Finland + +(2–3) France + + Germany + 3 Greece + +(6) Hong Kong + +(5) Hungary + 3–4 India + +(4–5) Ireland + +(3) Israel + + Italy + 3–4 Japan + 3–4 Jordan + + Korea + + Mexico + + the Netherlands + +(2–3) Norway + + Poland + 2–3 Portugal + +(3–5) Saudi Arabia + 4(3) Singapore + 3 South Africa + + Spain + + Sweden + 2–3 Switzerland + 3 Taiwan + + Turkey + + UK + 3 USA + +* Number in ( ) indicates customary number transferred *Transfer no more embryos than would lead to a more than 2% triplet rate CRYOPRESERVATION The cryopreservation of human gametes and embryos enables the yield of embryos from each pick-up to be maximized by storing embryos judged to be capable of implantation, and it may soon assist in the preimplantation diagnosis of genetic disease.

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Cryopreservation of supernumerary embryos has allowed patients undergoing in vitro fertilization to have the opportunity to achieve pregnancies from more than one embryo transfer without having to be subjected to controlled ovarian hyperstimulation and oocyte retrieval each time (Table 61.5). (1) Among the countries with statutory regulations (national legislation or other entity governing the use of ART) are Austria, Brazil, Czech Republic, Denmark, France, Germany, Hungary, Israel, Mexico, the Netherlands, Norway, Saudi Arabia, South Africa, Spain, Sweden, Switzerland, Taiwan, Turkey, and the UK. Embryo cryopreservation including pronuclear stage oocyte is allowed in all of the above, though in some countries it is necessary to meet certain requirements based on informed consent (for example, in France the informed consent is valid for 5 years, and both partners of the couple have to contact the center every year). In five there is no limit to the duration of the storage of cryopreserved prezygotes/embryos: Germany, Czech Republic, Saudi Arabia, the Netherlands, and South Africa. In the remaining countries, the limit of the duration of the storage varies between 1 and 5 years. Most countries establish a 5 year limit and in two cases (Hungary and the UK) the maximum duration of the storage allowed is 10 years. In all cases there are possibilities of modifications according to the circumstances. The statute allows for oocyte cryopreservation in many countries, namely: Brazil, Germany, France, Denmark, Czech Republic, Saudi Arabia, Austria, Israel, Hungary, Taiwan, Switzerland, and Sweden. Oocyte cryopreservation is forbidden by statute in Spain and Norway and it is not mentioned in the UK, the Netherlands, Mexico, Turkey, and South Africa. Regarding the time limit for the duration of the storage, seven out of 12 countries do not mention a limit, three establish a limit between 1 and 5 years, three do not practice it, and the limit is unclear in one case (Taiwan). Statutes allow sperm cryopreservation in most countries, and it is not mentioned only in the case of Turkey. In 60% there is no limit to the length of the storage. The limit to the duration of the storage varies very much in the rest of the countries: it is 1 year in Austria, 5 years in Switzerland and Spain, 10 years in Taiwan, until the age of 55 years in the UK, and it is not mentioned in two cases. (2) Among the countries that operate under guidelines by voluntary, religious or other organizations are Ireland, Egypt, Jordan, Australia, Argentina, Korea, Japan, Italy, USA, Singapore, and Poland. Embryo cryopreservation including pronuclear stage oocytes, is allowed in all countries, many of which establish in their informed consent certain requirements. In half of the countries, there is no limit to the duration of the storage: Ireland, Egypt, Jordan, Italy, and Poland. In Australia there is a limit or no limit depending on the decision of an ethics committee. No limit is mentioned in the case of Korea. Four countries have established a limit to the duration of the storage: Argentina, Japan, USA, and Singapore. The duration of the limits varies very much: between one and five years, reproductive life of the women, reproductive life of the donor,

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and between 5 and 10 years, respectively. According to the guidelines, oocyte cryopreservation is allowed in 80% of countries: Egypt, Australia, Argentina, Japan, Italy, Korea, USA, and Poland. In Australia, even though oocyte cryopreservation is allowed, the authorization for the embryo transfer is unclear. Oocyte cryopreservation is not allowed in Singapore, and it is not mentioned in the remaining two countries (Ireland, Jordan).

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There is no limit as regards the duration of the storage in five countries. There are three countries which have a time limit: until the death of the women in Egypt and Poland, and until the end of the reproductive life in Japan. Sperm cryopreservation is allowed in most countries, and it is not mentioned in the case of Japan. There is no time limit regarding the duration of the storage of frozen sperm in seven cases, while in Egypt and Poland the limit to the storage is the death of the

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patient. (3) Some countries have no statutes or voluntary guidelines governing the use of ART. Among those countries are: Greece, Finland, Canada, India, China, and Belgium. Embryo cryopreservation is used in 85.7% of countries. There is no consensus as to the duration of the storage in four out of the seven countries, namely: Greece, Canada, China and Belgium—even though in Canada several centers use 5 years. In the remaining countries such as Finland and India, the limit is 5 years. Oocyte cryopreservation is not used in any of these countries for clinical purposes. Sperm cryopreservation is used in seven countries. There is no limit as to the length of storage observed in most countries, except for India where they report a 2 year limit.

PRE-IMPLANTATION GENETIC DIAGNOSIS (PGD) Over the past 10 years, PGD has become established as an alternative to conventional prenatal diagnosis for both chromosomal and single gene defects.7 By the end of 1997 about 1200 cycles have been reported worldwide, resulting in over 160 pregnancies, approaching a pregnancy rate of 26%. The procedure is not without error. PGD was practiced in half of the nations surveyed, but the number of countries and centers involved is increasing every day (Table 61.6).

Table 61.6. Pre-implantation genetic diagnosis. Country Legislation Guidelines Argentina − + Australia (Remainder) − + Australia (South) + − Australia (Victoria) + − Australia (West) + − Austria + − Belgium − − Brazil + − Canada − − Czech Republic + − Denmark + − Egypt − + Finland − − France + −

Practiced −, not mentioned +, allowed +, allowed +, not mentioned −, not allowed −, not mentioned + +, allowed +/− −, allowed +, allowed1 +, allowed − +, allowed

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Germany − Greece − Hong Kong − Hungary + Country Legislation India − Ireland − Israel + Italy − Japan − Jordan − Korea − Mexico + the Netherlands + Norway + Poland − Portugal − Saudi Arabia + Singapore − South Africa + Spain + Sweden + Switzerland + Taiwan − Turkey + United Kingdom + 1 In restricted conditions

1196

− − − − Guidelines − − − + + + + + − − + + − + − − − − − − −

− − − −, allowed Practiced −, allowed −, not allowed +, allowed +, not mentioned −, not mentioned +, allowed +, not mentioned +, not allowed +, allowed −, allowed −, not mentioned − +, not mentioned +, not mentioned +, not mentioned +, allowed +, allowed − − −, not mentioned +, allowed

EMBRYO RESEARCH AND CLONING There is a need for research involving the human embryo to improve the success rate of IVF, to learn more about factors affecting implantation, to learn how to avoid fetal chromosomal abnormalities, and to discover more about maternal, paternal, and environmental factors that may affect early embryonic development. Animal models may sometimes permit the avoidance of human embryo research, but they are of limited value since there are so many interspecies differences concerning basic reproductive physiology. Animal studies are

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not sufficient for the proper understanding of human embryology. The ultimate aim of the research is to improve human reproduction. Many countries are unclear and have difficulty in formulating national approaches in these areas (Table 61.7). Embryo research may be allowed by law and/or controlled by local or national Ethics Committees, acting under national guidelines, or forbidden by law. Research may be licenced to an individual center or restricted by government control of research funding. In most of the countries where there is legislation, embryo research is forbidden. The UK is the major exception and may be considered as an example: the research license on human embryos (14 days) may be granted by the Human Fertilisation and Embryology Authority (HFEA) if the use of embryos is essential for the research intended for one or more of the following. • Promotion of advances in the treatment of infertility • Increase of knowledge about the causes of congenital diseases

Table 61.7. Embryo research. Country Legislation Argentina nil Australia (Remainder)

nil

Australia (West) Australia (South) Australia (Victoria)

yes

Guidelines yes, for therapeutic purposes. No specific approval yes, by NM&MRC Federal Dept of Health. Overview by Institutional Ethics Committee yes, NH&MRC

yes

yes, NH&MRC

Austria Belgium

Brazil Canada

Czech

yes. Forbidden, even on unfit yes, NH&MRC for transfer embryos. Q/A may be defined as Research yes. Forbidden nil use of MRC Warnock Committee Guidelines. Institutional Ethics Committee yes nil local Ethics Committee. Medical Research Council of Canada. No funding after 14 days yes Central Ethics Committee of

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Republic Denmark Egypt

yes, revised every 2 yrs nil

Finland

nil

France Germany Greece Hong Kong

yes. Reviewed 5 yearly. Forbidden yes. Forbidden nil nil

Hungary India Ireland Israel Italy

yes no no yes nil

Japan

nil

Jordan

yes

Korea

yes

Mexico the Netherlands Norway Poland Portugal Saudi Arabia Singapore

yes yes yes yes no yes yes

South Africa Spain

yes yes

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Czech Min of Health Central Ethics Committee Bioethics in Human Reproduction Research in Muslim World, Local EC, Therapeutic Research Voluntary Guidelines, Local Ethics Committee

no guidelines yes, Institutional Ethics Committee yes no not acceptable not acceptable yes (proposed by Italian Fertility Society) yes, by Japanese OB/Gyn Society Research registered no specific approval of research required ART Committee of Korean Association of O&G. Research not culturally acceptable guidelines legislation under construction not acceptable not acceptable guidelines not acceptable Ministry of Health guidelines approval MRC guidelines yes, licence required on viable embryos

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Table 61.7. Embryo research. Country Legislation Guidelines Sweden yes yes. Local Ethics Committee Switzerland yes yes Taiwan yes not acceptable Turkey yes no guidelines UK yes acceptable, but research submitted to HFEA USA yes ASRM Local Institutional Review Board. Federal Funding restrictions • Increase of knowledge about the causes of miscarriage • Development of more effective techniques of contraception • Development of methods for detecting the presence of gene or chromosomal abnormalities in embryos before implantation There are two kinds of cloning (Table 61.8):

Table 61.8. Human reproductive cloning. Country Legislation Guidelines Argentina + (decreet) + Australia (Remainder) − + Australia (South) + − Australia (Victoria) + − Australia (West) + − Austria + − Belgium − − Brazil + − Canada − + Czech Republic − + Denmark + − Egypt − + Finland − − France + − Germany + − Greece − − Hong Kong − − Hungary + − India − − Ireland − −

Practiced −, not mentioned −, not allowed −, not allowed −, not allowed −, not allowed −, not allowed − −, allowed − −, not allowed −, not allowed −, not allowed − −, not allowed −, not allowed − − −, not allowed − −, not allowed

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Israel Italy Japan Jordan Korea Country Mexico the Netherlands Norway Poland Portugal Saudi Arabia Singapore South Africa Spain Sweden Switzerland Taiwan Turkey United Kingdom USA

− − − − − Legislation + + + − − + − + + + + + + + −

+ + + + + Guidelines − − − + − − + − − − − − − − +

1200

−, not allowed −, not mentioned −, not allowed −, not allowed Practiced −, not allowed −, not mentioned −, not allowed −, not allowed − −, not allowed −, not mentioned −, not allowed −, not allowed −, not allowed −, not allowed −, not allowed −, not mentioned −, not allowed −, not mentioned

(1) The reproductive cloning: cloning a human being by somatic cell nuclear transfer, for example, would require a consenting person as a source of DNA, eggs to be enucleated and then fused with the DNA, a woman who would carry and deliver the child, and a person to raise the child. It should be clear that a cloned human may have the same appearance as his predecessor but not necessarily his nature, mental ability, character, or capacity of achievement. This form of cloning does not achieve the normal biological goal of a shared genetic message in reproduction. In no country is human reproductive cloning practiced. At present several countries have set legislation that prohibit its application. In other countries it is forbidden by guidelines. The American Society of Reproductive Medicine (ASRM) and the European Society for Human Reproduction and Embryology (ESHRE) are also against the encouragement of this procedure. (2) The cellular cloning could allow: • the understanding of mechanisms of genetic diseases; • the better production of transgenic animals; and • a clone of the cells of an individual to provide a source of stem cells or tissue for therapeutic benefit of the original donor. Animal cloning as well as non-reproductive cloning with human cells are already regulated by international ethical guidelines as those of the American Congress, European Commission, WHO, FIGO, and ESHRE.

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EMBRYO REDUCTION The frequency of multiple gestation has increased as a result of the widespread use of gonadotrophins and ART. The overall multiple pregnancy rate in ART is different between countries about 20–25% of all pregnancies are twins. Triplets and higher order rates are 4–7%. There is an increase of maternal complications together with higher perinatal morbidity and mortality. Embryo reduction has been established as an efficient and safe way to improve outcome of multiple pregnancies, especially those with four or more fetuses and, those carrying three or more fetuses. As the experience from the procedure continuously increases, it seems that reduction of triplets to twins can be offered to women with satisfactory results. Reduction of twins or higher gestations to singletons has not yet been established and it is applied only when medical reasons indicate (Table 61.9).

Table 61.9. Embryo reduction. Country Legislation1 Guidelines Practiced (−/+) Argentina − + −, not mentioned Australia (West) + + +, not mentioned Australia (South) + + +, not mentioned Australia (Victoria) − + do not know, not mentioned Australia (Remainder) − + +, not mentioned Austria + − +, not mentioned Belgium − − + Brazil − − +, not allowed Canada − − + Czech Republic + − +, allowed Denmark − − +, allowed Egypt − + +, allowed Finland − − + France − − +, not mentioned Germany + − +, not mentioned Greece − − + Hong Kong − − − Hungary + − +, allowed India − + + Ireland − + −, not mentioned Israel + + +, allowed Italy − + +, not mentioned

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Japan − + Jordan − + Korea − + Mexico + − the Netherlands − − Norway + − Poland − + Portugal − + Saudi Arabia + − Singapore − + South Africa − + Spain − + Sweden − − Switzerland + − Taiwan − − Turkey + − UK + − USA − + 1 Specifically concerning embryo reduction

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+, not allowed +, allowed +, not mentioned −, not allowed +, not mentioned −, not allowed +, allowed + +, not mentioned not known +, not mentioned + +, not mentioned +, not mentioned +, not mentioned +, not mentioned +, allowed +, not allowed

The procedure carries a risk of total loss of the pregnancy (around 10% depending on technics and authors). Long term follow up of the children from continuing sacs remains to be done, but there is a certain risk of premature births. Some women who have undergone embryo reduction have become psychologically disturbed. Pre-reduction psychological evaluation is important. Usually, the countries which allow legal abortion or therapeutic abortion are the ones where embryo reduction is practiced. In order to prevent the need for embryo reduction, the number of embryos transferred should be limited.

DONATION OF GAMETES AND EMBRYOS Donation of gametes may be considered in cases of absolute male or female sterility, as, for example, in azoospermia of testicular failure or premature menopause, respectively. It is also chosen by some parents as a way of avoiding the transmission of serious genetic disease. Thus both men and women are concerned, and in the rare cases where both partners in a couple are struck by irremediable infertility, embryo donation represents their only possibility to bear a child.

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Legislation is in place concerning gametes and embryo donation in a majority of countries, and guidelines in others. In several, a non-profit making transaction for the donors is required (for example, France, Spain, to be implemented in the UK). Identical guidelines are recommended by the American Society of Reproductive Medicine (ASRM) even though it is not the predominant practice in the USA. The consequences of paying gametes donors are twofold: this may coerce a potential donor who would not give free informed consent; this may also have an effect on the potential child, who would be seen more as a product than as a subject (Table 61.10). Another major area is consensus about screening of gamete donors in order to protect the recipients and prospective child, sometimes included in the regulations (France, UK), or guidelines (USA). Centers practising donation in ART may be working for instance under license (UK), or approved (France), or the procedure can be performed only by physicians (Germany). In France, gamete donation is only possible through licensed centers. It must be anonymous, free of charge, and respect quarantine for sexually transmittable diseases. The consequences of no systematic screening of donors are the dangers this represents both to recipients (particularly with HIV, or hepatitis B and C) and the future child. Worldwide, there are conflicting positions concerning those who oppose the rights and interests of the gamete donors to stay anonymous and the right or interest of the offspring to know one’s origins. A salient example is the 1985 law in Sweden, with mandatory information to the offspring of the donor’s identity, and the French example, where the more than 20 years old Centre d’Etude et de Conservation du Sperme (CECOS) tradition of anonymous gift from a fertile couple to a sterile couple was enshrined in legislation in 1994. We have no hard evidence of the consequences of either policy, although the majority of couples still prefer anonymity, while secrecy is becoming less common. There is also discrepancy about potential recipients’ eligibility (see “welfare of the child”), with France restricting treatment to married couples or those in a 2 year relationship. In Spain, however, all women have access to donor insemination (Dl). Finally there is a contract in several countries between the availability and legality of sperm donation, and the forbidding of oocyte donation (for example, Austria, Germany, Norway). The consequences of allowing one but not the other is the reinforcement of sexual discrimination. The banning of embryo donation may imply that some embryos will eventually be destroyed rather than achieving their potential of a pregnancy. The arguments against embryo donation are connected with its consequences for children and society. The arguments in favor are that it is preferable to adoption. In all countries where embryo donation is

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practised, informed consent is obtained both from the gamete donors and from the recipient patients.

Table 61.10. Donation of gametes and embryos. Country sperm oocyte embryo donation donation donation Argentina yes yes yes Australia yes yes yes (Remainder) Australia (W) yes yes Australia (S) Austria yes, not IVF no no Belgium

yes

yes

Brazil Canada

yes yes

yes yes

Czech Republic Denmark Egypt Finland France Germany Greece Hong Kong Hungary India Italy Ireland Israel

yes

yes

yes no yes yes yes yes yes yes yes yes yes, not IVF yes

yes no yes yes prohibited yes yes yes yes yes no yes, IVF patients no no yes yes yes

Japan Jordan Korea Mexico the Netherlands

yes, not IVF no yes yes yes

center specific yes no no yes prohibited yes yes yes

Donation/welfare no, couples yes, paramount married; >5 years paramount married; psycho/social tests depending on centers

stable no lesbians N/A yes implied (couples) no law

care till major

no no no no

no no stable

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Norway Poland Portugal Saudi Arabia Singapore South Africa Spain Sweden

yes, not IVF yes yes no

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prohibited no yes yes

no

no no no yes yes yes yes yes yes yes yes no 6 offspring yes (not IVF) prohibited no access to identity of written donor consent Switzerland yes, forbidden yes prohibited by Basel and Glarus Taiwan yes yes yes one live birth Turkey no no no UK yes yes yes yes, max 10 births USA yes yes yes Column 5 contains remarks that specifically pertain to the welfare of the child in the context of donation. In the table, the characters are bold where there is legislation; and bold and italic where there are guidelines. IVF SURROGACY IVF surrogacy is defined as the transfer of an embryo conceived by the gametes provided by both parties of the parenting couple into the uterus of another woman, who after the delivery of the baby is obliged to return it to the couple. This type of surrogacy is practiced in less than half of the countries studied, which reveals the difficulty of the international community in approaching this assisted method of reproduction. In Brazil and Hungary it is allowed only if a relative is willing to undergo this procedure. In Europe it is practised according to legislation only on two countries: UK and Israel. The rest of the nations studied either prohibit (IVF law) or simply do not use surrogacy, mainly as a result of cultural or religious attitudes (Table 61.11). The payment of surrogates raises special concern. This is the main concern for many jurisdictions.

Table 61.11. IVF surrogacy. Country Legislation Argentina −

Guidelines Practiced (−/+) − +

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Australia (Remainder) Australia (South) Australia (Victoria) Australia (West) Austria Belgium Brazil Canada Czech Republic Denmark Egypt Finland France Germany Greece Hong Kong Hungary India Ireland Israel Italy Japan Jordan Korea Mexico the Netherlands Norway Poland Portugal Country Saudi Arabia Singapore South Africa Spain Sweden Switzerland Taiwan Turkey

− + + + + − + − + + − − + + − − + − − + − − − − + + + − − Legislation + − + + + + + +

+ − − − − − − + − − + − − − − − − − + − + + + + − − − + + Guidelines − + − − − − − −

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+, allowed −, not allowed +, allowed −, not allowed −, not allowed + +, allowed +, allowed −, not allowed −, not allowed −, not allowed + −, not allowed −, not allowed + − +, allowed + −, not allowed +, allowed −, not allowed −, not allowed −, not allowed not mentioned +, not allowed +, allowed −, not allowed −, not allowed − Practiced (−/+) −, not allowed −, not allowed +, allowed −, not allowed −, not allowed −, not allowed −, not allowed −, not allowed

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United Kingdom USA

+ −

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− +

+, allowed +, allowed

WELFARE OF THE CHILD It must be pointed out that there is no really good definition of “welfare of the child,” even in the two countries where it is mentioned: considered “paramount” (Australia) and “taken into consideration” (UK). It is the most complex issue to assess scientifically and medically because of its large psychosocial component (Table 61.12).

Table 61.12. Does custom impose on the IVF program any consideration for the welfare of any resulting offspring? Country Regulation/Guidelines on welfare Argentina bill Australia NSW/T/NT/C man+woman Australia (W) no harm/Reprod Tech Act 91 Australia (S) fit proper person Austria anonymous right origins 14 Belgium no Brazil 1358/92 Canada stable Czech Republic <18 >45, married, stable Denmark stable relationship (3 years) Egypt yes, marriage Finland practice Nordic countries Code France married or 2 years, bisexual, alive Germany not posthumous, socially stable Greece no Hong Kong married/stable Hungary married/stable India law Italy Ireland no Israel posthumous 1 year only Country Regulation/Guidelines on welfare Japan Obstet/Gyn Society, married couple Jordan marriage

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Korea guidelines Mexico bill the Netherlands Norway Poland stable Portugal draft bill, stable couples Saudi Arabia Singapore married South Africa stable Spain good mental and physical health Sweden stable; physician responsibility Switzerland Dl, marriage, no other Taiwan married Turkey UK yes USA In the table, the characters are bold where there is legislation; and bold and italic where there are guidelines. In the UK, the statutory HFEA’s code of practice speaks of a “the importance of a stable and supportive environment for any child produced as a result of treatment.” It also enjoins to take “all reasonable steps to ascertain who would be legally responsible for any child as a result of the procedure and who it is intended will be bringing up the child.” Finally it lists the factors to “bear in mind” when taking into account the welfare of the child: “Commitment, age, medical histories, ability to meet the need of child or children, any risk to the child, including that of inherited disorders, and the effect on any existing child of the family”. The second column in the table thus describes and lists factors that, even if not specifically stated, may be surmised to be relative to the interest or welfare of the child (for example, parental “reproductive” age in French law). The requirement of marriage or stability in a couple is included in several European codes and legislation with a view to enhance the welfare of the child and his/her chances of appropriate care. What is illogical is the time considered necessary before qualifying for treatment between the 2 groups (for example, France), as it especially penalises women over the age of 35. The consideration of the welfare of the child in such circumstances may be assessed when obtaining consent implying a dual undertaking of responsibility towards the resulting child, legally symbolised by filiation.

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In most countries a child conceived by artificial insemination with donor sperm (AID) or sperm donation with the consent of the mother’s husband is treated under the law for all purposes as the legitimate child of the husband.

CONCLUSIONS In many countries ART has produced the desire for control and regulation of this form of infertility treatment. These regulations induce important implications for doctors, biologists, and patients. Such regulations remain open to questioning or discussion since there are not even two countries where regulations are similar. These different regulations lead to a “procreative tourism” as, for example, in Europe, where couples travel to a nearby country in order to accomplish their wish. These different medical protocols lead to different chances and risks for the couples as for example multiple pregnancies because of different number of embryos replaced. These different laws prevent in certain countries the development of fundamental research which is a prejudice to the whole human community. Because of all these reasons, medical and biological communities acting in ART should express minimum threshold of regulations. These common international guidelines, whether technical or ethical, should express for each given situation the minimal aims, means and risks as well as individual and social costs. Each individual and each government would then be able to make up its own mind and decide according to its own imperatives.

REFERENCES 1 Baird PA. The role of society in assisted reproduction in IVF and ART. Proceedings of the 10th World Congress on IVF. Gomel V, Leung P eds. Monduzzi Editore (1997); 1119–26. 2 Cohen J. Regulations of assisted reproductive technologies international comparisons in IVF and ART. Proceedings of the 10th World Congress on IVF. Gomel V, Leung P eds. Monduzzi Editore (1997); 1131–6. 3 Jones, Jr, HW. The time has come. Fertil Steril (1996); 65:1090. 4 Jones, Jr, HW. The many faces of morality. In: Frontiers in Endocrinology: perspectives on assisted reproduction. Proceedings of VIIIth World Congress of In Vitro Fertilization (1994), Tokyo. 5 Schenker J. Assisted reproductive practice in Europe: legal and ethical aspects. Hum Reprod Update (1997); 3,2:173–84.

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6 Jones H, Cohen J, eds. IFFS Surveillance 98. Fertil Steril (1999); 71:5:suppl 2. 7 Handyside A. PGDA ten year perspective. In: Jansen R, Mortimer D. Towards reproductive certainty. Parthenon Publishing Group (1999); 389.

62 Times of transition: modern ethical dilemmas Françoise Shenfield

In a time of transition from one century to another, and when the first born in vitro fertilization (IVF) child has reached what used to be the age of majority, we face a doubly symbolic appraisal of the dilemmas of assisted reproduction. Long since has the age of legal majority been lowered from 21 to 18 in many European countries and assisted reproductive technologies (ART) been integrated in the framework of our family structuring and our societies. There are numerous ethical dilemmas which cannot even be thoroughly discussed in books solely dedicated to the subject/ and the choice made below must per force be eclectic and reflect current concerns. However, one must always start at the beginning, and before explaining the choice made in this chapter, the introduction must mention the embryo. The necessity of embryo research and its implications for the status of the entity embryo are under less challenge than in the past decade, at a time when the therapeutic potential of ES cells gives further arguments to its necessity in a consequentialist fashion. Symbolically, in France where the legislation allowed embryo observation but not research unless for the benefit of the embryo with the consequence of the impossibility to apply the law, the Conseil d’Etat is about to make public a report that advises a reform allowing research at the time where the law is due to be revised. Other dilemmas will only be alluded to. They relate to the use in general of gamete donation, to the source of these gametes, and the way donors are recruited.2 Currently, the theme of identity, the social place of our children in a constantly changing (mostly) west European framework seems to be acutely relevant, and this is for two reasons. At a time when cloning has been in the news for over three years, the possible total upheaval of the relational links of generations which reproductive cloning would create and which some psychologists have compared to the taboo around incest must be considered. But also, in the context of gamete donation, current concern about the secrecy and anonymity3 of the process must be reflected upon. This will not be replaced, only displaced, and also enriched by the current acute concerns linked to more recent achievements of preimplantation genetic diagnosis (PGD) and cryopreservation of

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reproductive tissues. Thus these topics have been chosen to represent the modern ethical dilemmas of our times of transition. But the first issue raised is one of justice, a meta-ethical and societal concern, with many grave consequences. There is no doubt that this question should take precedent as it colors many other problems in the real world and in ART. Can our patients be offered totally autonomous choice when 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 attempts they can afford, especially at IVF? Are we really benevolent to our patient when we present success rates inflated by these multiple pregnancies, and can we ignore the price that the children of ART will pay in terms of morbidity, and their family in terms of stress?

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 criteria selection used by national legislation or local codes of practice (age, marriage, etc…), and the economic factors, whether funded or not 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’ population. Thus, striking differences between countries of similar populations and economic wealth are worth mentioning, within the context of a British report4 which estimated a need of 400 cycles per million population. For instance, France, the UK, Germany and Canada perform 489, 408, 228 cycles and 107 per 1000000 population respectively.5 Of note is the fact that two wealthy countries like the USA and Switzerland perform 104 and 133 cycles per 100000 respectively, whereas in Israel the astonishing level of 2000/100000 population is achieved. Whatever the precision of the method of calculation, France has proportionally the most IVF cycles in Europe, and, with a population practically similar in number to that of the UK, outnumbers access by about 17% more cycles. It is unlikely that this discrepancy should be linked to different needs of the patient population. It is more likely that it stems from the greater ease of access in France, where patients may have a minimum of four IVF cycles subsidised by their national health system. However, in the UK, access is either totally free through the NHS, or at maximum private cost, and this in respective proportion of 20/80%, compounded by unequal access between areas, with wide variations among regions responsible for their budget, which use heterogeneous criteria to select patients. Furthermore, infertility treatments are not

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reimbursed by private insurance. To this day, the UK still lacks a political will in order to systematically improve national 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, lack of political will is not the sole reason to the disparity of access between countries, as the proportion of cycles performed in Germany is about half that of France, with a theoretically equivalent system: sociocultural factors with a restrictive legislation on the embryo and the diktat of insurance companies, which generally pay 70% of costs, probably both play a part in this state of affairs. Furthermore the consequences of the inequity of access are grave as this has a part to play in the multiple pregnancy rate6 of IVF. Patients may be tempted to “maximize” the only attempt at IVF they can afford, and 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.7 Solutions may lie in both research in order to select the embryo most likely to implant and the way results are reported. For instance the report of “success” rate could be put in terms of likelihood of clinical pregnancy per cycle started, including the replacement of frozen thawed embryo transfers. This may diminish the replacement of multiple embryos in order to have a higher success rate per cycle, an inevitable consequence of the reporting methodology. The prevention of multiple pregnancies with their untoward consequences, both for the parents and for the potential children, has become a matter of grave concern to fertility specialists and patients alike.

RECENT ADVANCES AND THEIR ETHICAL IMPLICATIONS PGD The availability of multiple embryos created in vitro is essential in order to perform PGD for the couples in need. PGD may be described as the ultimate step in antenatal screening, retrogressing from the fetus in utero to the embryo in vitro. As far as PGD itself is concerned, it may be appropriate to introduce the term of pregravid diagnosis, as indeed the mother to be is not pregnant until (a) fertilized embryo(s) (after PGD or not) has/have been replaced and has implanted in utero following the necessary IVF. General dilemmas are not dissimilar to those of antenatal screening, and the UNESCO bioethics committee (International Bioethics

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Committee, or IBC) addressed similar concerns in its report on genetic screening and testing8: underlined were the problems of the limits of the technique (such as accuracy and quality control), those concerning information, privacy of the 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 (TOPs) 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. The IBC also stated that there must be careful monitoring of screening programmes, 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.9 In the IBC’s terms, heed must be paid to the gravity of the disorder (which is diagnosed or screened for), the age of onset of the disease, the availability of treatment, and the boundaries to be chosen. The “promotion of informed reproductive decisions”10 is indeed a caring and sensitive terminology to qualify this pregravid diagnosis, a deed sometimes branded as “genetic cleansing.” Let us not forget, however, that the danger at societal level is that those opting out will be portrayed as irresponsible and stigmatised. Needless to say counselling is of great importance in all these decisions.11 What is the most awe inspiring denominator of this complex equation? 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 entails recording the births and follow up of the children. Thus follow specific dilemmas, 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 psychological consequences of this intrusion for the children.12 The problem of confidentiality with regard to the child seems sometimes insoluble as it entails a parental, if not sometimes a state, decision, as is the case in the cessation of anonymity in gamete donation in Sweden. The eugenics debate must also be mentioned, as it has been argued that some couples would demand, after pre-implantation diagnosis, the assurance of a “perfect” baby.13 Actually, in practice, couples say they want a “normal” rather than a “perfect” baby.14 But is the basic philosophy of preconception and pre-implantation 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 PGD15 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. Furthermore, some writers would actually wish to find new terms rather than “eugenics” in view of the tainted historical background

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of the field, and in particular the radical eugenics movement of the beginning of the 20th century.16 One may also 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. The argument that sex selection is likely to reinforce sexist attitudes already too prevalent in most societies must be the most powerful one against whimsical sex selection.17 It thus seems clear that the unique advantage of PGD lies in 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 an alternative.18 This may also apply to a new technique, the cryopreservation of reproductive tissues for possible future use, further illustrating the difficulties at the interface between research and therapy, and of consenting on behalf of others. CRYOPRESERVATION OF GAMETES, EMBRYOS, AND REPRODUCTIVE TISSUES 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 counselling and consent of the man preserving his sperm in the UK,19 in French law, “assisted reproduction treatments are solely to be used for a couple’s parenting project,” and “the man and woman which constitute this couple, of a reproductive age, must be alive and give consent”.20 At the time where legislation was not yet in place, a refusal by the French Tribunal de Grande Instance to transfer two cryopreserved zygotes to a widow21 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. Furthermore, this happened when the French CCNE22 had proposed in their avis that such embryos would be available to a widow after about nine months of reflection to allow the immediate grief reaction to be worked through. Interestingly, the CCNE also recommended that this would not be allowed for cryopreserved sperm after the death of the man who had stored it before a terminal illness. The members of the CCNE made the point that the intention to become parents was necessarily stronger at the stage of embryos than at the stage of cryopreserved gametes, and thus deserved more respect from society at large by way of legislation. French legislators eventually decided otherwise. By contrast, in the UK posthumous treatment is allowed with explicit prior consent in writing and after the opportunity of counselling has been given to the gamete donor(s). After the Blood case23—where no consent

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had been available at the time of sperm retrieval due to the terminal illness of Mr Blood—a report24 published after a 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. 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 outcome 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 till 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 psychological nature. Although the burden of the disease process is often reflected in the compliance problems 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 United Kingdom 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 non-maleficent 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 his/her parents. It has been estimated that by 2000, one in 1000 adults will be a survivor of childhood cancer.25 It is of note that male children are particularly affected by alkylating agents and testicular irradiation, whereas female children who have radiation therapy to the abdomen have decreased fertility and an increased risk of adverse pregnancy outcome in terms of early miscarriages or preterm birth resulting from uterine

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dysfunction.26 Furthermore, the more recent possibility of preserving the reproductive outlook for women was followed with intense media exposure after the presentation of the first successful follicular development in autografted previously cryopreserved ovarian tissue.27 Other concerns over the years have been the possibility of damage to the offspring directly linked to cryopreservation itself. No extra risk has been observed in the children of adults treated for cancer (usually Hodgkin’s disease or testicular cancer) when they resulted from the use of such cryopreserved gametes, as well as post radiation/chemotherapy treatment.28 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. The technical difficulties concerning the cryopreservation of immature and mature oocytes have stimulated the development of the storage of ovarian tissue. For the female child/adolescent, the risk linked to the obtaining of gametes and tissues is of yet a different kind owing to the necessity of a laparoscopy to obtain the sample of ovarian tissue, which itself has to be of a fairly large size. In a large British survey, the risk of laparoscopy was estimated at 1:10000 for mortality and 4:10000 for significant morbidity. Thus research has developed in the field of in vitro maturation (IVM) of immature oocytes, as well as in vivo maturation in nude mice. Another alternative is the very topical freezing of ovarian tissue, with the possibility of later autografting when the young woman has recovered and wishes to procreate.29 However, there is at least a theoretical risk of transmitting malignant cells in autograft slices of ovaries.30 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, whereas for therapeutic research the proxy must be satisfied that on a reasonable assessment of the risk/benefit ratio of the procedure, it is in the best interest of the child to participate.31 Legal problems of consent for under age children fall within the remit of Gillick competence, jurisprudence which sets a precedent in English law. The child must be mature and understand the nature of the proposed treatment in order to give valid consent, which does not need to be validated by the parents. Complications do, however, exist with dissent or refusal to treatment by the child which, although the logical opposite of consent and subject to the same criteria of capacity and understanding for an adult, can be overridden by parents who may consent to the refused treatment on behalf of their underage child. 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 tissues. Thus while the treatment of males—or the repair, in the psychoanalytical sense of the couple’s infertility—by artificial insemination of frozen thawed sperm is current

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practice, it may be argued that cryopreservation of prepubertal testicular and ovarian 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 non-therapeutic research. The definition of therapeutic research is one which would benefit the patient, which is the case in our dilemma. But this is usually understood as a more or less immediate benefit for a current condition and not for the possible applications in the future as a consequence of current treatment for a disease (infertility) that is not life threatening. The duty of care of staff involved is first to the child. What if the autonomy of the child and of the parents, caring adults, conflict, as they may, for example, when the parents are keen for their child to undergo a procedure, implying either actual treatment of the present condition, or in order to protect future fertility, and the child refuses? In a recent case in London involving the imposition of HIV testing on a neonate against the parents’ wishes, the argument of best interest was used. Whatever the legal context, however, it may be very difficult to impose sperm preservation and ovarian cryopreservation in the face of the child’s or adolescent’s refusal. It may therefore be argued that the only acceptable solution would be the enrolment of patients in a prospective multicentre study. Meanwhile, while it is still difficult to freeze oocytes and there is no certainty that ovarian tissue may be used to obtain gametes or as an autograft, adult women or parents have enquired whether oocytes could be stimulated 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.32 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.33 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, including the need for a father, as put in the Human Fertilisation and Embryology Authority (HFEA) code of practice.34 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 he or she is in a couple, provisions must be made to deal with the outcome of frozen gametes or tissues. Might the parents have any access to the gametes which represent the only life potential of a dead child, in a time where 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 counselling and written consent, but the concept of procreation totally outside a couple and decided by future grandparents

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rather than parental figures does not seem to be compatible with the account to be taken of the welfare of the child as required both by the Human Fertilisation and Embryology (HFE) Act 1990 and the spirit of the Children Act 1989 which places emphasis on parental responsibility rather than parental rights. The Code of Practice of the HFEA lists factors to be taken into account with regards to the welfare of the child. These include (the parents’) commitment to having and bringing up a child or children; their ability to provide a stable and supportive environment for any child produced as a result of treatment; their medical histories and the medical histories of their families; and their age and future ability to look after or provide for a child’s need, among others. This latter requirement alone may well preclude grandparents commissioning, for example, a surrogate mother to be inseminated with their deceased son’s sperm. However, before addressing the eventual use of cryopreserved tissues, it seems appropriate to hope that the same storage regulations for immature as well as for mature gametes be applied in the UK, thus closing the present legal loophole in order to protect both patient and offspring.

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 reverse the order of the previous statement and aim at placing 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. This matter has the focus of many reproductive topics, perhaps best epitomized by the recent debates in the wake of the Dolly experiment and the theoretical possibility of achieving somatic reproductive cloning. It is possible that more than any other technique already practised, this would totally alter the social perceptions of reproduction. There has been so much international activity35 concerning cloning that it is difficult to summarize. Suffice it to say that it cannot be a per se objection as there is no objection to identical twins.36 Thus most of the objections must stem from the social interpretation of the burden we would impose on the offspring who would be more of a similar being to the genitor (or genitrix) than in our average reproductive endeavors. If society looks onto the clone as a freak, the clone will obviously have a very uncomfortable beginning. There is no way to predict how society might change in this aspect, but one feels that caution, again taking into account the responsibility we owe to the future child, should be our main rule. It seems fair to be wary of the experiment from the point of view of identity in the broad sense. Finally, of utmost importance is the fact that both the US and European reports on cloning stress the importance of educating the public in order to

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enable a more democratic process of decision making. The conclusion of the NABC37 report states: “While we have been able to agree on this and certain other recommended actions, we feel quite strongly that most of the legal and moral issues raised can only be resolved, even temporarily, by a great deal more widespread deliberation and education,” and the GAIEB report states that “further efforts must be made to inform the public to improve public awareness of potential risks and benefits of such technologies, and to foster informed opinion. The European Commission is “invited to stimulate the debate involving public, consumers, patients, environment…protection associations, and a well structured public debate should be set up at European level.” 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 optimistically be seen 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 Convention39 which expresses 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 certainly should strive for this communication to be achieved within all layers of society in order to share the ethical appraisal and assume our joint responsibility.

REFERENCES 1 Shenfield F, Sureau C, eds. Ethical dilemmas in assisted reproduction. New York and London: Parthenon, 1997. 2 Shenfield F. Gamete donation: ethical implications for donors. Human Fertility (1999); 2:98–101. 3 Shenfield F, Steele SJ. Information to the children of assisted reproduction. Human Reprod (1997); 12:393–5. 4 Effective Health Care. August 1992, no. 3, School of Public Health, University of Leeds, Centre for Health Economics, University of York, Research Unit, Royal College of Physicians . 5 Royal College of Obstetrics and Gynaecology. Evidence Based Infertility, London: RCOG 1999. 6 Jones WJ Jr. New reproductive technologies. In: Sureau C, Shenfield F, eds. Bailliere’s Clinical Obstetrics and Gynaecology, 1999; vol 13(3). 7 Nisand I, Shenfield F. Multiple pregnancies and embryo reduction: ethical and legal issues. In: Shenfield F and Sureau C, eds. Ethical dilemmas in assisted reproduction. London and New York: Parthenon, 1997.

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UNESCO International Bioethics Committee. Report of the subcommittee on genetic screening and testing, 1994. Paris: UNESCO. 9 Harper JC. Preimplantation diagnosis of inherited disease by embryo biopsy: an update of the world figures. J Assist Reprod Genet (1996); 13:90–5. 10 Modell B, Kuliev AM. Services for thalassaemia as a model for cost benefit analysis of genetic services. J Inher Metab Dis (1991); 14:640– 51. 11 Simpson SA, Simpson D, Haites NE. Genetic prediction of adult onset disease. BMJ (1994); 308:535. 12 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, London and New York: Parthenon, 1997; 51–9. 13 Every child a perfect child? New Scientist, 26 October 1995, 14–15. 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? Human Reprod (1995); 11(12):3086–90. 16 Missa JN (1998). Eugenics. In: Sureau C, Shenfield F, eds. Clinical Obstetrics and Gynaecology, Bailliere Tindall (1999); 13(3). 17 Shenfield F. Sex selection: why not! Human Reprod (1994); 9:569. 18 Pembrey M. Preimplantation diagnosis as an alternative to prenatal diagnosis. International conference on genetic diagnosis from prenatal to pre-implantation, 1998; Rennes, France. 19 Human Fertilisation and Embryology Act 1990. London: HMSO. 20 Loi no 94–654 du 29 Juillet 1994, Relative au Don, Assistance Médicale a la Procréation et Diagnostic Prenatal. Paris: Journal Officiel du 30 Juillet 1994. 21 Toulouse Tribunal de Grande Instance, Mme Veuve Pires v CECOS (1993). 22 Comité Consultatif National d’Ethique pour les Sciences de la vie et de la Santé, (1993), Avis sur le Tranfert d’Embryons Aprés Décès du Conjoint (ou du Concubin), no 40. 23 R v Human Fertilisation and Embryology Authority ex parte Diane Blood [1997] 2 All ER 687. 24 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. 25 Davies HA. Late problems faced by childhood cancer survivors, Br J Hosp Med (1993); 50:137–40. 26 Nicholson HS, Byrne J. Fertility and pregnancy after treatment for cancer during childhood and adolescence. Cancer (supplement) (1993); 71:10. 27 Oktay K, et al, American Society for Reproductive Medicine, press release, 27 September 1999.

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28 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. 29 Newton H, Aubard Y, Rutherford A, Sharma V, Gosden R. Low temperature storage and grafting of human ovarian tissue. Human Reprod (1996); 11:1487–91. 30 Gosden RG, Rutherford A, Norfolk DR. Debate: ovarian banking for cancer patients, transmission of malignant cells in ovarian grafts. Human Reprod (1997); 12:403. 31 Kennedy I, Grubb A. Research: the incompetent patient. In: Medical law, text with materials, 2nd edn. London: Butterworth, 1994:1052. 32 European Society for Human Reproduction and Embryology. Ethics and Law Special Interest Group taskforce on the embryo. Human Reprod (in press). 33 Englert Y. Particular requests in sperm donation. Human Reprod (1994); 9:1971–4. 34 HFEA Code of Practice, 1993, London: HFEA. 35 Shenfield F, ed. Cloning, societal, medical and ethical implications. Biomed (1998), DG 12. Brussels: European Commission. 36 European Commission: Opinion of the Group of Advisors on the Ethical Implications of Biotechnology to the European Commission, 28 May 1997: ethical aspects of cloning techniques, rapporteur: Dr Anne McLaren, Brussels. 37 Report and Recommendations of the National Bioethics Advisory Commission, Cloning human beings, Rockville, Maryland (USA): June 1997 38 Council of Europe, Convention for the protection of human rights and dignity of the human being with regard to the application of biology and medicine: Bioethics convention, Strasbourg, November 1996, Dir/Jur (96) 2.

Index α-subunit gene 448–9 A/B index 562 Aarskog-Scott syndrome 290 abdominal complications after OPU 656 ablation of endometriotic lesions 628 abnormal twin removal 686–7 abortion 746 abscess, tubo-ovarian 658 abstinence from sexual activity 62, 147, 409 accreditation 732 in Europe 47–59 in North America 35–45 ACE inhibitors 651 acellular debris 199 acid Tyrode’s 161, 189 acrosomes 71 actin 119 activin A 128 adhesion assay 343 adhesion molecules 347 adhesion phase 342 administration of ART 725–6 agarose gel 347 age and legal consent 757 age, gestational 687 age, maternal basal FSH levels and 528, 529 and CC challenge test 532–3 and fragmentation 175–7 and GIFT outcome 604 and IVF 397–8 and MFPR 683 and MNBs 303–4 and morbidity 667 and multiple pregnancy 663, 676 and oocyte donation 692, 693, 694 and ZIFT 614–15 age-related aneuploidies 339, 544 age-related trisomy 668 ageing of unfertilized oocyte 169 air handling system 31–3 air quality 139 and building work 22–4 and laboratory design 19–20

Index

1224

outgassing and 21 testing 31–3 albumin 650, 651–2 alignment of nucleoli 196–7 alkaline lysis buffer 327 allele dropout 185, 320, 321, 322 in polar bodies 335 reducing 326 allergic response to progesterone 519 American Association of Bioanalysts 36 American Society for Reproductive Medicine (ASRM) 39, 744 Ethics Committee 693 amino acid sequence of GnRH agonists 485 amino acids 205, 206–7, 210 amniocentesis 668 amplicon 319 amplification failure 320 amplification refractory mutation system 325 anaesthesia follicular fluid and 509 and oocyte retrieval 508–9, 599 and sperm retrieval 586 and ZIFT 610 analysis file 370–1 analysis of fertilization 193–201 anasarca 645 androgens during ovarian stimulation 461, 469 and PCOS 638 andrology laboratory 35 anejectory infertility 585–6 aneuploid cells 308, 311 aneuploidy 233, 241, 254 in embryo development 306, 309 aneuploidy rates 239, 302, 309 aneuploidy screening and PGD 292, 337, 339 animal models 741 see also Chinese hamster; hamster anonymity 695, 746, 755 antarelix 494 anti-adhesion molecules 348, 350 antiphospholipid antibodies 555 antisperm antibodies 61, 69–70, 410 see also sperm antibodies antithrombogenic therapy 555 antral follicles 111, 114, 130, 536 from xenografting 280, 281 apoptosis 170–1 apposition phase 342, 350 aromatase activity 428, 462, 639 ascites 651

Index

1225

aspirin, low dose 580, 694 for low responders 540 assisted ejaculation 585, 586 assisted hatching 160–4, 188 and cryopreservation 253 for low responder patients 540 in RIF 544, 548–9, 554 asthenospermia 289 atresia/atretic follicles 1, 113, 114, 427, 428 attention deficit and hyperactivity disorder 478 auditing of ART units 47–8 audits 52 autoclaves 28 autocrine factors 209–10 autocrine-paracrine regulation 343 autografted ovarian tissue 757, 758 autoimmunity 692 autologous cell coculture 546, 547, 548, 551–2, 553 autologous transplants 280, 283 azaline-B 494 azoospermia 285 algorithms for 586, 587 obstructive 287, 410 azoospermia factor (AZF) region 287 β-mercaptoethanol 126 β-subunit genes 448 for hCG 451 for hFSH 449–50 for hLH 450–1 banking of ovarian tissue 236, 279, 282, 283 Bardet-Biedl syndrome 290 basal FSH levels see under follicle-stimulating hormone basal medium for IVM 124, 129 additives 124–7 Bavister’s medium 93 Bayesian framework 391 Beckwith-Wieldemann syndrome 290 bench top incubator 142 bias 384, 390 bicarbonate buffer 90–1 bicarbonate buffered medium 209 bichorionic triplet pregnancy 685 bicornate uterus 407 binucleated blastomeres 308 bioactivity of LH 463, 469 bioassays 419, 434, 502 biochemical tests for sperm 72 biomarkers for acrosome reaction 71 clinical use of 361

Index

1226

endometrial 353–62 biopotency of rhFSH 433, 434 bipronucleated zygotes 312 birth abnormalities 207 rates 250–1, 633 for own v donor eggs 692 weights 682 births from frozen oocytes 240–1, 243 blastocoelic cavity 229 blastocyst biopsy 183 culture and transfer 211–12, 550–1, 554–6, 633 culture 216, 650 transfer 151, 153, 618 mixed embryo 669 single embryo 677 formation 177, 227 freezing 249, 251–2 grading system 228, 229 blastocysts 9, 349 blastocyte development 200, 214, 215 quality 677 blastocytes 343 chromosome analysis of 310 blastomere assessment 228–9 biopsy 545, 549 viability 189 bleeding intraperitoneal/retroperitoneal 655–6 in surrogate hosts 709 vaginal 655 and vaginal progesterone 521, 522 blinding in trials 384 block randomization 384 blood flow endometrial/subendometrial 579 intra-ovarian 569 blood: testis barrier 69 bone density evaluation 692 Bouin’s solution 276 Bourn Hall 11 Ethics Committee 705, 711, 715–16 breast feeding 706 British Human Fertilization and Embryology Act 17 British Medical Association 703 Brown, Louise 11 Bryan-Leishman stain 65 buccal cell controls 326

Index

1227

buffer systems 90–2 building works and materials 22–3 burning in new laboratories 23–4 buserelin 485, 496, 649 cadherin-11 358 caesarian section 699 calcitonin 358 calcium movement 140 calcium signaling 123 cameras 22 cancellation rate 497 cancer treatment as children 756 CAP/ASRM Reproductive Laboratory Accreditation Program 42–3 capacitation of sperm 1, 2, 71 case file, computerized system 56 catheter loading 624 placement, blind 625 transfer 601 Catholic Church 597, 598, 713 cavitation 178, 179 CECOS group 264 cell changes during freezing 244 lysis 327 numbers as criteria 225 recycling 324 cellular cloning 744 Centers for Disease Control and Prevention 39 centrifugation 77, 78, 585, 599 centriol, abnormal 314 cerebral palsy 664, 665 certification of embryo laboratories 39 certification procedures 96 cervical anatomy 614 cervical lavage 623 cervical mucus 624 cervicovaginal flora 656 cetrorelix 494, 495, 496, 497, 498 chaotic embryos 308 characterization of rhFSH protein 455–6 of rhLH protein 456 chelators 207 chemoattractants 346 chemokines 346, 350 chemotherapy 692 Childlessness Overcome Through Surrogacy (COTS) 705 Children Act 758 Chinese hamster ovary (CHO) cell line 447, 448, 451, 452, 453

Index

1228

chip DNA technology 297 Chlamydia trachomatis 44 chorionic villus sampling 668 chromosomal disarrangement 121 chromosome abnormalities 285 development to blastocyst stage 310–11 embryo development and 305–9 and fragmentation 170 in human embryos 297–314 and implantation failure 550 male factor 313–14 in MNBs 304 morphological traits and 302–5 and patient type 311–14 chromosome analysis 124 and testicular failure 410 chromosomes, freezing 238 chronic low dose protocol 647 chymotrypsin 63 classification of CCOCs 116 cleaning schedules 31, 32 clearance rates 415, 419 cleavage anomalies 177 cleavage arrest 124 cleavage characteristics 553 cleavage patterns 299 cleavage stage biopsy 183, 185–8 embryos 225, 301–9 freezing 250–1 Clinical Laboratory Improvement Amendments (CLIA’88) 35–9 clinical prenatal diagnosis 686 clinical problem, formulation of 381 clomiphene citrate 413–15, 496, 497, 628 challenge test 531–3, 693 and extrauterine pregnancy 659 cloning legislation 741–4 co-bedding of twins 672 CO2/bicarbonate buffered medium 208–9 CO2 gas tanks 28 coagulation of cord 685 coagulum 63 coasting 647 Cochrane Controlled Trials Register 387 Cochrane Library 430, 650 Cochrane review of clomiphene citrate 414 coculture 129, 189 of embryos 342, 343, 347 of frozen blastocysts 251, 252 in RIF 546, 551–2, 555 in ZIFT 608 Code of Practice of HFEA 758

Index

1229

cohabitation 734 College of American Pathologists (CAP) 28, 31, 36, 38 College of American Pathology 17 colloidal silica density gradient (CSDG) 80 compared with swim up 81, 82 colloids 652 colour Doppler 562 Comité Européen de normalisation (CEN) 48 commercial surrogacy 703–4 Commission on Office Laboratory Accreditation (COLA) 38 commissioning of facilities 24 compaction 178, 179 comparative genome hybridization 297 competence 108, 123 see also developmental; meiotic competitive block of GnRH receptors 493 complaints policy/procedure 53 complex chorionicity 670 complications, maternal 553–5, 656, 667 computer assisted semen analysis 67–9 condoms, silastic 62 confidentiality 755 confocal laser microscopy 122 conformity assessment system 48 congenital abnormalities with ART 155 congenital bilateral absence of vas deferens (CBAVD) 287, 290, 291, 410 conscious sedation 599, 602 consent 258 contact lasers 162 containers for sperm collection 63 containment systems 142 contamination in PCR 321 continuous peritoneovenous shunting 653 contrast optics 196 controlled drift 647 controlled ovarian hyperstimulation (COH) 413–22 and endometriosis 628 gonadotropin-only cycles 473–80 for PCOS 639 controlled ovarian hyperstimulation studies 477 controls in trials 383 cooling rates 275 cooling, slow 248–9 cord ligation 685 coronal cells 64, 108 corpus luteum 341, 428, 468 cortical granules 118–19, 121 and freezing 239 cost of 3D ultrasound 565 of GIFT v donor oocyte 603

Index

1230

and meta-analysis 391 of ZIFT v UET 607 cost effectiveness 489 of COH and IUI 628 of IVF 397, 398, 399 see also economic considerations Council of Europe Bioethics Convention 759 counseling 82, 508 psychoeducative 724 psychosocial 726 supportive 724–5 in surrogacy 706 therapeutic 723–4 counting chambers 63 creatinine phosphokinase 72 Creutzfeld-Jakob disease 555 Crinone 520, 521 critical timepoint 224, 226 cross-contamination 265 crossover studies 385–6 cryoflex tubing 265 cryogenic survival 234, 239–40, 243, 250 cryopreservation 10, 200, 228 and autologous coculture 552 blastocyst 218, 249 embryo 12, 215, 615 national restrictions 738–40 problems with 233 supernumerary 736 and ICSI 240, 252–3 legislation 738–40 oocyte donation 233–42 protective procedures 244–7 semen 79 sperm 78, 79, 148, 261–9 in anejaculation 586 stages of 233 vapour phase storage 267, 269 cryopreservation equipment 20, 21 cryoprotectants 237 and follicle size 279 and oocytes 234, 235, 236–8, 245–6 removal of 246, 247 and sperm 262–3, 268, 275 cryoprotection, chemical 245 cryptorchidia 588 crystalloids 651, 652 culture media 100, 140, 203–8 endotoxins in 30 oocyte 113, 601 preparation 20 quality control 29–30, 210

Index

1231

sequential 227, 252, 254, 551 in blastocyte culture 554 and mouse embryos 204, 205 in ZIFT 608 sperm 77, 83 sperm washing 276, 599 storage 210 transitional 90–3, 96 culture systems 208–11 cumulative conception rates 618–19 cumulative meta-analysis 390, 391 of IVF treatment 438, 439 cumulus cell controls 326 removal 101–2 cumulus coronal oocyte complexes (CCOC) 89, 115, 116 cumulus oophorus mass 99, 235 loss of 506 cyclic adenosine monophosphate 109 cyclin B 109 cysteamine 126 cysteine 126 cystic fibrosis 335, 410 and CBAVD 287, 290 cystine 126 cysts endometriotic 569 functional ovarian 568–9 ovarian 468, 487, 568 cytokine inhibitors 651 cytokines 346, 359, 657 cytokinesis 170, 628 failure of 187 cytoplasm 197–8 aspiration 150 cytoplasmic blebbing 169 cytoplasmic streaming 198 cytoplasmic transfer 735 cytoskeleton 119, 238 data tables files 369, 370, 378 database analysis 373 database systems 367 central design 371–2 data entry 374, 376–8 hard disk space 372–3 objectives 368 personnel costs 371 procedure types and numbering 373–4, 375 summary reports 378–80 table and field names 373

Index

1232

defects after IVM 123–4 degenerate oligonucleotide primed PCR 323, 328 deletion detection 320 delivery, mode of 671 delivery rates and ovarian volume 536 in surrogacy 708, 709 in ZIFT 607 density gradient method 80–1, 84–5 denuded oocytes, assessment of 102–3 depot preparations 485, 486, 489, 493, 495 depression 666–7 deslorelin 486 developmental blocks 546 developmental competence 107, 108, 305–6 and follicle size 115 and fragmentation 175 diagnostic tests, value of 403 diakinesis 2, 3, 4 Diff-Quik staining 65, 66 difficult uterine transfers 614 digyny 300 dilution of sperm 77, 79 dimethylsulfoxide (DMSO) 236, 238, 245, 248, 249 diploid/polyploid mosaics 307–8 diseases communicable agents and 44 transmission of 735–6 dispermy 299 disposal/destruction of embryos 258, 259 dissecting microscopes 22 disseminated intravascular coagulopathy 687 DM3 medium 207 DNA, construction of 448 DNA microarray technology 325 DNA of oocyte 118 DNA tests 323, 712 document control 51–2, 55 donation of gametes/embryos 746–7 donor egg programs 515 donor insemination pregnancy rate 264 donor oocytes 615, 633 donor uterine lavage 691 donor-recipient synchrony 523 Doppler frequency shift 562 Doppler ultrasound scan 406, 407, 569 of endometrium 561–4 of PCO 637, 641 techniques 561–4 double blind trials 384 down regulation 350, 494 Down’s syndrome 6, 312

Index

1233

drop lists 372–3, 376 Dutch Society of Obstetrics and Gynaecology 393, 394 duty of care 757 dyssynchrony in programmed cycles 516 Earle’s balanced salts 204, 205, 207 Echovist bolus method 609 economic considerations 754 of gonadotropin-only cycles 478–9 see also cost ectopic pregnancies 10, 618, 624, 658 ectopic pregnancy rates 603 education 383, 725 EGF growth factor family 360 egg donation see under oocytes EggCyte 369, 371, 374–8 eicosanoids 628 ejaculated spermatozoa and ICSI 153 electro-ejaculation 147, 585 electron microscopy of acrosomes 71 ELISA 534 elongated spermatids 78 elution profile of rhFSH 454 Embase 387 embryo banks, management of 258, 259 embryologists 18–19 intertech variation 31 embryology procedures proficiency testing 31 embryonic cell isolation 326–7 embryonic loss 543 embryonic regulation 341–50 embryos aspiration 683, 684 biopsy 184, 188–9 cryopreservation 12, 615 national restrictions 738–40 problems with 233 supernumerary 736 culture 55, 203–18 protocol 216–18 development 8, 151, 305–9 and oocyte competence 123 expulsion 609, 614 grouping 210 morphologies 199, 200 in endometriosis 630 number to transfer 675, 677–8 legislation 735–7 physiology, dynamics of 203–4 quality evaluation 223–31 replacement 151

Index

1234

research legislation 741–4 selection 200, 224–32, 254 chromosomal abnormalities 314 continuous grading system 199 extended culture 193, 194 ideal features 229 storage 247–8 thawing 247–8 transfer 12, 623–6 catheter type 624–5 difficult transfers 625 nutritional stress during 545 in surrogacy 708 timing of 211 variables affecting success 623, 624 viability 172 embryotrophic factors 546 emergencies, relocation during 21 emotional support 726 empty follicle syndrome 442 EN 4500 series 47 EN 45001 51 EN45001/ISO17025 48 Enclomiphene 413, 414 endocrines and ART cycles 459–69 and oocyte retrieval 113 endometrial adhesion molecules 347, 544 endometrial biomarkers 353 endometrial biopsy 404 endometrial cavity polyp 405 endometrial dating 354–5 endometrial-embronic interactions 342 endometrial epithelial cells (EEC) 342, 343, 347 monolayers 345, 348 endometrial pattern 577–8 endometrial receptivity 575–6, 676 endometrial stimulation 697 endometrial stromal cells 342 endometrial synchronicity 258 endometrial thickness 502, 575–6 endometriosis COH and oocyte retrieval 629–30 fertilization/embryo development 630–1 and GIFT 632–3 and GnRH antagonists 499 implantation and pregnancy 631–2 and infertility 627–8 and insemination 628 and IVF outcome 396 and ovulation induction 628 and surgery 633

Index

1235

endometrium 346, 347, 349, 353, 355 Doppler studies 578–80 embryo, prepared by 341 GnRH antagonist effect on 498 stabilization of 515 triphasic/multilayered 576, 577 ultrasound evaluation 575–80 endotoxemia 657 endotoxins 28, 30 endotracheal intubation 610 energy substrates 124–5 enzymes and sperm penetration 99 Epics Elite Flow Cytometer 345 epidermal growth factor 128 equipment 28, 54, 325 estradiol (E2) 127, 354 evaluation of secretion 469 levels in low responders 527 luteal supplementation 517–18, 522 during ovarian stimulation 465–7, 697 estrogen 428 measurement 501, 502 transdermal 697 ethical guidelines, international 744 ethical issues 13–14, 683, 688 ethical problems 555, 668, 753–9 cryopreservation 12, 755–8 justice for patients/children 753–4 in PGD 754–5 surrogacy 709 ethics and meta-analysis 391 ethidium bromide gel electrophoresis 329 ethnicity, disorders linked to 697 ethylene glycol 236, 245, 262 ethylene oxide 29 ethylenediaminetetraacetic acid 205, 207 eugenics 755 European co-operation for accreditation 47, 49 European Society of Human Reproduction and Embryology 401, 744 Andrology Special Interest Group 72–3 guidelines for IVF 57 evidence-based medicine 381–92 evidence-based practice 382–3 examination of sperm sample 63–7 exclusion criteria 384 exogenous FSH ovarian reserve test 535 expression of genes hCG 453 hFSH/rhFSH 451–2 hLH 452–3 extenders for sperm 262–3 external QC testing 56

Index

1236

extrauterine pregnancy 658–60 Fail-Safe N 390 failure to conceive 657 fallopian tubes cannulation 602 single patent 598, 617–18 ultrasound scan 569–71 falloposcopy 408, 602 female body mass index 401 Fertilitetscentrum 54–5, 57–8 Fertility Clinic Success Rate and Certification Act (FCSRCA) 39–42 fertility of sperm, post-thaw 264 fertilization 151, 194–5 abnormal, in ICSI 83 of IMV oocytes 122, 123 fertilization rates and freezing 239–40, 243 in ICSI 147, 150, 154 fetal abnormality 686 fetal anomaly screening 684 fetal loss 207 fetocide 668, 686 fetus as a patient 670 FF-MAS 127 fields, database 372 files, database 369–71 fine needle aspiration biopsy 81, 587 protocol 591–2 fixation 344 fixed effects model 389 flare-up effect 468, 469, 499 flow cytometry 70, 345, 347, 350 flow velocity waveform (FVW) 562, 578 fluorescence in situ hybridization (FISH) 124, 186, 297 in cell recycling 324 and chromosome abnormalities 298–9, 300 failure/misinterpretation 554 in hLH production 453 in PGD 545, 549, 550 in polar body removal 334 fluorescence PCR 322, 328–9, 335 fluorescent dyes 120 flushing of CCOCs 116 follicle-stimulating hormone (FSH) 2, 113, 114, 127 basal levels day 3 levels 528–9, 530–1 high 312, 548 intercycle variability 529 repetitive screening 529 and single ovary 529–30

Index

1237

biochemistry of 418 computer model 447 development of 417 and follicle development 426–8 in hMG 419 during ovarian stimulation 459–61, 469 and ovulation induction 429–36 in PCOS 639, 640, 641 purified 419–20 recombinant human (rhFSH) 447, 578 and cetrorelix 498 in ovarian stimulation 439–41 in PCOS 433, 434–5 production of 451 recombinant human (rhFSH)x 425–6 serum levels 406 surge 416 threshold 431, 459–60, 461, 529 and unifollicular response 640 window 460 follicles 7, 111 collapsed 508 development 426–43 monitoring form 573 diameter and volume 502, 506, 507 dominance 427 multiple 573 numbers 527, 536 recruitment 427 selection 427, 428 size 112, 115, 501, 573–4 see also antral; atresia; primordial follicular apoptosis 114 follicular aspirates 93 follicular fluid 509, 601 folliculogenesis 427, 430 effect of endometriosis 632 and FSH 460 and LH 461, 464, 465 multiplex PCR 439 follistatin 128 follitropin 425 Food and Drug Administration (FDA) 44, 413 forms files 369, 370 fragment removal 177–81, 555 fragmentation 229, 677 with autologous coculture 552, 553 degree and pattern 172–81 and endotoxins 30 in human embryo 169

Index

1238

mechanisms 170–2 methods and results 172–81 freezing biochemistry of 244 protocol 85–6 of sperm 263 freezing and thawing of embryos 53 freezing rates 234, 244 French National IVF registry (FIVNAT) 396 frozen semen method 85 frozen sperm 241 frozen-thawed embryo transfer 615–16 full surrogacy see surrogacy functional cysts 487 functional neuroteratology 478 funding policy and ICSI 395 funnel plot 390, 391 fusion of nucleoli 195 γ-glutamyl cycle 126 G1 medium 205 G1.2 medium 217, 218 G2 medium 205, 207 G2.2 medium 217, 218 gamete intra-fallopian transfer (GIFT) 10, 77, 95, 597, 605 controlled ovarian hyperstimulation 598 gamete separation 602 oocyte retrieval 599–601 patient selection 598 results 603–4 sperm collection and preparation 598–9 transfer 601–3 ganirelix 494, 495, 496, 498, 649 gas cylinders 21, 28–9 Gaucher disease 336 GE-80 microsyringe 143 gel electrophoresis 122 General requirements for the competence of testing laboratories ISO/IEC Guide 25 48 GeneScan 322, 329 genetic analysis of embryo 319–29, 685 genetic causes of male infertility 285–90 genetic conditions 82 genetic couple 704, 705, 706 genetic evaluation 290–1 genetic familial trait for twins 663 genetic mother 707 genetic screening for oocyte donors 696 v testing 292 genetic screening form 696–7

Index

1239

genetic testing of patient’s partner 287, 292 see also preimplantation genetic diagnosis genetically transmitted diseases 735 germinal vesicle (GV) 99 germinal vesicle oocytes 102, 112 gestational age 671, 687, 688 gestational diabetes 699 gestational surrogacy see surrogacy Gillick competence 757 gland/stroma dyssynchrony 356, 694 glassware 28, 326 glucocorticoids 687 glucose 125, 204, 205–6 glutamine 125 glutathione metabolism 125–6 glycerol 236, 245, 248, 251, 262 glycerol egg yolk citrate (GEYC) 262, 267 glyceryl trinitrate (GTN) 580 glycodelin 359 glycosylation of FSH 442, 455 GnRH-associated peptide (GAP) 420 gonadectomy, medical 484 gonadotropin in low responders 537 gonadotropin-only cycles 480 gonadotropin releasing hormone (GnRH) 111, 420–2, 494–6 agonistic analogs 421, 484–9 agonists 253, 415, 416, 483–9 administration route 485–6 in COH 462 dose and protocol 486–7, 488, 630 downregulation 537–9 effect on LH 463, 464, 468 effects on FSH secretion 461 in endometriosis 629 flare 538 as hCG substitute 647, 648–9, 650 long protocol 640 non-pulsatile administration of 473 optimal scheme 486–7 role in ART 474 stimulation test 535 v antagonists 499 analogs 420–2, 479, 483–4 antagonistic analogs 421–2 antagonists 465, 467, 483, 649, 650 action of 493 future developments 498–9, 633 LH surge, preventing 468 third generation 494 gonadotropins 127, 416–20 in HH 435

Index

1240

in PCOS 430–3 recombinant 447–56 structure of 417 in superovulation 436–42 Gonal-F 425, 447, 453–4, 646 gonosomal abnormalities 313 goserelin 486, 578 government policies on embryos 193, 200 government reporting requirements 379 gradient centrifugation of sperm 78 gradient separation procedures 80–1 grading of CCOCs 115, 118 granulosa cell coculture 551 granulosa cells 108, 418 and FSH 427, 428, 459 growth hormone receptors 539 Group of Advisers in Ethical Implications of Biotechnology 759 growth factors 359, 360 growth hormone 128, 540 guidelines 53 for ART 53, 57, 403, 715–16 laboratory safety 41 legislation and 53 personnel qualification 40 psychological evaluation 723 segregating patient material 20 half lives FSH 460 hCG 415–16 hMG 419 halo 197–8 Ham’s F-10 205, 207, 210, 262 hamster egg penetration assay (HEPA) 72 hamster epidydimal spermatozoa bioassay 30 see also Chinese hamster handling conditions 139–40 haploidy 306 harmonization of standards 48 hatching 159–60 assisted see assisted hatching heated stages 142, 218 hemi-zona assay 72 hemoconcentration 651 hemocrit 651 hemocytometer 63 hemodynamic monitoring 653, 655–6 hemophilia 336 heparin 92 hepatic dysfunction 645 hepatitis 267, 735

Index

1241

and coculture 555 and tissue quarantine 44 HEPES buffer 209 HEPES-buffered media 100 HEPES/MOPS buffered medium 218 heterogeneity, statistical, in meta-analysis 388 heterosexuality and ART 734 heterotopic pregnancy 660 heterotopic transplants 283 high order multiple pregnancies (HOMPs) 664, 665 births 671 HiGonavis assay 10, 11 histological dating of endometrium 575 histrelin 486 HIV 44, 266–7, 555 Hoechst staining 117, 118 Hoffman and Nomarski 22 Hoffman modulation contrast optics 94, 141 holding pipette 144 holistic approach 726 homeobox genes 360 homeostatic stress 545 homogenization 273–4 hormone replacement therapy (HRT) 344 human chorionic gonadotropin (hCG) 111, 354, 415–16 administration time 574 corpus luteum stimulated by 515 history of 2, 8 in luteal support 429, 522 monitoring 660 in OHSS 645, 648–9 recombinant (rhCG) 426, 434–5, 442 human cleavage stage embryo 187 “human embryo assay” 55–6 Human Fertilisation and Embryology Act 703, 712, 758 Human Fertilisation and Embryology Authority (HFEA) 223, 258, 259, 675, 741 Code of Practice 711, 715–16 on multiple embryo transfer 669 human menopausal gonadotropin (hMG) 103, 417, 419–20, 425 history of 6, 8 and ovulation induction 430, 435, 462 in PCOS 640, 641 human ovarian transplantation project 281–2 Human Reproductive Technology Act 259 human serum albumin 27, 92 human sperm preserving medium 262–3, 268 Human Tubal Fluid 205, 207 humidity 19, 23 hyaluronan extracellular matrix 89, 99 hyaluronic acid receptor 358 hyaluronidase 99, 100 hydrolytic enzymes 71

Index

1242

hydrosalpinges 398, 405, 543–4, 548 bilateral 571 in oocyte recipient 693 ultrasound visualization 569–71 hydrosalpinx fluid 544 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) 90, 91, 92 hyperactive sperm 67 hyperandrogenism, ovarian 469 hypo-omotic medium 86 hypo-osmotic swelling test 70, 409, 410 hypogonadotropic hypogonadism (HH) 429–30, 435–6, 442, 585 hyponatremia 651 hypophyseal desensitization 463, 467, 468, 469 see also pituitary desensitization hypophysectomy, medical 484 hysterosalpingography 408 hysterosapingo-contrast sonography 408 hysteroscopic tube transfers 613 hysteroscopy 409, 602 iatrogenic sterility 233, 755 IBM compatible computers 368 ice 275 ice nucleation 246–7 identification of CCOCs 115 IFFS Surveillance 98 732 immature gametes 758 immature oocytes 235 culture procedure 113 retrieval 114 immobilization of sperm 79, 150, 151 immune dysfunction 627–8 immunobead binding test 69 immunofluorescence 345 immunoglobulin, intravenous 555 immunohistochemical markers 357 immunohistochemistry 344, 345, 347, 349 immunometric determinations 463, 464, 467, 469 immunostaining 119, 120, 347 implantation 353–4 with autologous cell coculture 552 defects 353 failure, repeated 613–14, 693 therapeutic approach 543–56 potential 545, 614, 615 rates 177, 181, 215 and biopsy 189 with blastocyte transfer 211 and chromosomal abnormalities 311–12 in endometriosis 632 with ICSI 154

Index

1243

predictive tests 528–32 site 350, 354 in vitro fertilization (IVF) 1–14, 17, 111, 411 cascade 475 and gonadotropins 436 indications for 393–9 natural cycle 10, 11, 497–8 or GIFT 597 outcomes v infertility causes 395, 396 prognostic factors 397–8 repeated failures 312, 676 and sperm antibodies 70 and stress 720 in vitro maturation (IVM) 107–38, 399, 411, 757 and endometriosis 633 future studies 129–30 history of 1, 4 and ICSI 113 in vitro oocyte ageing 123 inaccessible ovaries 693 inclusion criteria for meta-analysis 387 for trial subjects 384 incubation chamber 208 incubation volume ratio 209–10 incubators, spare 20 indirect embryo screening 550 indomethacin 650 induction of superovulation 100 infection, risk of 259–60 infectious diseases 82, 735–6 screening 695 infertility anejectory 585–6 causes 396–7 and IVF outcomes 395, 396 and multiple pregnancy 676 counselors 722 duration of 398 male factor 156, 393, 395 and stress 720 management 397 treatment 417 tubal factor 631, 676 unexplained 396, 411 CC challenge test 532 inflammatory reaction, local 657 information as support 725 infrared lasers 188 inherited disease 183 inhibin B 533–4 inhibin levels 529

Index

1244

inner cell mass development 210, 229 insemination 94 donor 264 and ICSI 95 intracervical 261–2 intrauterine 70, 261, 411 insemination medium 95 inspections under CLIA’88 38 inspectors, paid, in JCAHO 43 insulin 127–8, 639 insulin-like growth factors 127–8 insurance 24 integrins 347, 544 Intenational Standardization Organization 48 interference optics 22 interleukin-1, embryonic 348 interleukin-1 family 359–60 interleukin-8 346, 347, 350 International Accreditation Format 50 International Bioethics Committee 755–6 International Federation on Fertility Societies (IFFS) 732 International Laboratory Accreditation Cooperation 50 interstitial pregnancy 659 intracardiac KCL injection 686 intracervical insemination (ICI) 261–2 intracytoplasmic sperm injection (ICSI) 13, 17, 100–4, 150, 151, 693 and abnormal semen 77 and cryopreservation 262 and endometriosis 633 and gonadotropins 436 and gonosomal abnormalities 313 and IMV 122 indications for 395 lack of guidelines 53 outcome 153, 154, 156 procedure 111 severe male factor 286, 291 for sperm abnormalities 81–2, 273 and sperm antibodies 70 and sperm evaluation 61 and stress 720 technical aspects 139–46 and ZIFT 615 intrathoracic KCl injection 684 intrauterine insemination (IUI) 70, 261, 411 intravascular volume depletion 651 intravenous infusion of LH releasing hormone 484 invasive evaluation of oocytes 117 inventory system 28 investigation of patient 401–11 irradiation 756 ISO 10011 52

Index

1245

ISO 17025 48, 50, 51–3, 54 ISO 9000 51 ISO 9000 series 47 ISO 9001 48 ISO/IEC Guide 25 48 Isolate 80, 262 IVF-ET efficiency of 193 in endometriosis 629 Joint Commission on Accreditation of Health Care Organizations (JCAHO) 43–4 inspections 38 Kallman’s syndrome 289, 290, 291 Kartagener syndrome 289 karyotype analysis 301–2 Kennedy syndrome 289, 291 key fields 371 linked 372 kinematics 67, 68 Klinefelter syndrome 285, 286, 290, 588 Kruger strict criteria 65, 66, 67, 148 labeling of samples 54, 63 laboratories 18 accreditation 35–45, 47–59 burning in 23–4 cleaning schedules 31 design 19–20 embryo 39–40 certification of 39–40 equipment 20–1 performance standards 31 personnel 17–18 quality management 27–33 safety guidelines 41 laboratory managers 52 laboratory procedures 39–40, 101 lactate 125, 204 laminaria 623 laparoscopy 409 diagnostic 509, 598 and endometriosis 627 in GIFT 597, 599, 600 “large offspring syndrome” 125 laser assisted hatching 162, 163, 164 LCHAD 336 leakage in liquid nitrogen 265 legal issues 709, 711–13 legislation 258, 731–51 cryopreservation 738–40

Index

1246

divergent laws and practices 731–3 donation of gametes/embryos 746–7 embryo numbers 735–7 embryo reduction 744–6 embryo research and cloning 741–4 legislation and guidelines 732–4 marital status 734 micromanipulation 734–5 PGD 740–1 surrogacy 748–9 welfare of offspring 745–50 leukocyte recruitment 346 leukocytosis 651 leuprolide 486, 649 linked polymorphic marker analysis 336 liquefaction of semen 63, 78 liquid nitrogen contamination 265 long protocol of GnRH agonists 487, 489 of GnRH antagonists 498 low dose regimens 433 low responder patients defining 527 evaluation of secretion 528–36 treatment 537–40 luteal phase 516–24 deficiency 355, 358, 359 luteal progesterone levels 516 luteal support 9 protocols 515–24 route of 518–20 timing 517 luteinizing hormone 2, 99, 111, 127, 355 biochemistry of 418–19 ceiling hypothesis 429, 462, 465 and follicle development 428–9 as hCG substitute 648 high tonic levels 498 in implantation 341 ovulation induction 429–36, 461–5 ovulation, test for 404 in PCOS 640–1 pharmacokinetics of 462 recombinant human (rhLH) 426, 434–5, 441, 448 rise, untimely 474, 494, 498 secretion 463, 464 serum levels in PCOS 638 suppression 463 surge 11, 415, 416, 428 prevention of 468, 495 surrogate for 415–16, 429 threshold 464, 465

Index

luteoplacental shift 515, 524 lutropin 418 Luveris 426, 448 lymphoblast cell lines 326 lymphocyte controls 326 lysins 160 macromolecules 205, 207–8 Makler counting chamber 63, 67, 148 male factor infertility 156, 393, 395 and stress 720 management reviews 52 manipulation plate setup 145–6 manipulators 140–1, 142 mannose binding assay 73 marital status 734 markers for human oocyte grading 103 masking in trials 384 masturbation 62, 78, 147 material safety data sheets 22–3 maternal factors and cryopreservation 253 maternal fetal medicine (MFM) 669 maternal physiology, dynamics of 204 maturation promoting factor 109, 110 maturation regulation 130 maturity evaluation 89, 94, 122 maturity grading 117, 130 McGill Reproductive Centre 401–2 MCP-1 346, 347 media see culture media medical screening 693 for oocyte donors 695 for surrogacy 705, 708 Medline database 387 meiosis 2, 99, 428 arrest of 107–8, 130–1 disruption of 313 meiotic competence 108–9, 122 meiotic maturation 107, 112 meiotic spindle 238 meiotic status markers 99 membrane integrity, loss of 177 menopause 693 menstrual cycles, normal 515, 529 mental health professionals 721 meta-analysis 386–92 of albumin trials 650 cumulative 390, 391 of effect of down-regulation 473 on FSH and hMG 430 on GnRH agonists 485

1247

Index

1248

with uFSH and hMG 438, 439 ZIFT v UET 610 metformin 650 method manual 53–4 methodological quality 389 methylene blue 623 Metrodin 419–20, 646–7 Metropin 417 MicroCell 64, 67 microdeletion on Y chromosome 287 microdose GnRH agonist flare 538–9 microfilaments 119, 121 microinjection in ICSI 149 micromanipulation 139, 145–6, 734–5 micromanipulators 20, 145–6, 149 see also manipulators microsatellites 323 microscopes 22, 94, 119, 141 Microsoft software 368–9, 380 microsurgical epidydimal sperm aspiration (MESA) 81, 85, 147, 287, 735 protocol 594–6 during scrotal examination 587 microsurgical fragment removal 177–81 microsyringe systems 142–4, 149 microtools 144, 145–6, 149 for assisted hatching 160 microtubules 120 depolymerization 140 Milli-Q water 28 minidose GnRH agonists 538 minilaparotomy 601 minipercoll technique 240 Minnesota Multiphasic Personality Inventory-2 722 miscarriage rates and endometrial thickness 576 and multiple pregnancy 677, 688 and PCO 641 in surrogacy 714 miscarriage, risk of 398, 402 miscarriages, repeated 704 misdiagnosis 321, 326, 335, 554 mitochondriae 120 aggregation of 198 in male infertility 290 mitochondrial mutations 175 mixed agglutination reaction 69 modular design of laboratory 20 Monash assay 529, 533 monitoring IVF cycles 501–2 cycles with GnRH antagonists 503 follicular maturation 502 ultrasound 501

Index

1249

monohormonal products 426 monosaccharide composition of FSH 455 monovulation 429 monozygotic twinning 161, 163 monozygotic twins 554 MOPS buffer 209 moral issues 695, 721 morphological defects of oocytes 102–3 morphological markers 224 morphology 225 and chromosome abnormalities 302 dominant blastomeres/giant eggs 304–5 fragmentation 303 multinucleation 303–4 mortality rates in HOMPs 681 morula stage 311, 314 mosaicism 187, 305, 306–7, 308–9 at blastocyte stage 310 and cancer 692 fate of mosaic cells 307–8 gonosomal 313 onset 298, 307 and pronuclei 299, 300 motility and cryopreservation 261, 264 motility stimulants 588 motivation to donate 696 mouse embryo bioassay 30 clinical aspects 55–6 MUC1 348–9, 350 mucification 99, 107 mucins 348, 358–9 multifetal pregnancy reduction (MFPR) 664, 665, 667, 670 legislation 744–6 mortality rates 666 outcome 684–5 psychological effect 685–6 spontaneous reduction 683 techniques 683–4 multinucleated blastomeres (MNBs) 303–4 multiple births with GIFT 603 multiple dose protocol of GnRH antagonists 494, 495–6 multiple embryo transfer 677–8 ethical problems 754 restrictive criteria 675, 735–7 multiple follicular cysts 566 multiple follicular development 429, 430, 436–42 multiple pregnancies 163, 188, 223, 430 embryo quality and 677 with GnRHa 493 iatrogenic 663–73 successful outcomes 672 increase in 663, 681

Index

1250

mortality rates 672 in PCOS 639 rates 477, 480, 744 and PCO 641 regulations 736–7 risk factors 676 spontaneous reduction 668, 669 with ZIFT 616–17 multiple zygote transfer 617 multiplex PCR 321, 328, 336 mutation analysis 324 mutation-free oocytes 333 myometrial contractions 625 myometrium 577 myotonic dystrophy 288, 291 Narishige IM 6 microsyringe 143 Narishige micromanipulator 140, 141 National Bioethics Advisory Commission 759 natural menstrual cycle 497–8 IVF 10, 11, 497–8 natural surrogacy 704, 709, 712–13 necklace sign 646 needles, aspiration 506–7 Neisseria gonorrhea 44 neonatal intensive care unit 671, 672 nested PCR conditions 327 nested primer PCR 319–20 Nomarski differential interference contrast optics 102 non-contact lasers 162–3, 188 non-English literature 387 non-invasive evaluation of CCOC culture 117 Noonan syndrome 290 Northern blot analysis 344, 347 Noyes dating criteria 355 nuchal translucency test 684, 685 nuclear maturation 194 nuclear membrane breakdown 228 nuclear staining 171 nuclear transfer 735 nucleolar precursor bodies 224, 228 distribution and morphology 301 nucleoli 196–8 nucleolus organizing regions 194 obesity in PCOS 638 occupational health and safety 47 off gassing 21, 29 oil overlay 210, 217, 231 “Oktay” modification 279 oligoastenoteratospermia 285, 288

Index

1251

oligoasthenospermy 395 oligoasthenozoospermia 311 oligospermia 67, 79, 85–6 oocyte cumulus coronal complexes (CCOC) 89, 115, 116 maturity evaluation 89, 94 oocyte meiotic inhibitor (OMI) 107 oocytes activation 194 ageing 123, 169 aspiration 505–13 risks 655–60, 691 births from frozen 240–1, 243 collection 149, 574 cryopreservation 243, 333, 738, 740, 758 denudation 100, 101–2, 102–3 DNA of 118 donation 12, 215, 691–9 and cryogenic survival 233–42, 633 donor recruitment 694–5 endometrial preparation 697–9 and endometriosis 615 and HRT 344 obstetrical outcome 699 and RIF 693 screening 693–7 donation programs 462, 535, 691–9 germinal vesicle 102, 112 grading 94, 95, 100, 103 handling 100–3 holding 95 immature 235 retrieval 113–22 invasive evaluation of 117 maturation 117, 122, 428 after superovulation 111 and gonadotropin stimulation 112 stimulus for 99 maturity, timing of 122 morphology 102, 102–3 mutation-free 333 recovery rates and follicle size 574 surrogacy and 710 recovery with tubal insemination (ORTI) 10 retrieval 93, 95 cumulus mass loss/damage 506, 507–8 damage to oocytes 506, 507 egg pickup technique 508–13 experimental and physical aspects 505 from immature follicles 113–22 laparoscopic 599, 600 structure 100, 238

Index

1252

in transition 90 treatment 89–97 viability of 334 volume and competence 108 oogenesis 107 oolemma 108, 150 and sperm motility 64 ooplasm penetration 150 “ooplasmic transplantation” 131 opioid analgesia 509 optics 141 optimal thawing rate 237 oral contraceptives 487 orchidometer, Prader 403 orthotopic transplants 283 osmotic pressure/properties 246 osmotic shock 234 outgassing 21, 29 ovarian artery 563 ovarian enlargement 645 ovarian function and cryopreservation 253 ovarian hyperstimulation 170 ovarian hyperstimulation syndrome (OHSS) 112, 241, 430, 441 after IVF 396 with GnRH agonists 493 with GnRH antagonists 496 and gonadotropin-only cycles 477, 480 and LH/FSH ratio 420 monitoring 503 and ovarian volume 571 and PCO 566, 567, 640, 641 in PCOS 639 risk factors 517, 646 severe 645, 646–53 ovarian markers 333 ovarian morphology 571–2, 637 ovarian parenchyma freezing technique 235–6 ovarian reserve 692–3 diminished 531–2 predictive tests 528–32 screening threshold values 534–5 ovarian response and multiple pregnancy 676 predictors of 467 ovarian responsiveness predictors 535–6 v basal FSH levels 531 ovarian senescence 398 ovarian steroids 354 ovarian stimulation 7, 8, 436–42 deliveries per cycle 479 and endometriosis 630

Index

1253

with GnRH antagonists 494 gonadotropin profiles 459–65, 465–9 protocols 10 “soft” protocols 496–7 steroid profiles 465–9 ovarian stromal perfusion 572 ovarian structure 571–2, 637 ovarian tissue cryopreservation “Oktay” modification 279 optimising 282 transplantation 279–83 animal models 280 clinical trials 281–2 ovarian transplantation project, human 281–2 ovarian volume 535–6, 567, 571–2 calculation 566 ovary, responsibilities of 425 overresponders to hormones 312 oversight, regulatory of laboratories 44 Ovidrel 426 Ovidrelle 448 oviduct fluids 206 ovulation induction 2, 149 endometriosis and 628 and FSH 429–36 with hMG 430, 435, 462 and LH 429–36, 461–5 ovum pick-up (OPU) 655, 656 oxygen tension in IVM 128–9 packaging sperm 263 packing for liquid nitrogen 265, 268 paediatric isolettes 216 paracentesis 652–3 paracervical block 509 paracrine autocrine signals 347 paraffin oil 274 parthenogenic activation 239, 298 partial surrogacy 704 partial zona dissection 161, 184 partner genetic testing 287, 292 paternal genome contamination 321 pathophysiology 381 patient test management 36–7 patients fetus as a patient 670 history 61 investigation 401–11 low responder defining 527

Index

1254

evaluation of secretion 528–36 treatment 537–40 pediatric 236 screening 266 selection 211, 215 criteria 174 support 719–28 future directions 727 methods 721–6 results 726–7 strategies 727 stress 719–20 payment in surrogacy 709, 710, 712, 748 peak systolic blood flow velocity 574 pediatric patients 236 peer evaluation of accreditation bodies 49–50 embryology procedures 32 and recruitment 18 peer inspection in CAP 43 pelvic examination 402–3 pelvic inflammatory disease (PID) 403 effect on IVF-ET outcome 656–7 and GIFT 597 mechanisms 656 treatment 657–8 pelvic structures 561 ultrasound assessment 563, 564 pelvic vascular perfusion 564 pelvic vascularization assessment 561 penile absorption of progesterone 521 pentoxifylline 86, 588 peptide hormones 358 Percoll 262 gradient centrifugation 599 percutaneous epidydimal sperm aspiration (PESA) 81, 85, 147, 587 protocol 590–1 performance measures under FCSRCA 41 Pergonal 417 perifollicular blood flow 574–5 perifollicular perfusion 574–5 perinatal mortality 681 periovulatory oviductal fluid 77 personnel assessment using database 368 costs 371 at Fertilitetscentrum 57 and laboratory set-up 17–18 qualification guidelines 40 and quality management 37–8 responsibilities 40, 50, 52–3 staff ratios 18

Index

1255

PGD legislation 740–1 pH colour standards 209 pH stability 208 oocyte 90, 117 pharmacotherapy for endometriosis 634 phase contrast microscopy 70 phenylketonuria (PKU) 336, 337 phosphate buffered saline (PBS) 91, 92 phosphoric acid buffer 90, 91 physical examination, pre-anaesthetic 508 physicians 669, 670 pinopods 356, 360–1 pituitary desensitization 467, 640 dose dependence 539 see also hypophyseal desensitization placebo 383 effect 384 placental steroids 515 plasma estradiol 466, 467 plasma expanders 651 plasma membrane damage 274 plasma membrane, sperm 70 plasticware testing 28 Pol-scope 22 polar bodies 1, 198–9, 333 abnormalities 198 placement 225 removal 334 shape and alignment 301 polar body analysis 545 polar body biopsy 183, 184–5, 339 and chromosomal disorders 337–9 and Mendelian disorders 334–7 v ovarian responsiveness embryo biopsy 554 polar body testing 549 polarized EEC 343 polycystic ovaries (PCO) 404, 405, 637–42 diagnosis 406, 566–8, 637–8 differentiated from PCOS 637–8 and ovarian volume 571 prevalence 638 response to stimulation 638–40 stromal blood flow in 572 polycystic ovary syndrome (PCOS) 103 diagnosis 406 ovarian morphology in 637 ovulation induction in 429, 430–5 treatment 497, 499 polymerase chain reaction (PCR) 287, 297, 319–22, 323 and polar body diagnosis 334 polymerase chain reaction product 319 polymorphic STRs 323–4

Index

1256

polyp 406, 548 polyploidy 305, 306 polyspermy 119, 159, 239 polyvinyl pyrollidone 80, 149 positive controls 326 postcoital test 403 posthumous treatment 756–7 postimplantation development 186 povidone-iodine and saline 657 Prader orchidometer 403 Prader-Willi syndrome 290 pre-eclampsia 699 pre-embryo research 735 prediction models 399 pregnancy from cryopreserved oocytes 240 and fragmentation 174–5 loss 746 and hydrosalpinges 546 increased with endometriosis 632 multifetal pregnancy 682, 684 outcomes 155, 437 termination 653 pregnancy/delivery characteristics 154–6 pregnancy-induced hypertension (PIH) 699, 709 pregnancy rates 189, 193, 227 with autologous cell coculture 552 and basal day 3 FSH 529 with blastocyte transfer 211 CC challenge test and 531–2, 533 and cleavage stage biopsy 185 donor insemination 264 and early cleavage 226 ectopic 603 and embryo numbers 223, 736 in endometriosis 632 with GnRH agonists 487 with GnRH antagonists 498 with ICSI 154 and light source 603 and ovarian volume 536 and PCO 641 with PGD 338 reported under Wyden bill 39 in surrogacy 708 with uFSH and hMG 438 uFSH v rhFSH 440 pregnant mares’ serum (PMS) 2 preimplantation genetic diagnosis (PGD) 183, 314, 339 for aneuploidy 337 basic principles 319–21 ethical problems 755–6

Index

1257

and ICSI 291, 292, 588 laboratory techniques 325–9 molecular methods, advanced 321–5 outcomes 185 pitfalls of PCR 320–1 in RIF 544–5, 549–50 prematuration system 130 premature LH peaks 474, 480 premature ovarian failure 579, 691–2 prematurity 671 preterm labor 681, 699 primary ciliary dyskinesia 289, 291 primary PCR conditions 327 primer extension preamplification 323, 328 primordial follicle isolation 281 primordial follicles 279, 280 procedures under FCSRCA 41 procreative tourism 751 production process for rhFSH 453 products detection 329 proficiency testing 31, 36 progesterone 357 intramuscular route 9, 518–19 luteal supplementation 517, 649 luteal support protocols 522–4 oral route 518, 519, 520 during ovarian stimulation 468–9 pharmacokinetics 520 serum half-life by route 520 timing in oocyte donation 698 vaginal route 518, 519–20, 641 peculiarities 521 programmable freezers/freezing 263, 269 programmed cell death 170–1 programmed cycles 516–17 progressive nature of fragmentation 172 PROH 235 Prolan 416 Prometrium 520 pronuclear abnormalities apronuclear zygotes 300 centrosome inheritance, abnormal 298 single pronucleate zygotes 298–9 tripronuclear zygotes 299–300 uneven/distant pronuclei 300–1 examination 228 junction 196 orientation 225 pronuclear stage transfer (PROST) 604, 608 pronucleate embryos 212, 217, 224–5 pronucleate mouse embryo bioassay 210

Index

1258

pronuclei 195–6 1,2-propanediol (PROH) 236, 245, 248 and sucrose 249, 251, 243 prophylactic antibiotics 657–8 prostatic enzymes 63 protein kinases 109 protein source in IVM 125 protein supplementation 27 proteinase K/SDS buffer 327 proteins 358, 359 psychogenic anejaculation 586 psychogenic infertility model 719 psychological effects of surrogacy 709 psychological evaluation 722, 746 guidelines 723 psychological problems and irradiation 756 morbidity 666, 667 psychological screening for oocyte donors 696 of recipient couples 693 for surrogacy 705 psychological sequelae model 719 psychosocial counseling 726 psychosocial problems with multiple pregnancies 493 publication bias 390 pulsatility of FSH 418–19, 460 of GnRH 420, 483 of LH 418–19 of progesterone 515 pulsatility index (PI) 562, 578 pulsed Doppler 562 PureSperm 80 purification of hFSH 453–4 of hLH and hCG 454–5 of rhLH 455 pyknosis 114 pyruvate 125, 204, 224–32 quadruplet pregnancies 681 quality management 638–40 document control 51–2 under FCSRCA 41 personnel responsibilities 37–8 QA/QC relationship 27–8 quality assurance (QA) 27–33, 38 between accreditation bodies 48–9 at Fertilitetscentrum 55 quality control (QC) 27–33, 37

Index

1259

culture media 210 in egg retrieval 90 of fertilization medium 95 using database 368 quality system 51 organization 52 quality manual 50 method manual 53–4 SOPs 50, 53, 54 quality policy 50, 51 Fertilitetscentrum 57–8 standard operating procedures (SOPs) 50 quantitative fluorescent PCR 322 quarantine regulations 44, 261, 746 queries in database analysis 373 radiation 244 treatment 692 random effects model 389 randomization 384 randomized controlled trials 383–5 strengths and weaknesses 385 randomized controlled trials, multicentre 495 RANTES 346, 347 real time data handling 368 receptive phase/window 341, 350, 353–4 reciprocal translocations 285, 286 recombinant DNA technology 447–56 recombinant gonadotropins 425–6 clinical relevance of 442 and ovarian stimulation 439–42 in PCOS 433–5 see also under follicle-stimulating hormone; luteinizing hormone record maintenance under FCSRCA 42 recrystallization 234 rectourine pouch 89 recurrent miscarriages 292, 311–12 reinitiation of meiosis 108, 110 relational databases 371 religious issues 597, 713, 721 repeated implantation failure (RIF) assisted hatching in 544, 548–9, 554 coculture in 546, 551–2, 555 and oocyte donation 693 and PGD 544–5, 549–50 and salpingectomy 543–4, 546–8, 553 and ZIFT 613–14 reproduction cloning 744 Reproductive Laboratory Accreditation Program 42–3 residual secretion 464

Index

1260

resistance index 562, 578 resistance to impedance of blood flow 562 responsibilities, management 50 responsibility, legal, for offspring 750 restriction endonuclease digestion 324–5, 329 results of assisted hatching 164 reverse transcription PCR 322, 346, 347 risk factors ror extrauterine pregnancy 659 risk testing for CF/CBAVD offspring 288 risk testing for CF or CBAVD offspring 291 RKH syndrome 710 RNA 129 RNA synthesis 194 Robertsonian translocations 285, 286 Royal College of Obstetricians and Gynaecologists 403 Royal College of Pathologists 265 rubber 79 safety guidelines 41 safety measures 325–6 saline installation 565 salpingectomy 570 salpingectomy, prophylactic in RIF complications 553 methods 543–4 results 546–8 salpingoscopy 408 sampling liquid nitrogen 265 sanctions under CLIA’88 39 under FCSRCA 42 scanning, 3-D 407 scanning electron microscopy 344 SCID mice 280 Science Citation Index 387 scientific research analysis 368 scoring assessment for blastocysts 227 Scott zygote grading system 194 screening for genetic conditions 82 patients and donors 266, 693–7 see also aneuploidy; genetic; medical; psychological screw actuated syringe 143 secondary necrosis 177 secondary zygote scoring 199 sedation 509 seeding 234, 235, 247, 250

Index

1261

selection of abnormal embryos 310–11 criteria 387, 753 of spermatozoon 150 strategy 228–31 selective assisted hatching 548 selective termination 686–7 semen 63, 64–5 abnormal 77 analysis 61, 62, 148, 409 computer assisted 67–9 collection 78–9, 147 preparation 261–2 quality 61 semen laboratory 20 see also andrology seminal collection devices 62 seminiferous tubules procedure 81 sequential analysis of development 228–31 sequential culture media 227, 252, 254, 551 in blastocyte culture 554 and mouse embryos 204, 205 in ZIFT 608 sequential PCR 324 sequential protocol 433 Sertoli cell only syndrome 588 Sertoli cells 69 serum 27, 204, 207 in cryopreservation 246, 250 serum markers 357 set-up station 141, 142 severe male factor 285–93 sex chromatin 6 sex chromosomal abnormalities 291 sex chromosomal aneuploidies 83 sex ratio 254 with ICSI 83 sex selection 755 sexually transmitted diseases 44, 266–7, 555, 735 short flare protocol of GnRH agonists 487 short tandem repeats (STRs) 323–4 signal transduction factors 110 single blind trials 384 single cell PCR 325 single dose protocol of GnRH antagonists 494–5 single embryo transfer 216, 223, 666, 677 slow freeze-rapid thaw procedure 237 smoking 402 social issues 721 Society for Assisted Reproductive Technology (SART) 39, 677 accreditation requirements 42–3 Sorenson’s phosphate buffer standards 209

Index

1262

SOSFS 1985:35 48 spectral imaging (SKY) 297 sperm abnormalities 61, 65 characteristics 77 collection 62, 78–9 concentration 63–4, 67 count variation 64 cryopreservation 738, 740 evaluation 61–76 function testing 61, 73 selection 240 extenders for 262–3 immobilization 79, 150, 151 incorporation 100 morphology 65–7 movement motility 64 progression 64–5 vitality 65 penetration 77–87, 99, 100 assay 72 preparation methods 83–4 viability 70 see also testicular sperm sperm antibodies 69–70, 79 see also antisperm antibodies sperm dependent complications 82–3 sperm motion parameters 67 sperm washing media 276, 599 spermatogenesis, focal 588 spermatozoa retrieval, surgical 585–96 spermatozoa selection 150, 314 SpermFreeze 263 sperm:zona pellucida binding 72, 73 spinal bulbar muscular atrophy 289 spinal injuries and sperm collection 585–6 spindle structure abnormalities 120 spongiform encephalopathies 555 spontaneous pregnancy chances 397 spreadsheet format 376 staff see personnel standard operating procedures (SOPs) 50, 53, 54 standards in ART 35–6, 48 standards in health care 47 Steelman-Pohley bioassay 434 Stein-Leventhal 637 stem cells 4 step-down protocol 432–3, 439, 441, 647 step-up protocol conventional dose 430, 431 low dose 431–2

Index

1263

stereomicroscope 94, 141 steroid-depleted medium 342 steroid hormones 357–8 steroidogenesis 462 steroids, placental 515 sterols, meiosis-activating 126–7, 131 stimulation tests 467 stimulatory agent and protocol 646–8 storage regulations 758 stress 719, 720, 721 string of pearls 646, 647 stromal vascularity in PCO 637 studies, prospective randomized 475–6 study designs 383–6 subgroup analysis 389–90 subject withdrawals from trials 385 submucosal fibroid 406 sucrose as cryoprotectant 235, 236, 243 suction devices, spare 20 suction in aspiration 508 summary reports files 369, 370 in EggCyte 378–9 supercooling 246–7 supernumerary embryos 666 superovulation 111, 411, 436–42 induction of 100 support see under patients supraphysiological hormones 516, 645 surgical sperm retrieval 81, 585–96 protocols 590–6 surgical spermatozoa retrieval 153 surrogacy 703–16 counseling 705, 706–7 definitions 704 future directions 710–11 guidelines 715–16 host 704, 707–8, 709 counselling 706 indications for 704–5 legal issues 711–13, 748–9 patient management 707–8 patient selection 705, 715 problems encountered 709–10 religious issues 713 results 708–9 Surrogacy Act, proposed 712 Surrogacy Arrangements Act 712 surrogacy review team 711 surrogate motherhood 668 survival potential, fetal 665 SWEDAC 48, 55 Swedish board for accreditation and conformity assessment (SWEDAC) 47

Index

1264

Swedish national board of health 47 swim-up method 78, 79–80, 84 compared with CSGD 81, 82 synchronization, donor-recipient 698 synchronous development 361 systematic reviews 386 table schemes 374, 375 take home baby rates 475, 477 TALP-HEPES medium 93 Tanner’s pubertal development scale 402 targeted drug delivery 520 technical manager, laboratory 52 Teflon-coated aspiraion needles 505 temperature 139–40 during burning in 23 in cryopreservation 233–4, 244, 246–8 and laboratory design 19 of media/equipment 92, 93, 96 in oocyte handling 101, 117 of sperm 77 temperature elevation 657 teratology of GnRH analogs 478 termination, selective 687–8 Testarik zygote grading system 194 TESTCY buffer system 263 testicular biopsy 81, 85, 410–11, 588 protocol 593–4 testicular biopsy, open 587, 592–3 testicular cancer 588 testicular parenchyma 273 testicular sampling 148 testicular sperm 273–6 cryopreservation 274, 275–6 loss on cryopreservation 274 preparation 273–4 retrieval 148 testicular sperm extraction (TESE) 273, 287, 735 testicular surgery, repeated 273 testicular tissue homogenate 274 testicular tubular atrophy 288 tests for ovulation 404–9 tests of tubal function 408 thalassemia 335, 336, 337 thaw-transfer cycles 523–4 thawing of sperm 264, 269 thawing rates 244 theca 108 theca cells 428 therapeutic terminations of pregnancies 755 thermocycler 319

Index

1265

threshold level of LH 427 timing of embryo entry into uterus 609 of embryo transfer 211 of ICSI 186 of insemination 94 of MFPR 683 of oocyte maturity 122 of thawing/replacement 258–9 tissue banking, ovarian 279–83 Tomcat catheter 624, 625 toxicity checks, equipment 79 toxicity, oil 210 toxicology of GnRH analogs 478 traceability of contact materials 90 transabdominal ultrasound guidance 625, 626 transabdominal ultrasound scan 561, 563 transcervical tubal transfer 612 transcervical UET, difficult 618–19 transfected plasmids 453 transfection plasmid, structure of beta-FSH gene 450 FSH gene 449 transfer medium 601 transitional media 90–3 translocations, maternally derived 337 transplantation 131, 279–83 autologous 280, 283 see also autografted ovarian tissue transport IVF systems 17 transvaginal follicle aspiration 474 transvaginal oocyte aspiration 655, 691 transvaginal oocyte retrieval 629 transvaginal ultrasound scan 561, 563 and endometrial thickness 576 and extrauterine pregnancy 660 in GIFT 599, 600 in OHSS 646 for oocyte donors 695 for oocyte retrieval 115 of uterus 565 transzonal projections 108, 121 Treponema pallidum 44 triplet pregnancies 681, 682 triptorelin 486, 489, 495 tromboxane levels 578 trophectoderm biopsy 188 trophectoderm grading 228, 229 trophinin 358 tubal disease 393, 659 tubal dysfunction 396 tubal embryo transfer 608, 616

Index

1266

tubal epithelial coculture 551 tubal factor infertility 631, 676 tubal pregnancy 617 tubing, microsyringe 144 twin to twin transfusion syndrome 685 twins 664, 665 two cell-two gonadotropin theory 461 typing see grading Tyrode’s solution 92, 184, 544 acid Tyrode’s 161, 189 UK Association of Clinical Embryologists standards 57 ultrarapid freezing 237, 249–50 ultrashort protocols of GnRH agonists 487 ultrasonographic evaluation 655–6 ultrasonographic follow-up 687 ultrasound and estrogens 503–4 ultrasound diagnosis 685 ultrasound guidance for embryo transfer 625 ultrasound scan 561–80 2-D 407 3-D, cost of 565 baseline 571–2 baseline results form 563 follicular characteristics with 573–5 of follicular growth 501, 502–3, 572 future developments 580 of PCO 637 pelvic structures 563, 564 pre-treatment scan endometrium 575–80 fallopian tubes 569–71 hydrosalpinges 569–71 ovaries 566–9 uterus 564–6 techniques Doppler 561–4 grey-scale 2-D 561 see also Doppler; transabdominal; transvaginal ultrastructural components 357 UNESCO bioethics committee 755 unexplained infertility 396, 411 and CC challenge test 532 unilateral oophoretomy 312 US Registry National Summary and Fertility Clinics Report 615 uterine artery 563, 578–9 circadian rhythm 564 uterine capacity 664 uterine cavity, depth and direction 623

Index

1267

uterine contractions 211, 624 uterine embryo transfer (UET) 607, 609–10, 618 uterine fibroids 566 uterine fluids 206 uterine morphology 407 uterine receptivity 354, 356 assessment of 353–62 biomarkers available 357–61 in ZIFT 609 uterine receptivity defects 361 uterine senescence 397 uterine septum 565 uterine vascularization, impaired 580 uterus, absence of 704, 710 uterus preparation 515 Utrogestan 520, 522 vacuum application to follicle 505–7 vacuum profiles 506 vaginal ultrasound guided puncture 149 vanishing twin syndrome 664 vas deferens aspiration 586 vascular endothelial growth factor (VEGF) 637, 639–40, 641 Vero cells 546, 551, 554 viability of embryo 203 loss in cryopreservation 261 of oocytes 334 vibration and micromanipulation 144 vibrostimulation, penile 585 viscosity of semen 78 vital stains 70 vitality stains 409 vitamins 210 vitrification 237–8, 248, 250 volatile organic compounds and laboratory design 19 and new equipment 24 Wallace catheter 624, 625 Wallace-Edwards catheter 626 warming rates 275 Warnock Committee 703 wash and swim up method 84 washout period in trials 385 water as lytic agent 327 water quality control problems 29–30 website, clinic’s 725 welfare of offspring 745–50 Wertheim’s hysterectomy 710 WHO criteria of sperm morphology 65

Index

1268

whole genome amplification 322–3 working manual see standard operating procedures World Health Organization (WHO) criteria for normal semen 409 guidelines on subfertility 403 Wright-Giesma stain 65 Wyden bill 39–42 xenografting 280, 281 zona pellucida 159 abnormal hardening 544 drilling 184, 185, 186, 188, 544 “flap” method 184 and freezing 239, 274 hardening 159, 608 penetration 184, 334 removal in HEPA 72 and sperm motility 64 thinning 159, 162, 544 Zuclomiphene 413, 414 zwitterionic buffer 90, 92 zygote freezing 251 morphology 612 scoring 195–9 application of 199–200 selection in ZIFT 608, 612 splitting following ART 665 zygote intra-fallopian transfer (ZIFT) 95, 607–19 advantages and disadvantages 608–9, 610 clinical/technical aspects 616–19 indications for 613–16 nomenclature 608 procedure 610, 612–16 world experience 609–10

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